Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor Calcium-release Channel

Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor... Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor Calcium-release Channel Don-On Daniel Mak, Sean M.J. McBride, Nataliya B. Petrenko, and J. Kevin Foskett Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104 abstract The inositol 1,4,5-trisphosphate (InsP ) receptor (InsP R), a Ca -release channel localized to the 3 3 endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca signals in many cell types. Three InsP R isoforms are expressed in different subcellular locations, at variable relative levels with heteromulti- mer formation in different cell types. A proposed reason for this diversity of InsP R expression is that the isoforms 2 2 are differentially inhibited by high cytoplasmic free Ca concentrations ([Ca ] ), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca ] in me- diating high [Ca ] inhibition of InsP R gating by studying single endogenous type 1 InsP R channels through i 3 3 patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentra- tions of a calmodulin antagonist nor overexpression of a dominant-negative Ca -insensitive mutant calmodulin 2 2 affected inhibition of gating by high [Ca ] . However, a novel, calmodulin-independent regulation of [Ca ] in- i i hibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with 2 2 [Ca ] 400 nM were inhibited by 290 M [Ca ] , exposure of the isolated nuclei to a bath solution with ultra- 2 2 low [Ca ] (5 nM, for 300 s) before the patch-clamp experiments reversibly relieved Ca inhibition, with 2 2 channel activities observed in [Ca ] up to 1.5 mM. Although InsP activates gating by relieving high [Ca ] inhi- i 3 i bition, it was nevertheless still required to activate channels that lacked high [Ca ] inhibition. Our observations suggest that high [Ca ] inhibition of InsP R channel gating is not regulated by calmodulin, whereas it can be dis- i 3 rupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca ] inhibition may not be an immutable property of different InsP R isoforms. Furthermore, these ob- i 3 2 2 servations support an allosteric model in which Ca inhibition of the InsP R is mediated by two Ca binding sites, only one of which is sensitive to InsP . key words: single-channel electrophysiology • patch clamp • calcium • Xenopus oocyte • nucleus INTRODUCTION dation that differ during different stages of cell devel- opment and in response to extracellular stimuli (Taylor The second messenger, inositol 1,4,5-trisphosphate et al., 1999). Furthermore, formation of hetero-tet- (InsP ), is generated in many cell types through the rameric channels is possible in cell types expressing hydrolysis of phosphatidylinositol 4,5-bisphosphate by more than one InsP R isoform (Joseph et al., 1995; membrane-bound phospholipase C activated by plasma Monkawa et al., 1995; Wojcikiewicz, 1995; Nucifora et membrane receptors responding to extracellular stim- al., 1996). Although this diversity of InsP R expression uli. InsP then diffuses through the cytoplasm to bind is impressive, its functional correlates and physiological to its receptor (InsP R) in the ER and activate it as a implications remain unclear. Studies of the single-chan- 2 2 Ca channel to release Ca stored in the ER lumen. nel properties of the various InsP R isoforms have re- Modulation of the cytoplasmic free Ca concentration vealed that whereas their permeation and conductance 2 2 ([Ca ] ) by InsP R-mediated Ca release is a ubiqui- i 3 properties are very similar (Mak et al., 2000; Ramos- tous intracellular signal transduction mechanism that Franco et al., 2000), their gating may be differentially regulates numerous processes (Berridge, 1993). inhibited by high [Ca ] (Bezprozvanny et al., 1991; Three isoforms of the InsP R, with spliced variants, Hagar et al., 1998; Mak et al., 1998; Ramos-Franco et have been identified (Joseph, 1996). Most mammalian al., 1998a,b, 2000; Boehning et al., 2001; Mak et al., cell types express multiple InsP R isoforms in distinct 2001a). Because high [Ca ] inhibition of InsP R i 3 and overlapping intracellular locations with their abso- channel gating may be a pivotal feedback mechanism lute and relative expression levels regulated by gene transcription, alternative splicing and receptor degra- Abbreviations used in this paper: InsP , inositol 1,4,5-trisphosphate; Address correspondence to J. Kevin Foskett, Department of Physiol- InsP R, InsP receptor; NCaS, regular [Ca ] bath solution; PCaS, 3 3 ogy, B39 Anatomy-Chemistry Bldg/6085, University of Pennsylvania, physiological [Ca ] bath solution; r-InsP R-3, rat type 3 InsP R; 3 3 Philadelphia, PA 19104-6085. Fax: (215) 573-6808; email: foskett@ CaM, calmodulin; ULCaS, ultra-low [Ca ] bath solution; X-InsP R-1, mail.med.upenn.edu Xenopus type 1 InsP R. 569 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2003/11/569/13 $8.00 Volume 122 November 2003 569–581 http://www.jgp.org/cgi/doi/10.1085/jgp.200308808 The Journal of General Physiology for the regulation of intracellular Ca signaling (Tay- and therefore may not be an invariant property of a lor, 1998), it has been suggested that differential inhibi- specific InsP R isoform. Furthermore, these observa- 2 2 tion by high [Ca ] of the different InsP R isoforms tions support an allosteric model in which Ca inhibi- i 3 2 2 may generate distinct Ca signals in different cell types tion of the InsP R is mediated by two Ca binding with different patterns of InsP R isoform expression, sites, only one of which is sensitive to InsP . 3 3 and that this may be a reason for the diversity of InsP R MATERIALS AND METHODS expression (Hagar et al., 1998). It has been suggested that high [Ca ] inhibition of Heterologous Expression of Calmodulin in Xenopus Oocytes the InsP R is mediated by calmodulin (CaM), a ubiqui- tous Ca -binding protein that binds to and regulates Maintenance of Xenopus laevis and surgical extraction of ovaries the functions of many proteins. CaM was found to bind were performed as described previously (Mak and Foskett, 1994, 1997, 1998). Oocytes were defolliculated as described (Jiang et to the InsP R-1 in the presence of free Ca to a single al., 1998). cRNA (1 g/l) of rat calmodulin (CaM), either wild- site in the regulatory domain (Maeda et al., 1991; Ya- type (w.t.) or a quadruple mutant (q.m.) containing a D→A mu- mada et al., 1995; Hirota et al., 1999). Purified InsP R-1 tation in each of the four EF hands so that Ca binding in all EF channels lacking bound CaM were not inhibited by hands was abolished (Xia et al., 1998; Keen et al., 1999), was syn- high [Ca ] , whereas addition of CaM restored inhibi- thesized in vitro from cDNA provided as a gift by Dr. John P. Adelman (Vollum Institute, Portland, OR). 23 nl of cRNA (either tion of channel gating by high [Ca ] (Hirota et al., w.t. or q.m.) was injected into the cytoplasm of oocytes 1 d after 1999; Michikawa et al., 1999). The notion that high defolliculation, as described (Mak et al., 2000). cRNA-injected Ca inhibition of channel gating was mediated by CaM and uninjected control oocytes were maintained under identical was reinforced by observations that the type 3 InsP R 3 conditions in individual wells in 96-well plates containing 200 l (InsP R-3) did not bind CaM (Yamada et al., 1995; of ASOS (100 mM NaCl, 2 mM KCl, 1 mM MgCl , 1.8 mM CaCl , 2 2 5 mM HEPES, pH adjusted to 7.6 with NaOH; with 3 mM Na Cardy and Taylor, 1998; Lin et al., 2000) and was not in- pyruvate, 100 g/ml gentamycin, and 100 M N-acetyl-Leu-Leu- hibited by high [Ca ] (Hagar et al., 1998). Neverthe- Norleucinal; Sigma-Aldrich). 80 l of ASOS in each well was less, other data suggest that the role of CaM in high changed daily. Nuclear patch clamp experiments and immuno- [Ca ] inhibition of InsP R channel gating is far from i 3 precipitations were performed 2–4 d after c-RNA injection when unequivocal. Despite the absence of detectable interac- the expression level of exogenous CaM was stable as determined by Western analysis. tion between CaM and a mutant InsP R-1 in which the putative CaM binding site was eliminated (Yamada et Western Analysis and Immunoprecipitation al., 1995), more recent studies have demonstrated that Western analysis was performed on oocyte extracts (cRNA- this mutant channel is nevertheless still inhibited by injected and uninjected), as described in Mak et al. (2000), to as- high [Ca ] (Zhang and Joseph, 2001; Nosyreva et al., certain the levels of endogenous and heterologously expressed 2002). Furthermore, whereas the InsP R-3 lacks the 3 CaM in the oocytes using a specific antibody (Upstate Biotech- CaM binding site present in the InsP R-1 and no inter- nology). Immunoprecipitation of InsP R (type 1) and CaM was 3 3 performed using oocyte lysates, as described in (Mak et al., action between InsP R-3 and CaM has been detected 2000), with a specific type 1 InsP R antibody (Joseph and Sa- (Yamada et al., 1995; Cardy and Taylor, 1998; Lin et al., manta, 1993; Joseph et al., 1995) and protein A agarose (GIBCO 2000), electrophysiological studies of the recombinant BRL), and an antibody to CaM and protein G agarose (GIBCO rat InsP R-3 in its native membrane environment dem- 3 BRL), respectively. onstrated that it is nevertheless inhibited by high Solutions for Patch Clamp Experiments [Ca ] (Mak et al., 2001a) with quantitative features similar to those of inhibition of the InsP R-1 in the All patch clamp experiments were per formed with solutions con- taining 140 mM KCl and 10 mM HEPES with pH adjusted to 7.1 same membrane (Mak et al., 1998). 2 2 with KOH. The free Ca concentration ([Ca ] ) of the pipette Here, we investigated the possible effects of CaM on solutions (to which the cytoplasmic side of the InsP R is exposed high [Ca ] inhibition of the gating of single endoge- in patch-clamp experiments) was tightly controlled by buffering nous InsP R-1 channels in their native membrane envi- 3 various amounts of added CaCl (40–400 M) with 500 M of ronment using nuclear membrane patch clamp electro- the high-affinity Ca chelator, BAPTA (1,2-bis(O-aminophe- noxy) ethane-N,N,N,N-tetraacetic acid; Molecular Probes) and physiology (Mak and Foskett, 1994). Our experiments 0.5 mM Na ATP (100 nM  [Ca ]  2.5 M); or 500 M of 2 i do not provide evidence supporting any role for CaM the low-affinity Ca chelator, 5,5-dibromo BAPTA (Molecular in this process. However, we discovered a novel regula- Probes) and 0.5 mM Na ATP (5 M  [Ca ]  15 M); or 0.5 2 i tion of high [Ca ] inhibition of InsP R-1 channel gat- 2 i 3 mM Na ATP alone (15 M  [Ca ]  300 M). Solutions with 2 i 2 2 ing. Inhibition of InsP R-1 gating by high [Ca ] can [Ca ]  300 M contained no Ca chelator for buffering. The 3 i i normal Ca bath solution (NCaS) contained 500 M BAPTA be reversibly abrogated by exposure of the channel to a and 250 M CaCl (free [Ca ]  400–500 nM), and the physio- bathing solution containing ultra-low [Ca ] (5 nM). logical Ca bath solution (PCaS) contained 500 M BAPTA and Our observations indicate that inhibition of InsP R-1 3 2 2 70 M CaCl (free [Ca ]  48  5 nM). The free [Ca ] of channel gating by high [Ca ] can be disrupted by en- 2 i these solutions was directly measured using Ca -selective mini- vironmental conditions experienced by the channel, electrodes (Baudet et al., 1994). The ultra-low Ca bath solution 570 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology (ULCaS) contained 1 mM BAPTA and no added CaCl . The con- for channel open probability (P ) evaluation. The number of 2 o taminating [Ca ] in the solution was determined by induction- channels in the membrane patch was assumed to be the maxi- coupled plasma mass spectrometry (Mayo Medical Laboratory) mum number of open channel current levels observed through- to be 6–10 M. Ca -selective minielectrodes were unable to out the current record. In experimental conditions with P  0.1, determine accurately the free [Ca ] in the ULCaS because of only current records with longer than 10 s of InsP R channel ac- the nonlinear response of the electrode in free [Ca ]  5 nM. tivities were used for determination of P , so there is little uncer- Free [Ca ] was calculated using the Maxchelator software (C. tainty in the number of channels in the current traces used. In Patton, Stanford University, Stanford, CA) to be 0.9–1.5 nM. experimental conditions with P  0.1, only current records ex- Unless specified otherwise, all pipette solutions contained a hibiting one open channel current level with InsP R channel ac- saturating concentration (10 M) of InsP (Mak and Foskett, tivities lasting longer than 30 s were used, to ensure that they 1994) from Molecular Probes. When specified, the pipette solu- were truly single-channel records (Mak et al., 2001a). The P data tions also contained 500 M W-7 (a CaM binding antagonist; shown for each set of experimental conditions are the means of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochlo- results from at least four separate patch-clamp experiments per- ride; Calbiochem), or 10 M purified bovine CaM (Calbio- formed under the same conditions. Error bars indicate the SEM. chem). All reagents were used with no further purification. RESULTS Oocyte Nucleus Isolation Protocols Lack of Effect of Calmodulin on Ca Inhibition of InsP R A stage V or VI oocyte was gently teased open mechanically in the isolation bathing solution, enabling the translucent nucleus to be Gating in Endoplasmic Reticulum Membrane isolated from the cytoplasmic material. The isolated nucleus was either directly transferred to the experimental bathing solution Previous single-channel patch-clamp studies of the en- (protocol Nd, Ld, and Pd, Fig. 1), or it was transferred through a dogenous Xenopus type 1 InsP R (X-InsP R-1) in its na- 3 3 series of culture dishes containing 4–5 ml of incubation bath so- tive ER membrane environment revealed a biphasic lutions (protocol L, LN, and LNL, in Fig. 1) before it was ulti- regulation by [Ca ] of the single-channel open proba- mately transferred to the experimental bath. The nucleus re- bility (P ) (Mak et al., 1998, 2001b). It has been sug- mained in each incubation bath for at least 20 min before the o next transfer, to ensure that the solution in the perinuclear lu- gested that calmodulin (CaM) bound to the channel men between the outer and inner nuclear envelope had attained mediates inhibition of InsP R-1 gating by high [Ca ] 3 i ionic equilibrium with the bath solution (Mak and Foskett, (Michikawa et al., 1999). We therefore investigated the 1994). Approximately 20 l of the previous bath solution accom- possibility that the high [Ca ] inhibition of X-InsP R-1 i 3 panied the nucleus to the new bath in a transfer. The culture channel gating observed in our previous studies was dish containing the nucleus in the experimental bath solution was finally moved onto the stage of the inverted microscope mediated by CaM. Oocyte nuclei were isolated and where patch clamp experiments were per formed. transferred directly into an experimental bath of NCaS for patch-clamp experiments (protocol Nd in Fig. 1). Acquisition and Analysis of Single-Channel Patch-clamp By repeated patch clamping over the surface of an iso- Current Records lated nucleus, regions on the outer nuclear envelope The isolated nucleus was gently immobilized as described previ- were identified in which the probability of detecting ously (Mak and Foskett, 1994) so that membrane patches could InsP R channel activities in membrane patches (P ) 3 d be repeatedly obtained from the same region (2 m) of the was high (Mak and Foskett, 1997). A series of patch- outer nuclear membrane (Mak and Foskett, 1997). Due to abrupt termination of channel activity (Mak and Foskett, 1994, clamp experiments was performed at these regions 1997), patch clamp experiments were per formed in “on-nucleus” with pipette solutions (to which the cytoplasmic side of configuration to maximize the duration of channel activities re- the InsP R was exposed) alternately containing either corded. To prevent contamination of the pipette solution by the bath solution (especially the Ca chelator in the bath solution) by diffusion through the pipette tip during the time when the pi- pette was immersed in the bath and before giga-Ohm seal forma- tion, a positive pressure (10 mmHg) was maintained inside the pipette until the pipette tip was properly positioned on the nu- clear membrane. Then suction was applied in the pipette to ob- tain the giga-Ohm seal. All experiments were per formed at room temperature with the pipette electrode at 20 mV relative to the reference bath electrode unless specifically stated otherwise. Each experiment recorded the InsP R channel activity at a spe- cific [Ca ] and [InsP ], with no change of the pipette or bath i 3 solutions during the experiment. Data acquisition was per- formed as previously described (Mak et al., 1998), with currents recorded with a filtering frequency of 1 kHz and a digitizing fre- quency of 5 kHz. The patch clamp current traces were analyzed using MacTac software (Bruxton) to identify channel-opening and -closing events using a 50% threshold. Current traces exhibiting one InsP R channel, or two InsP R channels determined to be identi- Figure 1. Schematic diagram showing the various protocols 3 3 cal and independently gated (Mak and Foskett, 1997), were used used to isolate oocyte nuclei for nuclear patch clamp experiments. 571 Mak et al. The Journal of General Physiology Figure 3. Typical current traces from nuclei in NCaS bath with pipette solutions containing 10 M InsP . Arrows indicate closed channel current levels. (A and B) Uninjected oocytes were used. InsP R channel activity was observed with [Ca ] of 755 nM (A, 3 i n  3), whereas no channel activity was observed in a membrane Figure 2. Western blots of oocyte lysates probed with a CaM anti- patch obtained from the same region of the same nucleus with body (both w.t. and q.m.). Lysates from oocytes injected with CaM [Ca ] of 290 M and the pipette solution containing 500 M W-7 cRNA (w.t. or q.m.) or uninjected oocytes were used as labeled. (B, n  5). (C and D) Oocytes injected with CaM q.m. cRNA were Oocytes used for lanes A and B or C and D were from the same used. InsP R channel activity was observed with [Ca ] of 2.1 M 3 i batches, respectively. Top arrow indicates wild-type CaM and the (C, n  4), whereas no channel activity was observed in a mem- bottom arrow indicates the quadruple mutant CaM. The slightly brane patch obtained from the same region of the same nucleus faster mobility of q.m. CaM is likely a reflection of the known Ca - with [Ca ] of 290 M (D, n  9). binding dependence of CaM mobility in gels (Xia et al., 1998). 2 2 [Ca ]  755 nM, or very high [Ca ] (290 M) with pression on SK channel gating provided evidence that i i 500 M of W-7, a CaM binding antagonist. The former endogenous CaM is tightly and constitutively (even in solution is one in which the channel gates with a high the absence of Ca ) associated with the SK channel P , thereby ascertaining the presence of functional and mediates the effects of Ca on SK channel gating. InsP R channels in the regions selected during the se- The ability of high Ca concentrations to inhibit ries of experiments. In contrast, the latter solution has InsP R channel gating in our in vitro electrophysiologi- [Ca ] sufficiently high to inhibit InsP R channel gat- cal studies can be observed for long times (up to 2 h) i 3 ing (Mak et al., 1998). Because CaM is endogenously after isolation of the nuclei (Mak et al., 1998; Boehning expressed in Xenopus oocytes (Fig. 2, Lane A and C), we et al., 2001; Mak et al., 2001b). Thus, if CaM mediates 2 2 reasoned that if CaM mediated the high [Ca ] inhibi- the effect of high [Ca ] , it must remain associated i i tion of InsP R channel gating, then inclusion of 500 with the channel in the isolated nuclei, and therefore M of W-7 in the pipette solution may block high must be tightly bound to the InsP R and not free to dif- [Ca ] inhibition by interfering with CaM binding to fuse away into the large experimental bath. We there- the InsP R channel (Michikawa et al., 1999), making fore explored the possibility that Ca inhibition of channel gating observable in the 290 M [Ca ] solu- InsP R channel gating was mediated by a constitutive i 3 tions. Nevertheless, no channel activity was detected in tight association of CaM with the channel, by examin- 2 2 any of the five patches with 500 M W-7 and [Ca ]  ing the effects of overexpression of the Ca -insensitive 290 M (Fig. 3 B), whereas InsP R channel activities quadruple mutant (q.m.) CaM on the Ca regulation were readily detected in five out of six patches with of the InsP R. [Ca ]  755 nM (Fig. 3 A). The q.m. CaM, which has all EF hands mutated and Whereas this result with W-7 is seemingly inconsistent therefore is Ca insensitive, was overexpressed in Xeno- with the hypothesis that CaM mediates Ca inhibition pus oocytes by cytoplasmic microinjection of cRNA. of InsP R gating, CaM-dependent regulation of the Western analysis (n  5) indicated that the exogenous small-conductance Ca -activated K (SK) channel gat- q.m. CaM was expressed to a level that was at least an ing is insensitive to W-7 and other CaM inhibitors (Xia order of magnitude higher than the endogenous wild- et al., 1998). However, overexpression of a mutant type CaM (Xia et al., 1998; Fig. 2). Patch-clamp experi- CaM, in which the Ca -binding EF hand motifs were ments using nuclei isolated by protocol Nd (Fig. 1) disabled, interfered with the Ca activation of the SK from q.m. CaM-expressing oocytes revealed that InsP R channel gating by competing with the endogenous channel gating was still inhibited by high [Ca ] : CaM for the interaction with the channels (Xia et al., InsP R channel activities were detected in 11 out of 11 1998; Keen et al., 1999). The effects of mutant CaM ex- patches with pipette solutions containing [Ca ]  2.1 572 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology 2 Figure 5. [Ca ] dependencies of the channel P of the InsP R i o 3 in oocyte nuclei isolated using various protocols (Nd, L, and LNL) and applied potentials (20 mV) as tabulated. All pipette solu- tions used contained 10 M InsP . The dashed curve is a simple ac- Figure 4. Typical current traces from nuclei in ULCaS bath iso- tivating Hill equation fit for the data from nuclei isolated with pro- lated by protocol L. Arrows indicate closed channel current levels. tocol L (large open circles). For comparison, the biphasic Hill Pipette solutions contained 10 M InsP and [Ca ] as tabulated. equation fit (continuous curve) for the data points from nuclei iso- 3 i The last current trace was obtained with 20 mV applied trans- lated directly into NCaS bath (small filled circles) obtained in a membrane potential. Other current traces were obtained with previous study (Mak et al., 1998) are also shown. The InsP R chan- 20 mV applied potential. nel P was lower in ULCaS than in NCaS at [Ca ]  100 nM. It is o i possible that this reflects some intrinsic properties of the InsP R af- ter exposure to the low bath [Ca ]. Alternately, this may only be an artifact as a result of the movement of free Ca ion across the M (Fig. 3 C), but no channel activity was detected in 2 open channel. With pipette [Ca ]  100 nM, when the oocyte any of 9 patches with pipette solutions containing 290 nucleus was in NCaS ([Ca ]  400–500 nM), the Nernst reversal 2 2 potential for Ca ions was 35 mV so Ca ions moved across the M [Ca ] (Fig. 3 D). These results therefore also did 2 open InsP R channel from the lumenal side to the cytoplasmic not support the hypothesis that Ca inhibition of side despite an applied transmembrane voltage of 20 mV. This InsP R channel gating is mediated by CaM. 3 2 2 could cause the effective [Ca ] at the activating Ca -binding The lack of effect of overexpression of the q.m. CaM sites on the cytoplasmic side of the channel to be higher than the 2 2 on Ca inhibition of gating may suggest that endoge- free [Ca ] in the bulk of the pipette solution if the Ca -binding sites are close enough to the ion conducting pore. Conversely, nous CaM is not normally associated with the InsP R. 2 2 when the nucleus was in ULCaS ([Ca ]  5 nM), Ca ions We examined the biochemical association between the moved across the open InsP R channel in the opposite direction, InsP R and CaM by coimmunoprecipitation. Using ly- down the electrical and chemical gradients, possibly lowering the sates prepared from cRNA-injected oocytes overex- 2 2 2 effective [Ca ] at the Ca -binding sites. In [Ca ]  250 nM, i i pressing either w.t. or q.m. CaM (Fig. 2), immunopre- the mean open channel duration (< >) of the InsP R increases o 3 2 2 with [Ca ] (Mak and Foskett, 1998). Therefore, if Ca flux cipitation of the endogenous type 1 InsP R with a spe- across the open InsP R channel caused the effective [Ca ] at the 3 i cific antibody did not coimmunoprecipitate either w.t. 2 2 activating Ca -binding sites to deviate from the free [Ca ] in the or q.m. CaM (n  4; unpublished data). In the con- bulk pipette solution, then channels in NCaS bath would have verse experiments, immunoprecipitation of CaM with longer < > and higher channel P than those in ULCaS bath, as o o an antibody that binds to both w.t. and q.m. forms did observed. On the other hand, in [Ca ]  300 nM, < > does not i o exhibit any dependence on [Ca ] although the mean closed not coimmunoprecipitate the InsP R (n  4; unpub- channel duration (< >) is still affected by [Ca ] (Mak and Fos- c i lished data). These results therefore do not provide evi- 2 2 kett, 1998). Deviation of effective [Ca ] at the Ca -binding sites dence of an association between CaM and the InsP R. 3 2 from the bulk free [Ca ] would dissipate quickly by diffusion In summary, our single-channel patch clamp experi- once the channel closed and therefore would not affect < >. ments revealed that neither high concentrations of a Thus, there would be no difference between the observed P of InsP R in ULCaS and NCaS bath in [Ca ]  300 nM, as ob- CaM antagonist, nor overexpression of a Ca -insensi- 3 i 2 served. tive q.m. CaM had any effect on [Ca ] inhibition of InsP R channel gating. In addition, coimmunoprecipi- tation failed to demonstrate an association between vitro patch clamp studies is mediated by CaM. These CaM and the InsP R. Thus, our investigations did not conclusions are therefore in agreement with those provide any evidence supporting the hypothesis that reached in some other studies (Zhang and Joseph, high [Ca ] inhibition of InsP R gating observed in in 2001; Nosyreva et al., 2002). i 3 573 Mak et al. The Journal of General Physiology 2 We previously demonstrated that the Ca depen- Abrogation of Ca -dependent Inhibition of dence of channel P in nuclei isolated by protocol Nd InsP R Channel Gating into NCaS was well fitted by a biphasic Hill equation Our experimental results suggested that CaM is not in- –1 –1 H H  act inh volved in the inhibition of InsP R channel gating by 2  2 P = P 1 +() K ⁄[] Ca 1C +() []a ⁄ K (1) i i o max act  inh high [Ca ] . However, it remained possible that a  different molecule may be involved, and that condi- with maximum channel open probability (P ) max tions could be identified which would strip such a puta- 0.81  0.02, half-maximal activating [Ca ] (K ) i act tive effector from the InsP R in the isolated nucleus, 210  20 nM, activation Hill coefficient (H )  1.9 2 act thereby rendering the InsP R insensitive to Ca inhibi- 0.3, half-maximal inhibitory [Ca ] (K )  54  3 i inh tion. We reasoned that the putative effector, as a sensor M, and inhibitory Hill coefficient (H )  3.9  0.7 2 2 inh of [Ca ] , might be dependent on normal [Ca ] for i i (Mak et al., 1998). Our new data indicated that the its association with the InsP R. We therefore incubated InsP R in nuclei isolated by protocol L into ULCaS ex- 2 3 the isolated nuclei in an ultra-low Ca bath solution 2 2 hibited no inhibition by high [Ca ] , so that the Ca (ULCaS) before using them for nuclear patch clamp dependence of channel P can be fitted by a simple ac- 2 o experiments to determine the Ca dependence of the tivating Hill equation InsP R gating. –1 In the first set of experiments, nuclei were isolated by H  act P = P 1 +() K ⁄[] Ca , (2) 2 o max act protocol L (Fig. 1) into a bath of ULCaS ([Ca ]  5  nM). In the presence of 10 M cytoplasmic (pipette) with maximum open probability P of 0.84  0.01, 2 max [InsP ] and [Ca ]  20 M, gating of the InsP R ex- 3 i 3 half-maximal activating [Ca ] (K ) of 280  30 nM, i act posed to the ULCaS was very similar to that of InsP R in and activation Hill coefficient (H ) of 2.7  0.3 (Fig. 5). act nuclei isolated directly into NCaS by protocol Nd (Fig. 4; Nuclei isolated directly into a ULCaS bath by proto- Mak et al., 1998). In both cases, channel P was low col Ld were used to determine the minimum duration (0.2) in [Ca ]  150 nM, it increased dramatically to of exposure to ULCaS bath required to relieve high 0.8 as [Ca ] was increased from 150 nM to 1M, and [Ca ] inhibition of InsP R gating. We found that i 3 then P remained at the maximum level of 0.8 when channel activities could be detected with a pipette solu- [Ca ] was further increased from 1 to 20 M (Fig. 5). tion containing 10 M InsP and 290 M [Ca ] no 3 i The InsP R in nuclei isolated by protocol Nd were inhib- earlier than 5 min after the nucleus was isolated into ited by [Ca ]  20 M (Mak et al., 1998) but, remark- the ULCaS bath. Thus, the process involved in the re- ably, InsP R in nuclei isolated into ULCaS by protocol L lief of Ca inhibition of InsP R channel gating by ex- 2 3 exhibited robust channel activities in [Ca ] as high as posure of the isolated nuclei to ULCaS is a slow one, re- 1.5 mM (Fig. 4) with no decrease in channel P (Fig. 5). quiring a few minutes. Thus, a 20-min exposure to the ULCaS containing 5 To determine if normal cytoplasmic [Ca ] (50 nM Ca caused the gating of InsP R channel to be no nM) is low enough to cause the relief of high [Ca ] 2 i longer inhibited by high [Ca ] . All of the InsP R chan- i 3 inhibition of InsP R gating, we isolated oocyte nuclei 2 3 nel activities observed in the ultra-low [Ca ] bath solu- directly in PCaS bath (protocol Pd, Fig. 1). In a series tion also terminated abruptly after 30 s, like those pre- of experiments performed in areas of the nuclear viously observed in the regular bath solution (Mak and membrane identified with very high P , using pipette Foskett, 1994, 1997). solutions with 10 M InsP and 0.5 mM ATP, contain- 2 3 Because of the difference between the free Ca con- ing alternately 630 nM or 221 M [Ca ] , InsP R chan- 2 i 3 centration in the high [Ca ] pipette solutions and ultra- nels were observed in seven out of seven patches with low [Ca ] bath solutions, it is possible that a potential 630 nM [Ca ] , but no InsP R channel activity was ob- i 3 difference may be established across the membrane and served in any of 11 patches with 221 M [Ca ] , even 2 i affect the high [Ca ] inhibition of the InsP R and thus i 3 when the nucleus was exposed to the PCaS bath for its P . We performed patch clamp experiments with 20 over 160 min. Thus, the normal resting [Ca ] of the mV applied potential, using high [Ca ] pipette solution cytoplasm (50 nM) is not sufficiently low to induce ([Ca ]  221 M) with nuclei isolated with protocol L. the relief of Ca inhibition observed in the ultra-low The InsP R channel P (9 out of 20 patches exhibited 3 d Ca condition. channel activity), gating kinetics (last current trace in Fig. 4), and P (Fig. 5) were not detectably different from that InsP Dependence of the InsP R in ULCaS Bath 3 3 recorded at 20 mV (P  6 out of 8 patches), indicating that the abrogation of high [Ca ] inhibition by expo- Our previous studies (Mak et al., 1998, 2001a) revealed sure to ULCaS is not due to simple electrostatic effects that InsP activates gating by relieving the Ca inhibi- that change the membrane potential. tion of the channel. InsP increases K , the inhibitory 3 inh 574 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology 2 half-maximal [Ca ] , with no effect on the values of the channel Ca activation parameters (K , H ) or act act P in Eq. 1. It seemed likely that this mode of InsP max 3 activation cannot operate if the channel is not inhib- ited by high [Ca ] as observed after the channel had been exposed to the ULCaS bath for a few minutes. We therefore examined whether InsP was still required to gate the InsP R under conditions that abrogated Ca inhibition of the channel. A series of experiments was performed using nuclei isolated by protocol L into ULCaS bath, patching in re- gions of the nuclei identified to exhibit high P , with pi- pette solutions alternately containing either 10 M 2 2 InsP and [Ca ]  755 nM, or no InsP and [Ca ] 3 i 3 i between 60 nM and 290 M. Again, the former solu- tion was used to ascertain the presence of functional InsP R channels in the regions of the isolated nuclei se- lected for our experiments for the entire duration of the series. InsP R channel activities were observed in 27 out of 30 membrane patches in the presence of InsP , but no channel activity was detected in any of the 10 patches without InsP (Fig. 6 A). Therefore, even though the InsP R was no longer inhibited by high Figure 6. (A and B) Typical current traces from nuclei isolated [Ca ] when the nucleus was isolated into ULCaS, with protocol L. Arrows indicate closed channel current levels. (A) InsP was nonetheless still necessary for channel gating. 2 3 The pipette solutions contained no InsP and 290 M [Ca ] as 3 i Because it seemed paradoxical that InsP activates tabulated. (B) The pipette solutions contained 10 nM InsP and 3 3 2 2 [Ca ] as tabulated. (C) [Ca ] dependence of the channel P of channel gating by modulating the ability of Ca to in- i i o the InsP R in the presence of various [InsP ] as tabulated. The 3 3 hibit the channel, and yet InsP R channels that exhibit number of channels used to evaluate each of the data points (n) is no high [Ca ] inhibition still require InsP for gating, i 3 tabulated next to the corresponding data point. Oocyte nuclei we examined the effects of subsaturating [InsP ] on 3 used were isolated using protocol L. The curves are simple activat- channel gating under conditions that abolish high ing Hill equation fits (Eq. 2) with the same K  280 nM and act H  2.7. The dashed, dotted, and continuous curves have P [Ca ] inhibition. It was shown previously that in the act max 0.18, 0.35, and 0.84 for [InsP ]  10 nM, 20 nM, and 10 M, re- presence of a subsaturating concentration of InsP spectively. (10–33 nM), InsP R channels isolated directly into NCaS (protocol Nd) were much more sensitive to Ca inhibi- tion than those exposed to higher [InsP ] (Mak et al., 2 2 Reversibility of the Regulation by Bath [Ca ] of Ca 1998). In contrast, we observed that channels in nuclei Inhibition of the InsP R Channel isolated into and incubated in ULCaS (protocol L), and activated by subsaturating concentrations of InsP (10– It is possible, as we stated before, that the inhibition of 2 2 20 nM) exhibited no Ca inhibition. Channel activities InsP R gating by high [Ca ] is mediated by some mol- 3 i were observed in 340 M [Ca ] , a normally inhibiting ecule that is tightly bound to the InsP R in the NCaS i 3 2 2 [Ca ] , as well as in 4.2 M [Ca ] (Fig. 6 B) with simi- bath, and that dissociates from the channel in the pres- i i lar channel P (Fig. 6 C). Importantly, the maximum P ence of extremely low [Ca ] in the ULCaS bath. Disso- o o observed in subsaturating [InsP ] was lower than that ciation of this putative effector from the InsP R chan- 3 3 observed in saturating [InsP ] (c.f. Fig. 4, [Ca ]  5.5 nel can then render the channel insensitive to inhibi- 3 i and 340 M, and Fig. 6 B). Within the subsaturating tion by high [Ca ] . Accordingly, after dissociation, the range, i.e., [InsP ] 100 nM, increasing [InsP ] af- putative effector molecule could possibly diffuse away 3 3 fected the channel activity mainly by tuning the value of into the essentially infinitely large volume of the bath. P in the simple activating Hill equation (Eq. 2) (Fig. If this model is correct, the loss of Ca inhibition max 6 C), instead of affecting the Ca inhibitory parame- should be irreversible. To explore the reversibility of ters (K or H ), but not P in the biphasic Hill the loss of Ca inhibition, we performed patch-clamp inh inh max equation (Eq. 1), as normally observed in the InsP R experiments on nuclei isolated from the same batch of channel exposed to NCaS (Mak et al., 1998). Thus, the oocytes using different isolation/incubation protocols. effect of InsP on the InsP R channel in ULCaS was dra- As described above, Ca inhibition was abrogated 3 3 matically different from that observed in NCaS. when the nuclei were isolated into ULCaS bath by pro- 575 Mak et al. The Journal of General Physiology Figure 7. Typical current traces obtained from nuclei isolated us- ing different protocols, all from the same batch of oocytes. Arrows indicate closed channel current levels. All pipette solutions con- tained 10 M InsP . (A) InsP R channel activity in 290 M [Ca ] 3 3 i in nuclei isolated by protocol L into ULCaS, n  5. (B) Absence of InsP R channel activity in 290 M [Ca ] in nuclei isolated by pro- 3 i tocol LN, n  11. (C) InsP R channel gating in 5.5 M [Ca ] 3 i (n  2) in the same nucleus as used in B. (D) InsP R channel activ- ity in 290 M [Ca ] in nuclei isolated by protocol LNL, n  4. tocol L (Fig. 7 A). However, when the nuclei were re- Figure 8. (A) Typical current traces obtained from nuclei iso- turned to the NCaS bath for 20 min before patch lated from uninjected oocytes using protocol L with pipette solu- clamping (protocol LN, Fig. 1), no InsP R channel ac- tion containing 10 M InsP , 290 M [Ca ] , and 10 M purified 3 3 i CaM, n  4. (B–D) Typical current traces obtained from nuclei iso- tivities were detected at [Ca ]  290 M (Fig. 7 B) in lated from oocytes expressing q.m. CaM with pipette solution con- any of the 11 patches obtained, even though channel taining 10 M InsP and 290 M [Ca ] . InsP R channel activity 3 i 3 gating was observed in 4 out of 5 patches using pipette was observed in 290 M [Ca ] in nuclei isolated by protocol L solutions with [Ca ]  5.5 M (Fig. 7 C). Thus, de- (B, n  6) or protocol LNL (D, n  8), but not in nuclei isolated spite prior exposure to ULCaS, normal Ca inhibition by protocol LN (C, n  9). Arrows indicate closed channel current levels. (E) Histogram of InsP R channel P at 10 M InsP and 290 of InsP R channel gating was restored when the nuclei 3 o 3 M [Ca ] observed in various nuclei under experimental condi- were transferred back into NCaS. This restoration of tions as tabulated. normal Ca inhibition was in turn reversible. Reexpo- sure of the nuclei to ULCaS (protocol LNL, Fig. 1) again eliminated normal Ca inhibition of gating (Fig. the bath [Ca ]. The working hypothesis was that addi- 7 D). The InsP R channels in nuclei isolated by proto- 3 tion of CaM would restore normal inhibition of chan- col LNL exhibited the same P (Fig. 5, filled square) as o nel gating by high [Ca ] after it had been relieved by those in nuclei isolated into ULCaS by protocol L with- exposure to the low [Ca ] bath. Patch-clamp experi- out ever being exposed to NCaS (Fig. 5, open circles). ments were performed on nuclei isolated by protocol L These experiments indicated, first, that abolition of into ULCaS bath, using a pipette solution containing Ca inhibition of channel gating by exposure of nuclei 10 M purified CaM with 10 M InsP and high [Ca ] 3 i to ultra-low bath [Ca ] was fully reversible, and sec- (290 M). Nevertheless, InsP R channel gating was ob- ond, that it was affected only by the [Ca ] of the bath- served in the presence of CaM (Fig. 8 A) that was indis- ing solution in which the patch-clamp experiments tinguishable (P  0.05, Fig. 8 E) from that observed un- were performed, independent of the history of bath der the same conditions without CaM (compare Fig. 4, [Ca ] to which the nuclei were previously exposed. [Ca ]  290 M). Thus, addition of CaM did not re- These results suggest either that the sensitivity of Ca constitute normal high [Ca ] inhibition of channel inhibition of the InsP R to the bath [Ca ] is an in- 3 gating. trinsic property of the InsP R channel, or that it is 3 We also performed a series of patch-clamp experi- mediated by some molecule that remains in a stable ments on nuclei isolated from oocytes expressing the complex with the channel throughout the multiple Ca -insensitive q.m. CaM. When the nuclei were iso- transfers of the nucleus into various baths containing lated by protocol L into ULCaS bath, InsP R channel different [Ca ]. activities were observed in high (290 M) [Ca ] (six of eight patches, Fig. 8 B) as frequently as in the nor- 2 2 Is CaM Involved in the Regulation by Bath [Ca ] of Ca mally “permissive” [Ca ] between 500 nM and 5.5 M Inhibition of InsP R Channel? (seven of eight patches). Furthermore, the channel P We explored the possible role of CaM in mediating the was the same as that observed in the channels in nuclei novel regulation of Ca inhibition of InsP R gating by isolated by protocol L from uninjected oocytes (P 576 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology 2 0.05, Fig. 8 E). In addition, expression of the q.m. CaM hibited by high [Ca ] in our nuclear patch-clamping had no effect on the reversibility of the low [Ca ] bath experiments (Fig. 3, A and B). Second, overexpression effect. Thus, no InsP R channels were detected in any in oocytes of a dominant-negative, Ca -insensitive q.m. of the nine patches from nuclei isolated from mutant CaM did not interfere with normal Ca inhibition of CaM-expressing oocytes by protocol LN (Fig. 8 C). the X-InsP R-1 in the oocyte nuclear envelope (Fig. 3, C Moreover, high [Ca ] inhibition of the channel was and D). Third, addition of CaM (10 M) to the pipette still completely abrogated in nuclei isolated from mu- solution did not reconstitute normal Ca inhibition of tant CaM-expressing oocytes by protocol LNL. Thus, InsP R channel after it was abrogated by exposure of channel activities were observed in 290 M [Ca ] the channel to ULCaS bath (Fig. 8 A). Fourth, overex- (Fig. 8 D) with P (8 of 13 patches) similar (P  0.05) pression of q.m. CaM in oocytes did not affect the abro- 2 2 to that in [Ca ] between 500 nM and 5.5 M (seven gation of Ca inhibition by exposure of the channel to of eight patches). InsP R channel P in these nuclei ULCaS bath, nor did it affect the restoration of Ca in- 3 o from mutant CaM-expressing oocytes was the same as hibition when the channel was placed back in NCaS that observed in nuclei isolated from uninjected oo- bath (Fig. 8, B–D). Furthermore, coimmunoprecipita- cytes by protocol L or LNL (P from t test was 0.05, tion experiments did not detect any association be- Fig. 8 E). Therefore, there were no differences between tween CaM and InsP R-1 in the Xenopus oocytes. There- 2 2 the Ca inhibition (or lack thereof) of InsP R channel fore, whereas CaM may regulate intracellular Ca sig- gating observed in nuclei isolated by various protocols naling through other mechanisms, our experimental from oocytes overexpressing the mutant CaM and that results, together with other recent publications (Zhang observed in nuclei isolated from uninjected oocytes, and Joseph, 2001; Nosyreva et al., 2002), indicate that it under all experimental conditions. does not regulate inhibition of InsP R-1 channel gating by high [Ca ] . What then could be the mechanism of Ca inhibi- DISCUSSION tion? The simplest hypothesis is that the Ca binding Is There a Role of CaM in Inhibition by High [Ca ] of sites responsible for Ca inhibition of channel gating InsP R-1 Gating? are contained within the structure of the InsP R pro- tein itself. Many regions of the protein have been Numerous investigations have explored the interac- shown to bind Ca in in vitro studies (Sienaert et al., tions between the InsP R and CaM, but their nature, 1996, 1997). One or more of these or as yet unidenti- regulation, and functional effects on intracellular Ca fied sites may play a role, although there are no data signaling are still far from clear. Although it was re- available that address this issue. Alternately, another ported that CaM binding regulates high [Ca ] inhi- molecule could perhaps be involved. The InsP R in- bition of the InsP R-1 channel (Hirota et al., 1999; teracts with other proteins (Patel et al., 1999; Yang et Michikawa et al., 1999), subsequent studies using mi- al., 2002). Of interest, a calmodulin-like protein, crosomal fluxes or reconstituted channels in lipid bilay- CaBP1, interacts with high affinity with the ligand- ers have provided contradictory evidence (Zhang and binding region of the channel (Yang et al., 2002). Joseph, 2001; Nosyreva et al., 2002). In this study, we in- Whereas it is highly unlikely that CaBP1 and its iso- vestigated the possible involvement of CaM in the high forms mediate Ca inhibition, since they are likely [Ca ] inhibition of single-channel InsP R-1 gating us- i 3 neurally restricted and have been shown to stimulate ing the nuclear patch clamp method (Mak and Foskett, channel gating (Yang et al., 2002), the identification 1994). This approach enables single-channel recording of noncalmodulin Ca -binding protein interactions of endogenous and recombinant InsP R channels in with the receptor lends credence to the notion that a their native membrane environment. Similar biphasic Ca -binding protein could possibly be involved in regulation by [Ca ] of both the endogenous Xenopus mediating Ca responses of the channel. Because type 1 channel and the recombinant rat type 3 InsP R Ca inhibition of channel activity has been observed channel have been observed in previous nuclear patch in a number of distinct experimental systems from dif- clamp studies (Mak et al., 1998, 2001a). In this study, ferent species, such a putative effector would need to we directly explored the role of CaM in high [Ca ] in- be ubiquitously expressed and tightly associated with hibition of InsP R channel gating. We have found no the channel. evidence to support the hypothesis that inhibition of InsP R-1 channel activities by high [Ca ] is mediated 3 i A Novel Regulation of [Ca ] Inhibition of InsP R-1 i 3 by direct interaction between the InsP R channel and Channel Gating CaM. First, in the presence of 500 M W-7, a CaM an- Our investigations have revealed a novel CaM-indepen- tagonist that was previously reported to alleviate Ca dent regulation of the InsP R-1 channel: abrogation of inhibition of InsP R-1 channels reconstituted into bilay- high [Ca ] inhibition of InsP R-1 channel gating by ers (Michikawa et al., 1999), the X-InsP R-1 was still in- i 3 577 Mak et al. The Journal of General Physiology 2 2 2 exposure of the channel to ultra-low bath [Ca ] (5 Ca . It could only be observed when bath [Ca ] was 2 2 nM). The physical location of the low [Ca ] -sens- reduced to very low levels. Thus, if the [Ca ] -sens- bath bath ing mechanism on the InsP R protein is unknown. The ing mechanism is located on the cytoplasmic side of the 2 2 abrogation could possibly be caused by low [Ca ] in channel, it is likely to be a set of cooperative Ca -bind- the perinuclear space between the inner and outer ing sites. Further studies are necessary to distinguish nuclear envelope, to which the lumenal side of the whether cytoplasmic or lumenal [Ca ] is being sensed InsP R-1 channel is exposed. In this case, exposure to in the disruption of high [Ca ] inhibition of the 3 i 2 2 the ultra-low bath [Ca ] causes the lumenal [Ca ] to InsP R, and to determine the molecular mechanisms fall to low levels due to uncompensated Ca leak; and involved in that process. the [Ca ] sensing mechanism responsible for switch- Mechanism of Regulation of High [Ca ] Inhibition of ing high [Ca ] inhibition of InsP R-1 channel on and i 3 InsP R-1 Channel Gating by Exposure to Low [Ca ] Bath off is located on the lumenal side of the InsP R chan- nel. The existence of a Ca -binding site on the lume- A novel allosteric model, developed in the accompany- nal side of the InsP R-1 channel has been reported ing manuscript, can account for the effect of ultra-low (Sienaert et al., 1996). Our previous studies indicated [Ca ] bath exposure on the abrogation of high that the ionic composition of the solution in the peri- [Ca ] inhibition as well as the effect of InsP to modu- i 3 nuclear space of the isolated oocyte nucleus is likely to late maximum channel P , rather than K , under o inh be similar to that of the bath solution (Mak and Fos- these conditions. In brief, this model accounts for our kett, 1997). The long lag time (300 s) between the iso- results by postulating the existence of two functional in- 2 2 lation of the nucleus into the ultra-low [Ca ] bath so- hibitory Ca binding sites associated with each mono- lution and the earliest detection of InsP R channel ac- mer of the tetrameric channel. One site is only inhibi- tivities that could not be inhibited by high [Ca ] may tory when the channel is not liganded with InsP , i 3 reflect the time required for the solution in the perinu- because InsP binding relieves the Ca inhibition clear space to become fully equilibrated with the bath imposed by this site. In contrast, the properties of the solution, or the time taken for Ca bound to the lume- other inhibitory site are not affected by InsP binding. 2 2 2 nal Ca -binding sites of the InsP R channel to dissoci- In normal physiological [Ca ] conditions, Ca bind- 3 i ate from the sites after the drop in lumenal [Ca ], or a ing to this InsP -insensitive site provides the observed combination of the two. high [Ca ] inhibition (K 50 M) of the fully i inh Alternately, the [Ca ]-sensing mechanism could pos- InsP -liganded channel. The ability of this InsP -insensi- 3 3 sibly be located on the cytoplasmic side of the chan- tive site to be inhibitory is reversibly lost after exposure nel. In this case, the long lag time (300 s) between ex- of the channel for 5 min to an ultra-low bath [Ca ] posure of the channel to ultra-low [Ca ] and the abro- (5 nM). Thus, the observed abrogation of high 2 2 gation of high [Ca ] inhibition would imply that [Ca ] inhibition of channel activity in saturating i i dissociation of Ca from the sensing mechanism is [InsP ] can be accounted for by the fact that there is no 1 2 slow (rate 0.003 s ). Although such a sensing mecha- longer any functional inhibitory Ca -binding site. On nism would be exposed to high [Ca ] in the pipette the other hand, in the absence of InsP , the InsP -sensi- 3 3 solution as soon as the giga-ohm seal was formed, tive Ca inhibition site is functional and keeps the InsP R channel activities were nevertheless observed channel closed. Thus, the channel still requires InsP 3 3 for typically 10 s when the channel was exposed to to gate open even when the InsP -insensitive site has 2 2 [Ca ] 290 M before the activities abruptly termi- been disrupted by exposure to ultra-low bath [Ca ]. A nated (Mak and Foskett, 1997; Boehning et al., 2001). detailed description of this model, which can account Thus, binding of Ca to the sensing mechanism to re- for these and many other features of ligand regulation store normal high [Ca ] inhibition must also be a very of the channel observed in nuclear patch clamp experi- 1 2 slow process (rate 0.1 s ). If the [Ca ] sensing ments, is developed in the accompanying manuscript mechanism is in equilibrium with the cytoplasmic solu- (Mak et al., 2003, in this issue). tion, the forward rate constant (k ) for Ca dissocia- Are Different Sensitivities to Inhibition by High [Ca ] a 2 1 tion from the [Ca ]-sensing mechanism is 0.003 s Fundamental Distinguishing Feature among the and the reverse rate constant (k ) is such that 0.1 s InsP R Isoforms? k 290 M. If the [Ca ]-sensing mechanism is a sim- ple Ca binding site, then the equilibrium constant K The three isoforms of InsP R have complicated pat- (k /k ) for Ca dissociation from the site should then terns of expression in various tissues with complex reg- f r be 10 M. However, abrogation of channel inhibition ulation by various mechanisms (Taylor et al., 1999). Be- was not observed in our normal bath solutions that cause the permeation and conductance properties of contain 300–500 nM Ca (Mak et al., 1998), or in our the InsP R isoforms are very similar (Mak et al., 2000; physiological Ca bath solution containing 50 nM free Ramos-Franco et al., 2000), differences among the iso- 578 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology forms in localization and channel gating and its regula- note, this InsP dependence of maximum P is very sim- 3 o tion are likely to be reasons for the existence of InsP R ilar to the observed effect of InsP on the P of InsP R-2 3 3 o 3 diversity. A review of published single-channel studies channels reconstituted into lipid bilayers (Ramos- of various InsP R isoforms suggests that different sensi- Franco et al., 1998b). These observations raise the in- tivities to inhibition by high [Ca ] may be one distin- triguing possibility that the observed differences in the guishing functional feature among the various InsP R sensitivities to Ca inhibition of various InsP R iso- 3 3 isoforms. Nevertheless, it is not clear whether such dif- forms may be a consequence of the different environ- ferences are intrinsic to the channels, or whether they ment and/or isolation conditions to which the chan- are perhaps artificially generated by the different ex- nels were exposed, rather than the result of differences perimental protocols used for studying InsP R channel in fundamental intrinsic characteristics of the individ- activity. ual isoforms. For example, the InsP R-1 and InsP R-3 3 3 In the presence of 1 M InsP , native and recombi- channel isoforms exhibited very similar inhibition by nant InsP R-1 channels (including various splice vari- high [Ca ] when they are studied in a native ER mem- 3 i ants) reconstituted into lipid bilayers exhibited similar brane environment (Mak et al., 1998, 2001a), but they strong inhibition by [Ca ] with half-maximal inhibi- behaved differently in reconstitution systems. We sug- tory [Ca ] of 0.1–2 M (Bezprozvanny et al., 1991; Ra- gest that it is worth considering the possibility that pro- mos-Franco et al., 1998a,b; Tu et al., 2002), whereas na- cedures employed in the isolation and reconstitution tive Xenopus and recombinant rat InsP R-1 channels and recording of the InsP R-3 used in (Hagar et al., 3 3 studied in their native membrane environment using 1998) disrupted the normal high [Ca ] inhibition of nuclear patch clamp techniques exhibited inhibition the InsP R-3, causing the observed lack of Ca inhibi- by high [Ca ] , but with a significantly higher half- tion, in very much the same way that exposure to a 2 2 maximal inhibitory [Ca ] of 50 M (Mak et al., ULCaS bath abrogated the high [Ca ] inhibition of i i 1998; Boehning et al., 2001). When reconstituted into InsP R-1 observed in this study. Whereas the proce- planar bilayers, Ca inhibition of InsP R-1 could be al- dures used in the isolation and reconstitution and re- leviated by very high [InsP ] (180 M) (Kaftan et al., cording of InsP R-1 by themselves did not eliminate 3 3 2 2 1997; Moraru et al., 1999), whereas Ca inhibition of high [Ca ] inhibition of the channel (Bezprozvanny InsP R-1 studied in the native membrane environment et al., 1991; Ramos-Franco et al., 1998a,b; Tu et al., was not further affected by [InsP ] once the channel 2002), they may account for the ability, observed only was saturated with [InsP ] 100 nM (Mak et al., 1998). in the reconstituted systems, of extremely high [InsP ] 3 3 InsP R-2 channels reconstituted in lipid bilayers ex- to abrogate high [Ca ] inhibition (Kaftan et al., 1997; 3 i hibited variable but low sensitivity to inhibition by high Moraru et al., 1999). By the same token, it is possible 2 2 2 [Ca ] , with a half-maximal [Ca ] of 400 M for that the very low sensitivity to high [Ca ] inhibition of i i i recombinant InsP R-2 channels (Ramos-Franco et al., the InsP R-2 channel isoform reconstituted in lipid bi- 3 3 2000) and 1 mM for native channels (Ramos-Franco layers (Ramos-Franco et al., 1998b, 2000) was induced et al., 1998b, 2000) in 1 M InsP . by the isolation and reconstitution and recording pro- Native type 3 InsP R channels reconstituted into lipid tocols. Obviously, these issues will need to be resolved bilayers exhibited no detectable inhibition by high in future studies, for example, of the Ca responses of [Ca ] and its P remained at its maximum value type 2 InsP R channels in the native ER membrane en- i o 3 (0.05) in [Ca ] between 1 and 100 M in the pres- vironment, under the same experimental conditions as ence of 2 M InsP (Hagar et al., 1998). In marked con- those used for the types 1 and 3 InsP R isoforms; and of 3 3 trast, recombinant r-InsP R-3 in the nuclear membrane the sensitivities of Ca inhibition of the other InsP R 3 3 2 2 of oocytes is inhibited by high [Ca ] in an InsP -depen- isoforms to exposure to ultra-low bath [Ca ]. Never- i 3 dent manner very similar to that for X-InsP R-1 under theless, our identification in this study of conditions identical experimental conditions (Mak et al., 2001a). that can radically alter the [Ca ] inhibition properties How can we account for such divergent results? Our of the channel suggests that careful consideration of studies here demonstrate that Ca inhibition of the the isolation protocols and other conditions to which X-InsP R-1 channel in its native membrane environment InsP R channels are exposed before they are examined 3 3 can be completely, specifically and reversibly abrogated will be warranted in future studies. under certain experimental conditions (after exposure This work was supported by grants to J.K. Foskett from the NIH to a nominally Ca -free bath). Associated with this ef- (MH59937, GM56328) and to D.-O.D. Mak from the American fect, the InsP dependence of the channel P was also 3 o Heart Association (9906220U). changed—normally, InsP affects the apparent affinity Olaf S. Andersen served as editor. of the inhibitory Ca -binding sites of the channel (Mak et al., 1998), whereas after ULCaS bath exposure, Submitted: 27 Januar y 2003 InsP affects the maximum P observed (Fig. 6 C). Of Accepted: 2 September 2003 3 o 579 Mak et al. The Journal of General Physiology REFERENCES Mak, D.-O.D., S. McBride, and J.K. Foskett. 1998. Inositol 1,4,5-tris- Baudet, S.B., L. Hove-Madsen, and D.M. Bers. 1994. How to make phosphate activation of inositol trisphosphate receptor Ca and use calcium-specific mini- and microelectrodes. In A Practi- channel by ligand tuning of Ca inhibition. Proc. Natl. Acad. Sci. cal Guide to the Study of Calcium in Living Cells. 1st ed. R. Nuc- USA. 95:15821–15825. citelli, editor. Academic Press, San Diego. 94–114. Mak, D.-O.D., S. McBride, V. Raghuram, Y. Yue, S.K. Joseph, and Berridge, M.J. 1993. Inositol trisphosphate and calcium signalling. J.K. Foskett. 2000. Single-channel properties in endoplasmic Nature. 361:315–325. reticulum membrane of recombinant type 3 inositol trisphos- Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Bell-shaped cal- phate receptor. J. Gen. Physiol. 115:241–256. cium-response curves of Ins(1,4,5)P - and calcium-gated chan- Mak, D.-O.D., S. McBride, and J.K. Foskett. 2001a. Regulation by nels from endoplasmic reticulum of cerebellum. Nature. 351: Ca and inositol 1,4,5-trisphosphate (InsP ) of single recombi- 751–754. nant type 3 InsP receptor channels. Ca activation uniquely dis- Boehning, D., S.K. Joseph, D.-O.D. Mak, and J.K. Foskett. 2001. Sin- tinguishes types 1 and 3 InsP receptors. J. Gen. Physiol. 117:435– gle-channel recordings of recombinant inositol trisphosphate re- 446. ceptors in mammalian nuclear envelope. Biophys. J. 81:117–124. Mak, D.-O.D., S. McBride, and J.K. Foskett. 2001b. ATP-dependent Cardy, T.J., and C.W. Taylor. 1998. A novel role for calmodulin: adenophostin activation of inositol 1,4,5-trisphosphate receptor Ca -independent inhibition of type-1 inositol trisphosphate re- channel gating: kinetic implications for the durations of calcium ceptors. Biochem. J. 334:447–455. puffs in cells. J. Gen. Physiol. 117:299–314. Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. Mak, D.-O.D., S. McBride, and J.K. Foskett. 2003. Spontaneous 1998. Type III InsP receptor channel stays open in the presence channel activity of the inositol 1,4,5-triphosphate (InsP ) recep- 3 3 of increased calcium. Nature. 396:81–84. tor (InsP R). Application of allosteric modeling to calcium and Hirota, J., T. Michikawa, T. Natsume, T. Furuichi, and K. Mikoshiba. InsP regulation of InsP R single-channel gating. J. Gen. Physiol. 3 3 1999. Calmodulin inhibits inositol 1,4,5-trisphosphate-induced 122:583–603. calcium release through the purified and reconstituted inositol Michikawa, T., J. Hirota, S. Kawano, M. Hiraoka, M. Yamada, T. Fu- 1,4,5- trisphosphate receptor type 1. FEBS Lett. 456:322–326. ruichi, and K. Mikoshiba. 1999. Calmodulin mediates calcium- Jiang, Q., D.-O.D. Mak, S. Devidas, E.M. Schwiebert, A. Bragin, Y. dependent inactivation of the cerebellar type 1 inositol 1,4,5-tris- Zhang, W.R. Skach, W.B. Guggino, J.K. Foskett, and J.F. Engel- phosphate receptor. Neuron. 23:799–808. hardt. 1998. Cystic fibrosis transmembrane conductance regula- Monkawa, T., A. Miyawaki, T. Sugiyama, H. Yoneshima, M. Yama- tor-associated ATP release is controlled by a chloride sensor. J. moto-Hino, T. Furuichi, T. Saruta, M. Hasegawa, and K. Miko- Cell Biol. 143:645–657. shiba. 1995. Heterotetrameric complex formation of inositol Joseph, S.K., and S. Samanta. 1993. Detergent solubility of the ino- 1,4,5-trisphosphate receptor subunits. J. Biol. Chem. 270:14700– sitol trisphosphate receptor in rat brain membranes. Evidence 14704. for association of the receptor with ankyrin. J. Biol. Chem. 268: Moraru, I.I., E.J. Kaftan, B.E. Ehrlich, and J. Watras. 1999. Regula- 6477–6486. tion of type 1 inositol 1,4,5-trisphosphate-gated calcium channels Joseph, S.K., C. Lin, S. Pierson, A.P. Thomas, and A.R. Maranto. by InsP and calcium: Simulation of single channel kinetics based 1995. Heteroligomers of type-I and type-III inositol trisphosphate on ligand binding and electrophysiological analysis. J. Gen. Phys- receptors in WB rat liver epithelial cells. J. Biol. Chem. 270:23310– iol. 113:837–849. 23316. Nosyreva, E., T. Miyakawa, Z. Wang, L. Glouchankova, A. Mizu- Joseph, S.K. 1996. The inositol trisphosphate receptor family. Cell. shima, M. Iino, and I. Bezprozvanny. 2002. The high affinity cal- Signal. 8:1–7. cium-calmodulin-binding site does not play a role in modulation Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. Inositol 1,4,5-tris- of type 1 inositol (1,4,5)-trisphosphate receptor function by cal- phosphate (InsP ) and calcium interact to increase the dynamic cium and calmodulin. Biochem. J. 365:659–667. range of InsP receptor-dependent calcium signaling. J. Gen. Nucifora, F.C., Jr., A.H. Sharp, S.L. Milgram, and C.A. Ross. 1996. Physiol. 110:529–538. Inositol 1,4,5-trisphosphate receptors in endocrine cells: localiza- Keen, J.E., R. Khawaled, D.L. Farrens, T. Neelands, A. Rivard, C.T. tion and association in hetero- and homotetramers. Mol. Biol. Bond, A. Janowsky, B. Fakler, J.P. Adelman, and J. Maylie. 1999. Cell. 7:949–960. Domains responsible for constitutive and Ca -dependent inter- Patel, S., S.K. Joseph, and A.P. Thomas. 1999. Molecular properties actions between calmodulin and small conductance Ca -acti- of inositol 1,4,5-trisphosphate receptors. Cell Calcium. 25:247– vated potassium channels. J. Neurosci. 19:8830–8838. 264. Lin, C., J. Widjaja, and S.K. Joseph. 2000. The interaction of cal- Ramos-Franco, J., S. Caenepeel, M. Fill, and G. Mignery. 1998a. Sin- modulin with alternatively spliced isoforms of the type-I inositol gle channel function of recombinant type-1 inositol 1,4,5-tris- trisphosphate receptor. J. Biol. Chem. 275:2305–2311. phosphate receptor ligand binding domain splice variants. Bio- Maeda, N., T. Kawasaki, S. Nakade, N. Yokota, T. Taguchi, M. Kasai, phys. J. 75:2783–2793. and K. Mikoshiba. 1991. Structural and functional character- Ramos-Franco, J., M. Fill, and G.A. Mignery. 1998b. Isoform-spe- ization of inositol 1,4,5- trisphosphate receptor channel from cific function of single inositol 1,4,5-trisphosphate receptor mouse cerebellum. J. Biol. Chem. 266:1109–1116. channels. Biophys. J. 75:834–839. Mak, D.-O.D., and J.K. Foskett. 1994. Single-channel inositol 1,4,5- Ramos-Franco, J., D. Bare, S. Caenepeel, A. Nani, M. Fill, and G. trisphosphate receptor currents revealed by patch clamp of iso- Mignery. 2000. Single-channel function of recombinant type 2 lated Xenopus oocyte nuclei. J. Biol. Chem. 269:29375–29378. inositol 1,4, 5- trisphosphate receptor. Biophys. J. 79:1388–1399. Mak, D.-O.D., and J.K. Foskett. 1997. Single-channel kinetics, inac- Sienaert, I., H. De Smedt, J.B. Parys, L. Missiaen, S. Vanlingen, H. tivation, and spatial distribution of inositol trisphosphate (IP ) Sipma, and R. Casteels. 1996. Characterization of a cytosolic and receptors in Xenopus oocyte nucleus. J. Gen. Physiol. 109:571– a luminal Ca binding site in the type I inositol 1,4,5-trisphos- 587. phate receptor. J. Biol. Chem. 271:27005–27012. Mak, D.-O.D., and J.K. Foskett. 1998. Effects of divalent cations on Sienaert, I., L. Missiaen, H. De Smedt, J.B. Parys, H. Sipma, and R. single-channel conduction properties of Xenopus IP receptor. Casteels. 1997. Molecular and functional evidence for multiple Am. J. Physiol. 275:C179–C188. Ca -binding domains in the type 1 inositol 1,4,5-trisphosphate 580 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology receptor. J. Biol. Chem. 272:25899–25906. Xia, X.M., B. Fakler, A. Rivard, G. Wayman, T. Johnson-Pais, J.E. Taylor, C.W. 1998. Inositol trisphosphate receptors: Ca -modu- Keen, T. Ishii, B. Hirschberg, C.T. Bond, S. Lutsenko, et al. 1998. lated intracellular Ca channels. Biochim. Biophys. Acta. 1436:19– Mechanism of calcium gating in small-conductance calcium-acti- 33. vated potassium channels. Nature. 395:503–507. Taylor, C.W., A.A. Genazzani, and S.A. Morris. 1999. Expression of Yamada, M., A. Miyawaki, K. Saito, T. Nakajima, M. Yamamoto- inositol trisphosphate receptors. Cell Calcium. 26:237–251. Hino, Y. Ryo, T. Furuichi, and K. Mikoshiba. 1995. The calmodu- Tu, H., T. Miyakawa, Z. Wang, L. Glouchankova, M. Iino, and I. Bez- lin-binding domain in the mouse type 1 inositol 1,4,5-trisphos- prozvanny. 2002. Functional characterization of the Type 1 inosi- phate receptor. Biochem. J. 308:83–88. tol 1,4,5-trisphosphate receptor coupling domain SII(/ ) Yang, J., S. McBride, D.O. Mak, N. Vardi, K. Palczewski, F. Haese- splice variants and the Opisthotonos mutant form. Biophys. J. 82: leer, and J.K. Foskett. 2002. Identification of a family of calcium 1995–2004. sensors as protein ligands of inositol trisphosphate receptor Ca Wojcikiewicz, R.J. 1995. Type I, II, and III inositol 1,4,5-trisphos- release channels. Proc. Natl. Acad. Sci. USA. 99:7711–7716. phate receptors are unequally susceptible to down-regulation Zhang, X., and S.K. Joseph. 2001. Effect of mutation of a calmodu- and are expressed in markedly different proportions in different lin binding site on Ca regulation of inositol trisphosphate re- cell types. J. Biol. Chem. 270:11678–11683. ceptors. Biochem. J. 360:395–400. 581 Mak et al. The Journal of General Physiology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of General Physiology Pubmed Central

Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor Calcium-release Channel

Mak, Don-On Daniel; McBride, Sean M.J.; Petrenko, Nataliya B.; Foskett, J. Kevin
The Journal of General Physiology , Volume 122 (5) – Nov 1, 2003
Download PDF
13 pages

Loading next page...
 
/lp/pubmed-central/novel-regulation-of-calcium-inhibition-of-the-inositol-1-4-5-Enllrw58VW

References (45)

Publisher
Pubmed Central
Copyright
Copyright © 2003, The Rockefeller University Press
ISSN
0022-1295
eISSN
1540-7748
DOI
10.1085/jgp.200308808
Publisher site
See Article on Publisher Site

Abstract

Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor Calcium-release Channel Don-On Daniel Mak, Sean M.J. McBride, Nataliya B. Petrenko, and J. Kevin Foskett Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104 abstract The inositol 1,4,5-trisphosphate (InsP ) receptor (InsP R), a Ca -release channel localized to the 3 3 endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca signals in many cell types. Three InsP R isoforms are expressed in different subcellular locations, at variable relative levels with heteromulti- mer formation in different cell types. A proposed reason for this diversity of InsP R expression is that the isoforms 2 2 are differentially inhibited by high cytoplasmic free Ca concentrations ([Ca ] ), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca ] in me- diating high [Ca ] inhibition of InsP R gating by studying single endogenous type 1 InsP R channels through i 3 3 patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentra- tions of a calmodulin antagonist nor overexpression of a dominant-negative Ca -insensitive mutant calmodulin 2 2 affected inhibition of gating by high [Ca ] . However, a novel, calmodulin-independent regulation of [Ca ] in- i i hibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with 2 2 [Ca ] 400 nM were inhibited by 290 M [Ca ] , exposure of the isolated nuclei to a bath solution with ultra- 2 2 low [Ca ] (5 nM, for 300 s) before the patch-clamp experiments reversibly relieved Ca inhibition, with 2 2 channel activities observed in [Ca ] up to 1.5 mM. Although InsP activates gating by relieving high [Ca ] inhi- i 3 i bition, it was nevertheless still required to activate channels that lacked high [Ca ] inhibition. Our observations suggest that high [Ca ] inhibition of InsP R channel gating is not regulated by calmodulin, whereas it can be dis- i 3 rupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca ] inhibition may not be an immutable property of different InsP R isoforms. Furthermore, these ob- i 3 2 2 servations support an allosteric model in which Ca inhibition of the InsP R is mediated by two Ca binding sites, only one of which is sensitive to InsP . key words: single-channel electrophysiology • patch clamp • calcium • Xenopus oocyte • nucleus INTRODUCTION dation that differ during different stages of cell devel- opment and in response to extracellular stimuli (Taylor The second messenger, inositol 1,4,5-trisphosphate et al., 1999). Furthermore, formation of hetero-tet- (InsP ), is generated in many cell types through the rameric channels is possible in cell types expressing hydrolysis of phosphatidylinositol 4,5-bisphosphate by more than one InsP R isoform (Joseph et al., 1995; membrane-bound phospholipase C activated by plasma Monkawa et al., 1995; Wojcikiewicz, 1995; Nucifora et membrane receptors responding to extracellular stim- al., 1996). Although this diversity of InsP R expression uli. InsP then diffuses through the cytoplasm to bind is impressive, its functional correlates and physiological to its receptor (InsP R) in the ER and activate it as a implications remain unclear. Studies of the single-chan- 2 2 Ca channel to release Ca stored in the ER lumen. nel properties of the various InsP R isoforms have re- Modulation of the cytoplasmic free Ca concentration vealed that whereas their permeation and conductance 2 2 ([Ca ] ) by InsP R-mediated Ca release is a ubiqui- i 3 properties are very similar (Mak et al., 2000; Ramos- tous intracellular signal transduction mechanism that Franco et al., 2000), their gating may be differentially regulates numerous processes (Berridge, 1993). inhibited by high [Ca ] (Bezprozvanny et al., 1991; Three isoforms of the InsP R, with spliced variants, Hagar et al., 1998; Mak et al., 1998; Ramos-Franco et have been identified (Joseph, 1996). Most mammalian al., 1998a,b, 2000; Boehning et al., 2001; Mak et al., cell types express multiple InsP R isoforms in distinct 2001a). Because high [Ca ] inhibition of InsP R i 3 and overlapping intracellular locations with their abso- channel gating may be a pivotal feedback mechanism lute and relative expression levels regulated by gene transcription, alternative splicing and receptor degra- Abbreviations used in this paper: InsP , inositol 1,4,5-trisphosphate; Address correspondence to J. Kevin Foskett, Department of Physiol- InsP R, InsP receptor; NCaS, regular [Ca ] bath solution; PCaS, 3 3 ogy, B39 Anatomy-Chemistry Bldg/6085, University of Pennsylvania, physiological [Ca ] bath solution; r-InsP R-3, rat type 3 InsP R; 3 3 Philadelphia, PA 19104-6085. Fax: (215) 573-6808; email: foskett@ CaM, calmodulin; ULCaS, ultra-low [Ca ] bath solution; X-InsP R-1, mail.med.upenn.edu Xenopus type 1 InsP R. 569 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2003/11/569/13 $8.00 Volume 122 November 2003 569–581 http://www.jgp.org/cgi/doi/10.1085/jgp.200308808 The Journal of General Physiology for the regulation of intracellular Ca signaling (Tay- and therefore may not be an invariant property of a lor, 1998), it has been suggested that differential inhibi- specific InsP R isoform. Furthermore, these observa- 2 2 tion by high [Ca ] of the different InsP R isoforms tions support an allosteric model in which Ca inhibi- i 3 2 2 may generate distinct Ca signals in different cell types tion of the InsP R is mediated by two Ca binding with different patterns of InsP R isoform expression, sites, only one of which is sensitive to InsP . 3 3 and that this may be a reason for the diversity of InsP R MATERIALS AND METHODS expression (Hagar et al., 1998). It has been suggested that high [Ca ] inhibition of Heterologous Expression of Calmodulin in Xenopus Oocytes the InsP R is mediated by calmodulin (CaM), a ubiqui- tous Ca -binding protein that binds to and regulates Maintenance of Xenopus laevis and surgical extraction of ovaries the functions of many proteins. CaM was found to bind were performed as described previously (Mak and Foskett, 1994, 1997, 1998). Oocytes were defolliculated as described (Jiang et to the InsP R-1 in the presence of free Ca to a single al., 1998). cRNA (1 g/l) of rat calmodulin (CaM), either wild- site in the regulatory domain (Maeda et al., 1991; Ya- type (w.t.) or a quadruple mutant (q.m.) containing a D→A mu- mada et al., 1995; Hirota et al., 1999). Purified InsP R-1 tation in each of the four EF hands so that Ca binding in all EF channels lacking bound CaM were not inhibited by hands was abolished (Xia et al., 1998; Keen et al., 1999), was syn- high [Ca ] , whereas addition of CaM restored inhibi- thesized in vitro from cDNA provided as a gift by Dr. John P. Adelman (Vollum Institute, Portland, OR). 23 nl of cRNA (either tion of channel gating by high [Ca ] (Hirota et al., w.t. or q.m.) was injected into the cytoplasm of oocytes 1 d after 1999; Michikawa et al., 1999). The notion that high defolliculation, as described (Mak et al., 2000). cRNA-injected Ca inhibition of channel gating was mediated by CaM and uninjected control oocytes were maintained under identical was reinforced by observations that the type 3 InsP R 3 conditions in individual wells in 96-well plates containing 200 l (InsP R-3) did not bind CaM (Yamada et al., 1995; of ASOS (100 mM NaCl, 2 mM KCl, 1 mM MgCl , 1.8 mM CaCl , 2 2 5 mM HEPES, pH adjusted to 7.6 with NaOH; with 3 mM Na Cardy and Taylor, 1998; Lin et al., 2000) and was not in- pyruvate, 100 g/ml gentamycin, and 100 M N-acetyl-Leu-Leu- hibited by high [Ca ] (Hagar et al., 1998). Neverthe- Norleucinal; Sigma-Aldrich). 80 l of ASOS in each well was less, other data suggest that the role of CaM in high changed daily. Nuclear patch clamp experiments and immuno- [Ca ] inhibition of InsP R channel gating is far from i 3 precipitations were performed 2–4 d after c-RNA injection when unequivocal. Despite the absence of detectable interac- the expression level of exogenous CaM was stable as determined by Western analysis. tion between CaM and a mutant InsP R-1 in which the putative CaM binding site was eliminated (Yamada et Western Analysis and Immunoprecipitation al., 1995), more recent studies have demonstrated that Western analysis was performed on oocyte extracts (cRNA- this mutant channel is nevertheless still inhibited by injected and uninjected), as described in Mak et al. (2000), to as- high [Ca ] (Zhang and Joseph, 2001; Nosyreva et al., certain the levels of endogenous and heterologously expressed 2002). Furthermore, whereas the InsP R-3 lacks the 3 CaM in the oocytes using a specific antibody (Upstate Biotech- CaM binding site present in the InsP R-1 and no inter- nology). Immunoprecipitation of InsP R (type 1) and CaM was 3 3 performed using oocyte lysates, as described in (Mak et al., action between InsP R-3 and CaM has been detected 2000), with a specific type 1 InsP R antibody (Joseph and Sa- (Yamada et al., 1995; Cardy and Taylor, 1998; Lin et al., manta, 1993; Joseph et al., 1995) and protein A agarose (GIBCO 2000), electrophysiological studies of the recombinant BRL), and an antibody to CaM and protein G agarose (GIBCO rat InsP R-3 in its native membrane environment dem- 3 BRL), respectively. onstrated that it is nevertheless inhibited by high Solutions for Patch Clamp Experiments [Ca ] (Mak et al., 2001a) with quantitative features similar to those of inhibition of the InsP R-1 in the All patch clamp experiments were per formed with solutions con- taining 140 mM KCl and 10 mM HEPES with pH adjusted to 7.1 same membrane (Mak et al., 1998). 2 2 with KOH. The free Ca concentration ([Ca ] ) of the pipette Here, we investigated the possible effects of CaM on solutions (to which the cytoplasmic side of the InsP R is exposed high [Ca ] inhibition of the gating of single endoge- in patch-clamp experiments) was tightly controlled by buffering nous InsP R-1 channels in their native membrane envi- 3 various amounts of added CaCl (40–400 M) with 500 M of ronment using nuclear membrane patch clamp electro- the high-affinity Ca chelator, BAPTA (1,2-bis(O-aminophe- noxy) ethane-N,N,N,N-tetraacetic acid; Molecular Probes) and physiology (Mak and Foskett, 1994). Our experiments 0.5 mM Na ATP (100 nM  [Ca ]  2.5 M); or 500 M of 2 i do not provide evidence supporting any role for CaM the low-affinity Ca chelator, 5,5-dibromo BAPTA (Molecular in this process. However, we discovered a novel regula- Probes) and 0.5 mM Na ATP (5 M  [Ca ]  15 M); or 0.5 2 i tion of high [Ca ] inhibition of InsP R-1 channel gat- 2 i 3 mM Na ATP alone (15 M  [Ca ]  300 M). Solutions with 2 i 2 2 ing. Inhibition of InsP R-1 gating by high [Ca ] can [Ca ]  300 M contained no Ca chelator for buffering. The 3 i i normal Ca bath solution (NCaS) contained 500 M BAPTA be reversibly abrogated by exposure of the channel to a and 250 M CaCl (free [Ca ]  400–500 nM), and the physio- bathing solution containing ultra-low [Ca ] (5 nM). logical Ca bath solution (PCaS) contained 500 M BAPTA and Our observations indicate that inhibition of InsP R-1 3 2 2 70 M CaCl (free [Ca ]  48  5 nM). The free [Ca ] of channel gating by high [Ca ] can be disrupted by en- 2 i these solutions was directly measured using Ca -selective mini- vironmental conditions experienced by the channel, electrodes (Baudet et al., 1994). The ultra-low Ca bath solution 570 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology (ULCaS) contained 1 mM BAPTA and no added CaCl . The con- for channel open probability (P ) evaluation. The number of 2 o taminating [Ca ] in the solution was determined by induction- channels in the membrane patch was assumed to be the maxi- coupled plasma mass spectrometry (Mayo Medical Laboratory) mum number of open channel current levels observed through- to be 6–10 M. Ca -selective minielectrodes were unable to out the current record. In experimental conditions with P  0.1, determine accurately the free [Ca ] in the ULCaS because of only current records with longer than 10 s of InsP R channel ac- the nonlinear response of the electrode in free [Ca ]  5 nM. tivities were used for determination of P , so there is little uncer- Free [Ca ] was calculated using the Maxchelator software (C. tainty in the number of channels in the current traces used. In Patton, Stanford University, Stanford, CA) to be 0.9–1.5 nM. experimental conditions with P  0.1, only current records ex- Unless specified otherwise, all pipette solutions contained a hibiting one open channel current level with InsP R channel ac- saturating concentration (10 M) of InsP (Mak and Foskett, tivities lasting longer than 30 s were used, to ensure that they 1994) from Molecular Probes. When specified, the pipette solu- were truly single-channel records (Mak et al., 2001a). The P data tions also contained 500 M W-7 (a CaM binding antagonist; shown for each set of experimental conditions are the means of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochlo- results from at least four separate patch-clamp experiments per- ride; Calbiochem), or 10 M purified bovine CaM (Calbio- formed under the same conditions. Error bars indicate the SEM. chem). All reagents were used with no further purification. RESULTS Oocyte Nucleus Isolation Protocols Lack of Effect of Calmodulin on Ca Inhibition of InsP R A stage V or VI oocyte was gently teased open mechanically in the isolation bathing solution, enabling the translucent nucleus to be Gating in Endoplasmic Reticulum Membrane isolated from the cytoplasmic material. The isolated nucleus was either directly transferred to the experimental bathing solution Previous single-channel patch-clamp studies of the en- (protocol Nd, Ld, and Pd, Fig. 1), or it was transferred through a dogenous Xenopus type 1 InsP R (X-InsP R-1) in its na- 3 3 series of culture dishes containing 4–5 ml of incubation bath so- tive ER membrane environment revealed a biphasic lutions (protocol L, LN, and LNL, in Fig. 1) before it was ulti- regulation by [Ca ] of the single-channel open proba- mately transferred to the experimental bath. The nucleus re- bility (P ) (Mak et al., 1998, 2001b). It has been sug- mained in each incubation bath for at least 20 min before the o next transfer, to ensure that the solution in the perinuclear lu- gested that calmodulin (CaM) bound to the channel men between the outer and inner nuclear envelope had attained mediates inhibition of InsP R-1 gating by high [Ca ] 3 i ionic equilibrium with the bath solution (Mak and Foskett, (Michikawa et al., 1999). We therefore investigated the 1994). Approximately 20 l of the previous bath solution accom- possibility that the high [Ca ] inhibition of X-InsP R-1 i 3 panied the nucleus to the new bath in a transfer. The culture channel gating observed in our previous studies was dish containing the nucleus in the experimental bath solution was finally moved onto the stage of the inverted microscope mediated by CaM. Oocyte nuclei were isolated and where patch clamp experiments were per formed. transferred directly into an experimental bath of NCaS for patch-clamp experiments (protocol Nd in Fig. 1). Acquisition and Analysis of Single-Channel Patch-clamp By repeated patch clamping over the surface of an iso- Current Records lated nucleus, regions on the outer nuclear envelope The isolated nucleus was gently immobilized as described previ- were identified in which the probability of detecting ously (Mak and Foskett, 1994) so that membrane patches could InsP R channel activities in membrane patches (P ) 3 d be repeatedly obtained from the same region (2 m) of the was high (Mak and Foskett, 1997). A series of patch- outer nuclear membrane (Mak and Foskett, 1997). Due to abrupt termination of channel activity (Mak and Foskett, 1994, clamp experiments was performed at these regions 1997), patch clamp experiments were per formed in “on-nucleus” with pipette solutions (to which the cytoplasmic side of configuration to maximize the duration of channel activities re- the InsP R was exposed) alternately containing either corded. To prevent contamination of the pipette solution by the bath solution (especially the Ca chelator in the bath solution) by diffusion through the pipette tip during the time when the pi- pette was immersed in the bath and before giga-Ohm seal forma- tion, a positive pressure (10 mmHg) was maintained inside the pipette until the pipette tip was properly positioned on the nu- clear membrane. Then suction was applied in the pipette to ob- tain the giga-Ohm seal. All experiments were per formed at room temperature with the pipette electrode at 20 mV relative to the reference bath electrode unless specifically stated otherwise. Each experiment recorded the InsP R channel activity at a spe- cific [Ca ] and [InsP ], with no change of the pipette or bath i 3 solutions during the experiment. Data acquisition was per- formed as previously described (Mak et al., 1998), with currents recorded with a filtering frequency of 1 kHz and a digitizing fre- quency of 5 kHz. The patch clamp current traces were analyzed using MacTac software (Bruxton) to identify channel-opening and -closing events using a 50% threshold. Current traces exhibiting one InsP R channel, or two InsP R channels determined to be identi- Figure 1. Schematic diagram showing the various protocols 3 3 cal and independently gated (Mak and Foskett, 1997), were used used to isolate oocyte nuclei for nuclear patch clamp experiments. 571 Mak et al. The Journal of General Physiology Figure 3. Typical current traces from nuclei in NCaS bath with pipette solutions containing 10 M InsP . Arrows indicate closed channel current levels. (A and B) Uninjected oocytes were used. InsP R channel activity was observed with [Ca ] of 755 nM (A, 3 i n  3), whereas no channel activity was observed in a membrane Figure 2. Western blots of oocyte lysates probed with a CaM anti- patch obtained from the same region of the same nucleus with body (both w.t. and q.m.). Lysates from oocytes injected with CaM [Ca ] of 290 M and the pipette solution containing 500 M W-7 cRNA (w.t. or q.m.) or uninjected oocytes were used as labeled. (B, n  5). (C and D) Oocytes injected with CaM q.m. cRNA were Oocytes used for lanes A and B or C and D were from the same used. InsP R channel activity was observed with [Ca ] of 2.1 M 3 i batches, respectively. Top arrow indicates wild-type CaM and the (C, n  4), whereas no channel activity was observed in a mem- bottom arrow indicates the quadruple mutant CaM. The slightly brane patch obtained from the same region of the same nucleus faster mobility of q.m. CaM is likely a reflection of the known Ca - with [Ca ] of 290 M (D, n  9). binding dependence of CaM mobility in gels (Xia et al., 1998). 2 2 [Ca ]  755 nM, or very high [Ca ] (290 M) with pression on SK channel gating provided evidence that i i 500 M of W-7, a CaM binding antagonist. The former endogenous CaM is tightly and constitutively (even in solution is one in which the channel gates with a high the absence of Ca ) associated with the SK channel P , thereby ascertaining the presence of functional and mediates the effects of Ca on SK channel gating. InsP R channels in the regions selected during the se- The ability of high Ca concentrations to inhibit ries of experiments. In contrast, the latter solution has InsP R channel gating in our in vitro electrophysiologi- [Ca ] sufficiently high to inhibit InsP R channel gat- cal studies can be observed for long times (up to 2 h) i 3 ing (Mak et al., 1998). Because CaM is endogenously after isolation of the nuclei (Mak et al., 1998; Boehning expressed in Xenopus oocytes (Fig. 2, Lane A and C), we et al., 2001; Mak et al., 2001b). Thus, if CaM mediates 2 2 reasoned that if CaM mediated the high [Ca ] inhibi- the effect of high [Ca ] , it must remain associated i i tion of InsP R channel gating, then inclusion of 500 with the channel in the isolated nuclei, and therefore M of W-7 in the pipette solution may block high must be tightly bound to the InsP R and not free to dif- [Ca ] inhibition by interfering with CaM binding to fuse away into the large experimental bath. We there- the InsP R channel (Michikawa et al., 1999), making fore explored the possibility that Ca inhibition of channel gating observable in the 290 M [Ca ] solu- InsP R channel gating was mediated by a constitutive i 3 tions. Nevertheless, no channel activity was detected in tight association of CaM with the channel, by examin- 2 2 any of the five patches with 500 M W-7 and [Ca ]  ing the effects of overexpression of the Ca -insensitive 290 M (Fig. 3 B), whereas InsP R channel activities quadruple mutant (q.m.) CaM on the Ca regulation were readily detected in five out of six patches with of the InsP R. [Ca ]  755 nM (Fig. 3 A). The q.m. CaM, which has all EF hands mutated and Whereas this result with W-7 is seemingly inconsistent therefore is Ca insensitive, was overexpressed in Xeno- with the hypothesis that CaM mediates Ca inhibition pus oocytes by cytoplasmic microinjection of cRNA. of InsP R gating, CaM-dependent regulation of the Western analysis (n  5) indicated that the exogenous small-conductance Ca -activated K (SK) channel gat- q.m. CaM was expressed to a level that was at least an ing is insensitive to W-7 and other CaM inhibitors (Xia order of magnitude higher than the endogenous wild- et al., 1998). However, overexpression of a mutant type CaM (Xia et al., 1998; Fig. 2). Patch-clamp experi- CaM, in which the Ca -binding EF hand motifs were ments using nuclei isolated by protocol Nd (Fig. 1) disabled, interfered with the Ca activation of the SK from q.m. CaM-expressing oocytes revealed that InsP R channel gating by competing with the endogenous channel gating was still inhibited by high [Ca ] : CaM for the interaction with the channels (Xia et al., InsP R channel activities were detected in 11 out of 11 1998; Keen et al., 1999). The effects of mutant CaM ex- patches with pipette solutions containing [Ca ]  2.1 572 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology 2 Figure 5. [Ca ] dependencies of the channel P of the InsP R i o 3 in oocyte nuclei isolated using various protocols (Nd, L, and LNL) and applied potentials (20 mV) as tabulated. All pipette solu- tions used contained 10 M InsP . The dashed curve is a simple ac- Figure 4. Typical current traces from nuclei in ULCaS bath iso- tivating Hill equation fit for the data from nuclei isolated with pro- lated by protocol L. Arrows indicate closed channel current levels. tocol L (large open circles). For comparison, the biphasic Hill Pipette solutions contained 10 M InsP and [Ca ] as tabulated. equation fit (continuous curve) for the data points from nuclei iso- 3 i The last current trace was obtained with 20 mV applied trans- lated directly into NCaS bath (small filled circles) obtained in a membrane potential. Other current traces were obtained with previous study (Mak et al., 1998) are also shown. The InsP R chan- 20 mV applied potential. nel P was lower in ULCaS than in NCaS at [Ca ]  100 nM. It is o i possible that this reflects some intrinsic properties of the InsP R af- ter exposure to the low bath [Ca ]. Alternately, this may only be an artifact as a result of the movement of free Ca ion across the M (Fig. 3 C), but no channel activity was detected in 2 open channel. With pipette [Ca ]  100 nM, when the oocyte any of 9 patches with pipette solutions containing 290 nucleus was in NCaS ([Ca ]  400–500 nM), the Nernst reversal 2 2 potential for Ca ions was 35 mV so Ca ions moved across the M [Ca ] (Fig. 3 D). These results therefore also did 2 open InsP R channel from the lumenal side to the cytoplasmic not support the hypothesis that Ca inhibition of side despite an applied transmembrane voltage of 20 mV. This InsP R channel gating is mediated by CaM. 3 2 2 could cause the effective [Ca ] at the activating Ca -binding The lack of effect of overexpression of the q.m. CaM sites on the cytoplasmic side of the channel to be higher than the 2 2 on Ca inhibition of gating may suggest that endoge- free [Ca ] in the bulk of the pipette solution if the Ca -binding sites are close enough to the ion conducting pore. Conversely, nous CaM is not normally associated with the InsP R. 2 2 when the nucleus was in ULCaS ([Ca ]  5 nM), Ca ions We examined the biochemical association between the moved across the open InsP R channel in the opposite direction, InsP R and CaM by coimmunoprecipitation. Using ly- down the electrical and chemical gradients, possibly lowering the sates prepared from cRNA-injected oocytes overex- 2 2 2 effective [Ca ] at the Ca -binding sites. In [Ca ]  250 nM, i i pressing either w.t. or q.m. CaM (Fig. 2), immunopre- the mean open channel duration (< >) of the InsP R increases o 3 2 2 with [Ca ] (Mak and Foskett, 1998). Therefore, if Ca flux cipitation of the endogenous type 1 InsP R with a spe- across the open InsP R channel caused the effective [Ca ] at the 3 i cific antibody did not coimmunoprecipitate either w.t. 2 2 activating Ca -binding sites to deviate from the free [Ca ] in the or q.m. CaM (n  4; unpublished data). In the con- bulk pipette solution, then channels in NCaS bath would have verse experiments, immunoprecipitation of CaM with longer < > and higher channel P than those in ULCaS bath, as o o an antibody that binds to both w.t. and q.m. forms did observed. On the other hand, in [Ca ]  300 nM, < > does not i o exhibit any dependence on [Ca ] although the mean closed not coimmunoprecipitate the InsP R (n  4; unpub- channel duration (< >) is still affected by [Ca ] (Mak and Fos- c i lished data). These results therefore do not provide evi- 2 2 kett, 1998). Deviation of effective [Ca ] at the Ca -binding sites dence of an association between CaM and the InsP R. 3 2 from the bulk free [Ca ] would dissipate quickly by diffusion In summary, our single-channel patch clamp experi- once the channel closed and therefore would not affect < >. ments revealed that neither high concentrations of a Thus, there would be no difference between the observed P of InsP R in ULCaS and NCaS bath in [Ca ]  300 nM, as ob- CaM antagonist, nor overexpression of a Ca -insensi- 3 i 2 served. tive q.m. CaM had any effect on [Ca ] inhibition of InsP R channel gating. In addition, coimmunoprecipi- tation failed to demonstrate an association between vitro patch clamp studies is mediated by CaM. These CaM and the InsP R. Thus, our investigations did not conclusions are therefore in agreement with those provide any evidence supporting the hypothesis that reached in some other studies (Zhang and Joseph, high [Ca ] inhibition of InsP R gating observed in in 2001; Nosyreva et al., 2002). i 3 573 Mak et al. The Journal of General Physiology 2 We previously demonstrated that the Ca depen- Abrogation of Ca -dependent Inhibition of dence of channel P in nuclei isolated by protocol Nd InsP R Channel Gating into NCaS was well fitted by a biphasic Hill equation Our experimental results suggested that CaM is not in- –1 –1 H H  act inh volved in the inhibition of InsP R channel gating by 2  2 P = P 1 +() K ⁄[] Ca 1C +() []a ⁄ K (1) i i o max act  inh high [Ca ] . However, it remained possible that a  different molecule may be involved, and that condi- with maximum channel open probability (P ) max tions could be identified which would strip such a puta- 0.81  0.02, half-maximal activating [Ca ] (K ) i act tive effector from the InsP R in the isolated nucleus, 210  20 nM, activation Hill coefficient (H )  1.9 2 act thereby rendering the InsP R insensitive to Ca inhibi- 0.3, half-maximal inhibitory [Ca ] (K )  54  3 i inh tion. We reasoned that the putative effector, as a sensor M, and inhibitory Hill coefficient (H )  3.9  0.7 2 2 inh of [Ca ] , might be dependent on normal [Ca ] for i i (Mak et al., 1998). Our new data indicated that the its association with the InsP R. We therefore incubated InsP R in nuclei isolated by protocol L into ULCaS ex- 2 3 the isolated nuclei in an ultra-low Ca bath solution 2 2 hibited no inhibition by high [Ca ] , so that the Ca (ULCaS) before using them for nuclear patch clamp dependence of channel P can be fitted by a simple ac- 2 o experiments to determine the Ca dependence of the tivating Hill equation InsP R gating. –1 In the first set of experiments, nuclei were isolated by H  act P = P 1 +() K ⁄[] Ca , (2) 2 o max act protocol L (Fig. 1) into a bath of ULCaS ([Ca ]  5  nM). In the presence of 10 M cytoplasmic (pipette) with maximum open probability P of 0.84  0.01, 2 max [InsP ] and [Ca ]  20 M, gating of the InsP R ex- 3 i 3 half-maximal activating [Ca ] (K ) of 280  30 nM, i act posed to the ULCaS was very similar to that of InsP R in and activation Hill coefficient (H ) of 2.7  0.3 (Fig. 5). act nuclei isolated directly into NCaS by protocol Nd (Fig. 4; Nuclei isolated directly into a ULCaS bath by proto- Mak et al., 1998). In both cases, channel P was low col Ld were used to determine the minimum duration (0.2) in [Ca ]  150 nM, it increased dramatically to of exposure to ULCaS bath required to relieve high 0.8 as [Ca ] was increased from 150 nM to 1M, and [Ca ] inhibition of InsP R gating. We found that i 3 then P remained at the maximum level of 0.8 when channel activities could be detected with a pipette solu- [Ca ] was further increased from 1 to 20 M (Fig. 5). tion containing 10 M InsP and 290 M [Ca ] no 3 i The InsP R in nuclei isolated by protocol Nd were inhib- earlier than 5 min after the nucleus was isolated into ited by [Ca ]  20 M (Mak et al., 1998) but, remark- the ULCaS bath. Thus, the process involved in the re- ably, InsP R in nuclei isolated into ULCaS by protocol L lief of Ca inhibition of InsP R channel gating by ex- 2 3 exhibited robust channel activities in [Ca ] as high as posure of the isolated nuclei to ULCaS is a slow one, re- 1.5 mM (Fig. 4) with no decrease in channel P (Fig. 5). quiring a few minutes. Thus, a 20-min exposure to the ULCaS containing 5 To determine if normal cytoplasmic [Ca ] (50 nM Ca caused the gating of InsP R channel to be no nM) is low enough to cause the relief of high [Ca ] 2 i longer inhibited by high [Ca ] . All of the InsP R chan- i 3 inhibition of InsP R gating, we isolated oocyte nuclei 2 3 nel activities observed in the ultra-low [Ca ] bath solu- directly in PCaS bath (protocol Pd, Fig. 1). In a series tion also terminated abruptly after 30 s, like those pre- of experiments performed in areas of the nuclear viously observed in the regular bath solution (Mak and membrane identified with very high P , using pipette Foskett, 1994, 1997). solutions with 10 M InsP and 0.5 mM ATP, contain- 2 3 Because of the difference between the free Ca con- ing alternately 630 nM or 221 M [Ca ] , InsP R chan- 2 i 3 centration in the high [Ca ] pipette solutions and ultra- nels were observed in seven out of seven patches with low [Ca ] bath solutions, it is possible that a potential 630 nM [Ca ] , but no InsP R channel activity was ob- i 3 difference may be established across the membrane and served in any of 11 patches with 221 M [Ca ] , even 2 i affect the high [Ca ] inhibition of the InsP R and thus i 3 when the nucleus was exposed to the PCaS bath for its P . We performed patch clamp experiments with 20 over 160 min. Thus, the normal resting [Ca ] of the mV applied potential, using high [Ca ] pipette solution cytoplasm (50 nM) is not sufficiently low to induce ([Ca ]  221 M) with nuclei isolated with protocol L. the relief of Ca inhibition observed in the ultra-low The InsP R channel P (9 out of 20 patches exhibited 3 d Ca condition. channel activity), gating kinetics (last current trace in Fig. 4), and P (Fig. 5) were not detectably different from that InsP Dependence of the InsP R in ULCaS Bath 3 3 recorded at 20 mV (P  6 out of 8 patches), indicating that the abrogation of high [Ca ] inhibition by expo- Our previous studies (Mak et al., 1998, 2001a) revealed sure to ULCaS is not due to simple electrostatic effects that InsP activates gating by relieving the Ca inhibi- that change the membrane potential. tion of the channel. InsP increases K , the inhibitory 3 inh 574 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology 2 half-maximal [Ca ] , with no effect on the values of the channel Ca activation parameters (K , H ) or act act P in Eq. 1. It seemed likely that this mode of InsP max 3 activation cannot operate if the channel is not inhib- ited by high [Ca ] as observed after the channel had been exposed to the ULCaS bath for a few minutes. We therefore examined whether InsP was still required to gate the InsP R under conditions that abrogated Ca inhibition of the channel. A series of experiments was performed using nuclei isolated by protocol L into ULCaS bath, patching in re- gions of the nuclei identified to exhibit high P , with pi- pette solutions alternately containing either 10 M 2 2 InsP and [Ca ]  755 nM, or no InsP and [Ca ] 3 i 3 i between 60 nM and 290 M. Again, the former solu- tion was used to ascertain the presence of functional InsP R channels in the regions of the isolated nuclei se- lected for our experiments for the entire duration of the series. InsP R channel activities were observed in 27 out of 30 membrane patches in the presence of InsP , but no channel activity was detected in any of the 10 patches without InsP (Fig. 6 A). Therefore, even though the InsP R was no longer inhibited by high Figure 6. (A and B) Typical current traces from nuclei isolated [Ca ] when the nucleus was isolated into ULCaS, with protocol L. Arrows indicate closed channel current levels. (A) InsP was nonetheless still necessary for channel gating. 2 3 The pipette solutions contained no InsP and 290 M [Ca ] as 3 i Because it seemed paradoxical that InsP activates tabulated. (B) The pipette solutions contained 10 nM InsP and 3 3 2 2 [Ca ] as tabulated. (C) [Ca ] dependence of the channel P of channel gating by modulating the ability of Ca to in- i i o the InsP R in the presence of various [InsP ] as tabulated. The 3 3 hibit the channel, and yet InsP R channels that exhibit number of channels used to evaluate each of the data points (n) is no high [Ca ] inhibition still require InsP for gating, i 3 tabulated next to the corresponding data point. Oocyte nuclei we examined the effects of subsaturating [InsP ] on 3 used were isolated using protocol L. The curves are simple activat- channel gating under conditions that abolish high ing Hill equation fits (Eq. 2) with the same K  280 nM and act H  2.7. The dashed, dotted, and continuous curves have P [Ca ] inhibition. It was shown previously that in the act max 0.18, 0.35, and 0.84 for [InsP ]  10 nM, 20 nM, and 10 M, re- presence of a subsaturating concentration of InsP spectively. (10–33 nM), InsP R channels isolated directly into NCaS (protocol Nd) were much more sensitive to Ca inhibi- tion than those exposed to higher [InsP ] (Mak et al., 2 2 Reversibility of the Regulation by Bath [Ca ] of Ca 1998). In contrast, we observed that channels in nuclei Inhibition of the InsP R Channel isolated into and incubated in ULCaS (protocol L), and activated by subsaturating concentrations of InsP (10– It is possible, as we stated before, that the inhibition of 2 2 20 nM) exhibited no Ca inhibition. Channel activities InsP R gating by high [Ca ] is mediated by some mol- 3 i were observed in 340 M [Ca ] , a normally inhibiting ecule that is tightly bound to the InsP R in the NCaS i 3 2 2 [Ca ] , as well as in 4.2 M [Ca ] (Fig. 6 B) with simi- bath, and that dissociates from the channel in the pres- i i lar channel P (Fig. 6 C). Importantly, the maximum P ence of extremely low [Ca ] in the ULCaS bath. Disso- o o observed in subsaturating [InsP ] was lower than that ciation of this putative effector from the InsP R chan- 3 3 observed in saturating [InsP ] (c.f. Fig. 4, [Ca ]  5.5 nel can then render the channel insensitive to inhibi- 3 i and 340 M, and Fig. 6 B). Within the subsaturating tion by high [Ca ] . Accordingly, after dissociation, the range, i.e., [InsP ] 100 nM, increasing [InsP ] af- putative effector molecule could possibly diffuse away 3 3 fected the channel activity mainly by tuning the value of into the essentially infinitely large volume of the bath. P in the simple activating Hill equation (Eq. 2) (Fig. If this model is correct, the loss of Ca inhibition max 6 C), instead of affecting the Ca inhibitory parame- should be irreversible. To explore the reversibility of ters (K or H ), but not P in the biphasic Hill the loss of Ca inhibition, we performed patch-clamp inh inh max equation (Eq. 1), as normally observed in the InsP R experiments on nuclei isolated from the same batch of channel exposed to NCaS (Mak et al., 1998). Thus, the oocytes using different isolation/incubation protocols. effect of InsP on the InsP R channel in ULCaS was dra- As described above, Ca inhibition was abrogated 3 3 matically different from that observed in NCaS. when the nuclei were isolated into ULCaS bath by pro- 575 Mak et al. The Journal of General Physiology Figure 7. Typical current traces obtained from nuclei isolated us- ing different protocols, all from the same batch of oocytes. Arrows indicate closed channel current levels. All pipette solutions con- tained 10 M InsP . (A) InsP R channel activity in 290 M [Ca ] 3 3 i in nuclei isolated by protocol L into ULCaS, n  5. (B) Absence of InsP R channel activity in 290 M [Ca ] in nuclei isolated by pro- 3 i tocol LN, n  11. (C) InsP R channel gating in 5.5 M [Ca ] 3 i (n  2) in the same nucleus as used in B. (D) InsP R channel activ- ity in 290 M [Ca ] in nuclei isolated by protocol LNL, n  4. tocol L (Fig. 7 A). However, when the nuclei were re- Figure 8. (A) Typical current traces obtained from nuclei iso- turned to the NCaS bath for 20 min before patch lated from uninjected oocytes using protocol L with pipette solu- clamping (protocol LN, Fig. 1), no InsP R channel ac- tion containing 10 M InsP , 290 M [Ca ] , and 10 M purified 3 3 i CaM, n  4. (B–D) Typical current traces obtained from nuclei iso- tivities were detected at [Ca ]  290 M (Fig. 7 B) in lated from oocytes expressing q.m. CaM with pipette solution con- any of the 11 patches obtained, even though channel taining 10 M InsP and 290 M [Ca ] . InsP R channel activity 3 i 3 gating was observed in 4 out of 5 patches using pipette was observed in 290 M [Ca ] in nuclei isolated by protocol L solutions with [Ca ]  5.5 M (Fig. 7 C). Thus, de- (B, n  6) or protocol LNL (D, n  8), but not in nuclei isolated spite prior exposure to ULCaS, normal Ca inhibition by protocol LN (C, n  9). Arrows indicate closed channel current levels. (E) Histogram of InsP R channel P at 10 M InsP and 290 of InsP R channel gating was restored when the nuclei 3 o 3 M [Ca ] observed in various nuclei under experimental condi- were transferred back into NCaS. This restoration of tions as tabulated. normal Ca inhibition was in turn reversible. Reexpo- sure of the nuclei to ULCaS (protocol LNL, Fig. 1) again eliminated normal Ca inhibition of gating (Fig. the bath [Ca ]. The working hypothesis was that addi- 7 D). The InsP R channels in nuclei isolated by proto- 3 tion of CaM would restore normal inhibition of chan- col LNL exhibited the same P (Fig. 5, filled square) as o nel gating by high [Ca ] after it had been relieved by those in nuclei isolated into ULCaS by protocol L with- exposure to the low [Ca ] bath. Patch-clamp experi- out ever being exposed to NCaS (Fig. 5, open circles). ments were performed on nuclei isolated by protocol L These experiments indicated, first, that abolition of into ULCaS bath, using a pipette solution containing Ca inhibition of channel gating by exposure of nuclei 10 M purified CaM with 10 M InsP and high [Ca ] 3 i to ultra-low bath [Ca ] was fully reversible, and sec- (290 M). Nevertheless, InsP R channel gating was ob- ond, that it was affected only by the [Ca ] of the bath- served in the presence of CaM (Fig. 8 A) that was indis- ing solution in which the patch-clamp experiments tinguishable (P  0.05, Fig. 8 E) from that observed un- were performed, independent of the history of bath der the same conditions without CaM (compare Fig. 4, [Ca ] to which the nuclei were previously exposed. [Ca ]  290 M). Thus, addition of CaM did not re- These results suggest either that the sensitivity of Ca constitute normal high [Ca ] inhibition of channel inhibition of the InsP R to the bath [Ca ] is an in- 3 gating. trinsic property of the InsP R channel, or that it is 3 We also performed a series of patch-clamp experi- mediated by some molecule that remains in a stable ments on nuclei isolated from oocytes expressing the complex with the channel throughout the multiple Ca -insensitive q.m. CaM. When the nuclei were iso- transfers of the nucleus into various baths containing lated by protocol L into ULCaS bath, InsP R channel different [Ca ]. activities were observed in high (290 M) [Ca ] (six of eight patches, Fig. 8 B) as frequently as in the nor- 2 2 Is CaM Involved in the Regulation by Bath [Ca ] of Ca mally “permissive” [Ca ] between 500 nM and 5.5 M Inhibition of InsP R Channel? (seven of eight patches). Furthermore, the channel P We explored the possible role of CaM in mediating the was the same as that observed in the channels in nuclei novel regulation of Ca inhibition of InsP R gating by isolated by protocol L from uninjected oocytes (P 576 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology 2 0.05, Fig. 8 E). In addition, expression of the q.m. CaM hibited by high [Ca ] in our nuclear patch-clamping had no effect on the reversibility of the low [Ca ] bath experiments (Fig. 3, A and B). Second, overexpression effect. Thus, no InsP R channels were detected in any in oocytes of a dominant-negative, Ca -insensitive q.m. of the nine patches from nuclei isolated from mutant CaM did not interfere with normal Ca inhibition of CaM-expressing oocytes by protocol LN (Fig. 8 C). the X-InsP R-1 in the oocyte nuclear envelope (Fig. 3, C Moreover, high [Ca ] inhibition of the channel was and D). Third, addition of CaM (10 M) to the pipette still completely abrogated in nuclei isolated from mu- solution did not reconstitute normal Ca inhibition of tant CaM-expressing oocytes by protocol LNL. Thus, InsP R channel after it was abrogated by exposure of channel activities were observed in 290 M [Ca ] the channel to ULCaS bath (Fig. 8 A). Fourth, overex- (Fig. 8 D) with P (8 of 13 patches) similar (P  0.05) pression of q.m. CaM in oocytes did not affect the abro- 2 2 to that in [Ca ] between 500 nM and 5.5 M (seven gation of Ca inhibition by exposure of the channel to of eight patches). InsP R channel P in these nuclei ULCaS bath, nor did it affect the restoration of Ca in- 3 o from mutant CaM-expressing oocytes was the same as hibition when the channel was placed back in NCaS that observed in nuclei isolated from uninjected oo- bath (Fig. 8, B–D). Furthermore, coimmunoprecipita- cytes by protocol L or LNL (P from t test was 0.05, tion experiments did not detect any association be- Fig. 8 E). Therefore, there were no differences between tween CaM and InsP R-1 in the Xenopus oocytes. There- 2 2 the Ca inhibition (or lack thereof) of InsP R channel fore, whereas CaM may regulate intracellular Ca sig- gating observed in nuclei isolated by various protocols naling through other mechanisms, our experimental from oocytes overexpressing the mutant CaM and that results, together with other recent publications (Zhang observed in nuclei isolated from uninjected oocytes, and Joseph, 2001; Nosyreva et al., 2002), indicate that it under all experimental conditions. does not regulate inhibition of InsP R-1 channel gating by high [Ca ] . What then could be the mechanism of Ca inhibi- DISCUSSION tion? The simplest hypothesis is that the Ca binding Is There a Role of CaM in Inhibition by High [Ca ] of sites responsible for Ca inhibition of channel gating InsP R-1 Gating? are contained within the structure of the InsP R pro- tein itself. Many regions of the protein have been Numerous investigations have explored the interac- shown to bind Ca in in vitro studies (Sienaert et al., tions between the InsP R and CaM, but their nature, 1996, 1997). One or more of these or as yet unidenti- regulation, and functional effects on intracellular Ca fied sites may play a role, although there are no data signaling are still far from clear. Although it was re- available that address this issue. Alternately, another ported that CaM binding regulates high [Ca ] inhi- molecule could perhaps be involved. The InsP R in- bition of the InsP R-1 channel (Hirota et al., 1999; teracts with other proteins (Patel et al., 1999; Yang et Michikawa et al., 1999), subsequent studies using mi- al., 2002). Of interest, a calmodulin-like protein, crosomal fluxes or reconstituted channels in lipid bilay- CaBP1, interacts with high affinity with the ligand- ers have provided contradictory evidence (Zhang and binding region of the channel (Yang et al., 2002). Joseph, 2001; Nosyreva et al., 2002). In this study, we in- Whereas it is highly unlikely that CaBP1 and its iso- vestigated the possible involvement of CaM in the high forms mediate Ca inhibition, since they are likely [Ca ] inhibition of single-channel InsP R-1 gating us- i 3 neurally restricted and have been shown to stimulate ing the nuclear patch clamp method (Mak and Foskett, channel gating (Yang et al., 2002), the identification 1994). This approach enables single-channel recording of noncalmodulin Ca -binding protein interactions of endogenous and recombinant InsP R channels in with the receptor lends credence to the notion that a their native membrane environment. Similar biphasic Ca -binding protein could possibly be involved in regulation by [Ca ] of both the endogenous Xenopus mediating Ca responses of the channel. Because type 1 channel and the recombinant rat type 3 InsP R Ca inhibition of channel activity has been observed channel have been observed in previous nuclear patch in a number of distinct experimental systems from dif- clamp studies (Mak et al., 1998, 2001a). In this study, ferent species, such a putative effector would need to we directly explored the role of CaM in high [Ca ] in- be ubiquitously expressed and tightly associated with hibition of InsP R channel gating. We have found no the channel. evidence to support the hypothesis that inhibition of InsP R-1 channel activities by high [Ca ] is mediated 3 i A Novel Regulation of [Ca ] Inhibition of InsP R-1 i 3 by direct interaction between the InsP R channel and Channel Gating CaM. First, in the presence of 500 M W-7, a CaM an- Our investigations have revealed a novel CaM-indepen- tagonist that was previously reported to alleviate Ca dent regulation of the InsP R-1 channel: abrogation of inhibition of InsP R-1 channels reconstituted into bilay- high [Ca ] inhibition of InsP R-1 channel gating by ers (Michikawa et al., 1999), the X-InsP R-1 was still in- i 3 577 Mak et al. The Journal of General Physiology 2 2 2 exposure of the channel to ultra-low bath [Ca ] (5 Ca . It could only be observed when bath [Ca ] was 2 2 nM). The physical location of the low [Ca ] -sens- reduced to very low levels. Thus, if the [Ca ] -sens- bath bath ing mechanism on the InsP R protein is unknown. The ing mechanism is located on the cytoplasmic side of the 2 2 abrogation could possibly be caused by low [Ca ] in channel, it is likely to be a set of cooperative Ca -bind- the perinuclear space between the inner and outer ing sites. Further studies are necessary to distinguish nuclear envelope, to which the lumenal side of the whether cytoplasmic or lumenal [Ca ] is being sensed InsP R-1 channel is exposed. In this case, exposure to in the disruption of high [Ca ] inhibition of the 3 i 2 2 the ultra-low bath [Ca ] causes the lumenal [Ca ] to InsP R, and to determine the molecular mechanisms fall to low levels due to uncompensated Ca leak; and involved in that process. the [Ca ] sensing mechanism responsible for switch- Mechanism of Regulation of High [Ca ] Inhibition of ing high [Ca ] inhibition of InsP R-1 channel on and i 3 InsP R-1 Channel Gating by Exposure to Low [Ca ] Bath off is located on the lumenal side of the InsP R chan- nel. The existence of a Ca -binding site on the lume- A novel allosteric model, developed in the accompany- nal side of the InsP R-1 channel has been reported ing manuscript, can account for the effect of ultra-low (Sienaert et al., 1996). Our previous studies indicated [Ca ] bath exposure on the abrogation of high that the ionic composition of the solution in the peri- [Ca ] inhibition as well as the effect of InsP to modu- i 3 nuclear space of the isolated oocyte nucleus is likely to late maximum channel P , rather than K , under o inh be similar to that of the bath solution (Mak and Fos- these conditions. In brief, this model accounts for our kett, 1997). The long lag time (300 s) between the iso- results by postulating the existence of two functional in- 2 2 lation of the nucleus into the ultra-low [Ca ] bath so- hibitory Ca binding sites associated with each mono- lution and the earliest detection of InsP R channel ac- mer of the tetrameric channel. One site is only inhibi- tivities that could not be inhibited by high [Ca ] may tory when the channel is not liganded with InsP , i 3 reflect the time required for the solution in the perinu- because InsP binding relieves the Ca inhibition clear space to become fully equilibrated with the bath imposed by this site. In contrast, the properties of the solution, or the time taken for Ca bound to the lume- other inhibitory site are not affected by InsP binding. 2 2 2 nal Ca -binding sites of the InsP R channel to dissoci- In normal physiological [Ca ] conditions, Ca bind- 3 i ate from the sites after the drop in lumenal [Ca ], or a ing to this InsP -insensitive site provides the observed combination of the two. high [Ca ] inhibition (K 50 M) of the fully i inh Alternately, the [Ca ]-sensing mechanism could pos- InsP -liganded channel. The ability of this InsP -insensi- 3 3 sibly be located on the cytoplasmic side of the chan- tive site to be inhibitory is reversibly lost after exposure nel. In this case, the long lag time (300 s) between ex- of the channel for 5 min to an ultra-low bath [Ca ] posure of the channel to ultra-low [Ca ] and the abro- (5 nM). Thus, the observed abrogation of high 2 2 gation of high [Ca ] inhibition would imply that [Ca ] inhibition of channel activity in saturating i i dissociation of Ca from the sensing mechanism is [InsP ] can be accounted for by the fact that there is no 1 2 slow (rate 0.003 s ). Although such a sensing mecha- longer any functional inhibitory Ca -binding site. On nism would be exposed to high [Ca ] in the pipette the other hand, in the absence of InsP , the InsP -sensi- 3 3 solution as soon as the giga-ohm seal was formed, tive Ca inhibition site is functional and keeps the InsP R channel activities were nevertheless observed channel closed. Thus, the channel still requires InsP 3 3 for typically 10 s when the channel was exposed to to gate open even when the InsP -insensitive site has 2 2 [Ca ] 290 M before the activities abruptly termi- been disrupted by exposure to ultra-low bath [Ca ]. A nated (Mak and Foskett, 1997; Boehning et al., 2001). detailed description of this model, which can account Thus, binding of Ca to the sensing mechanism to re- for these and many other features of ligand regulation store normal high [Ca ] inhibition must also be a very of the channel observed in nuclear patch clamp experi- 1 2 slow process (rate 0.1 s ). If the [Ca ] sensing ments, is developed in the accompanying manuscript mechanism is in equilibrium with the cytoplasmic solu- (Mak et al., 2003, in this issue). tion, the forward rate constant (k ) for Ca dissocia- Are Different Sensitivities to Inhibition by High [Ca ] a 2 1 tion from the [Ca ]-sensing mechanism is 0.003 s Fundamental Distinguishing Feature among the and the reverse rate constant (k ) is such that 0.1 s InsP R Isoforms? k 290 M. If the [Ca ]-sensing mechanism is a sim- ple Ca binding site, then the equilibrium constant K The three isoforms of InsP R have complicated pat- (k /k ) for Ca dissociation from the site should then terns of expression in various tissues with complex reg- f r be 10 M. However, abrogation of channel inhibition ulation by various mechanisms (Taylor et al., 1999). Be- was not observed in our normal bath solutions that cause the permeation and conductance properties of contain 300–500 nM Ca (Mak et al., 1998), or in our the InsP R isoforms are very similar (Mak et al., 2000; physiological Ca bath solution containing 50 nM free Ramos-Franco et al., 2000), differences among the iso- 578 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology forms in localization and channel gating and its regula- note, this InsP dependence of maximum P is very sim- 3 o tion are likely to be reasons for the existence of InsP R ilar to the observed effect of InsP on the P of InsP R-2 3 3 o 3 diversity. A review of published single-channel studies channels reconstituted into lipid bilayers (Ramos- of various InsP R isoforms suggests that different sensi- Franco et al., 1998b). These observations raise the in- tivities to inhibition by high [Ca ] may be one distin- triguing possibility that the observed differences in the guishing functional feature among the various InsP R sensitivities to Ca inhibition of various InsP R iso- 3 3 isoforms. Nevertheless, it is not clear whether such dif- forms may be a consequence of the different environ- ferences are intrinsic to the channels, or whether they ment and/or isolation conditions to which the chan- are perhaps artificially generated by the different ex- nels were exposed, rather than the result of differences perimental protocols used for studying InsP R channel in fundamental intrinsic characteristics of the individ- activity. ual isoforms. For example, the InsP R-1 and InsP R-3 3 3 In the presence of 1 M InsP , native and recombi- channel isoforms exhibited very similar inhibition by nant InsP R-1 channels (including various splice vari- high [Ca ] when they are studied in a native ER mem- 3 i ants) reconstituted into lipid bilayers exhibited similar brane environment (Mak et al., 1998, 2001a), but they strong inhibition by [Ca ] with half-maximal inhibi- behaved differently in reconstitution systems. We sug- tory [Ca ] of 0.1–2 M (Bezprozvanny et al., 1991; Ra- gest that it is worth considering the possibility that pro- mos-Franco et al., 1998a,b; Tu et al., 2002), whereas na- cedures employed in the isolation and reconstitution tive Xenopus and recombinant rat InsP R-1 channels and recording of the InsP R-3 used in (Hagar et al., 3 3 studied in their native membrane environment using 1998) disrupted the normal high [Ca ] inhibition of nuclear patch clamp techniques exhibited inhibition the InsP R-3, causing the observed lack of Ca inhibi- by high [Ca ] , but with a significantly higher half- tion, in very much the same way that exposure to a 2 2 maximal inhibitory [Ca ] of 50 M (Mak et al., ULCaS bath abrogated the high [Ca ] inhibition of i i 1998; Boehning et al., 2001). When reconstituted into InsP R-1 observed in this study. Whereas the proce- planar bilayers, Ca inhibition of InsP R-1 could be al- dures used in the isolation and reconstitution and re- leviated by very high [InsP ] (180 M) (Kaftan et al., cording of InsP R-1 by themselves did not eliminate 3 3 2 2 1997; Moraru et al., 1999), whereas Ca inhibition of high [Ca ] inhibition of the channel (Bezprozvanny InsP R-1 studied in the native membrane environment et al., 1991; Ramos-Franco et al., 1998a,b; Tu et al., was not further affected by [InsP ] once the channel 2002), they may account for the ability, observed only was saturated with [InsP ] 100 nM (Mak et al., 1998). in the reconstituted systems, of extremely high [InsP ] 3 3 InsP R-2 channels reconstituted in lipid bilayers ex- to abrogate high [Ca ] inhibition (Kaftan et al., 1997; 3 i hibited variable but low sensitivity to inhibition by high Moraru et al., 1999). By the same token, it is possible 2 2 2 [Ca ] , with a half-maximal [Ca ] of 400 M for that the very low sensitivity to high [Ca ] inhibition of i i i recombinant InsP R-2 channels (Ramos-Franco et al., the InsP R-2 channel isoform reconstituted in lipid bi- 3 3 2000) and 1 mM for native channels (Ramos-Franco layers (Ramos-Franco et al., 1998b, 2000) was induced et al., 1998b, 2000) in 1 M InsP . by the isolation and reconstitution and recording pro- Native type 3 InsP R channels reconstituted into lipid tocols. Obviously, these issues will need to be resolved bilayers exhibited no detectable inhibition by high in future studies, for example, of the Ca responses of [Ca ] and its P remained at its maximum value type 2 InsP R channels in the native ER membrane en- i o 3 (0.05) in [Ca ] between 1 and 100 M in the pres- vironment, under the same experimental conditions as ence of 2 M InsP (Hagar et al., 1998). In marked con- those used for the types 1 and 3 InsP R isoforms; and of 3 3 trast, recombinant r-InsP R-3 in the nuclear membrane the sensitivities of Ca inhibition of the other InsP R 3 3 2 2 of oocytes is inhibited by high [Ca ] in an InsP -depen- isoforms to exposure to ultra-low bath [Ca ]. Never- i 3 dent manner very similar to that for X-InsP R-1 under theless, our identification in this study of conditions identical experimental conditions (Mak et al., 2001a). that can radically alter the [Ca ] inhibition properties How can we account for such divergent results? Our of the channel suggests that careful consideration of studies here demonstrate that Ca inhibition of the the isolation protocols and other conditions to which X-InsP R-1 channel in its native membrane environment InsP R channels are exposed before they are examined 3 3 can be completely, specifically and reversibly abrogated will be warranted in future studies. under certain experimental conditions (after exposure This work was supported by grants to J.K. Foskett from the NIH to a nominally Ca -free bath). Associated with this ef- (MH59937, GM56328) and to D.-O.D. Mak from the American fect, the InsP dependence of the channel P was also 3 o Heart Association (9906220U). changed—normally, InsP affects the apparent affinity Olaf S. Andersen served as editor. of the inhibitory Ca -binding sites of the channel (Mak et al., 1998), whereas after ULCaS bath exposure, Submitted: 27 Januar y 2003 InsP affects the maximum P observed (Fig. 6 C). Of Accepted: 2 September 2003 3 o 579 Mak et al. The Journal of General Physiology REFERENCES Mak, D.-O.D., S. McBride, and J.K. Foskett. 1998. Inositol 1,4,5-tris- Baudet, S.B., L. Hove-Madsen, and D.M. Bers. 1994. How to make phosphate activation of inositol trisphosphate receptor Ca and use calcium-specific mini- and microelectrodes. In A Practi- channel by ligand tuning of Ca inhibition. Proc. Natl. Acad. Sci. cal Guide to the Study of Calcium in Living Cells. 1st ed. R. Nuc- USA. 95:15821–15825. citelli, editor. Academic Press, San Diego. 94–114. Mak, D.-O.D., S. McBride, V. Raghuram, Y. Yue, S.K. Joseph, and Berridge, M.J. 1993. Inositol trisphosphate and calcium signalling. J.K. Foskett. 2000. Single-channel properties in endoplasmic Nature. 361:315–325. reticulum membrane of recombinant type 3 inositol trisphos- Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Bell-shaped cal- phate receptor. J. Gen. Physiol. 115:241–256. cium-response curves of Ins(1,4,5)P - and calcium-gated chan- Mak, D.-O.D., S. McBride, and J.K. Foskett. 2001a. Regulation by nels from endoplasmic reticulum of cerebellum. Nature. 351: Ca and inositol 1,4,5-trisphosphate (InsP ) of single recombi- 751–754. nant type 3 InsP receptor channels. Ca activation uniquely dis- Boehning, D., S.K. Joseph, D.-O.D. Mak, and J.K. Foskett. 2001. Sin- tinguishes types 1 and 3 InsP receptors. J. Gen. Physiol. 117:435– gle-channel recordings of recombinant inositol trisphosphate re- 446. ceptors in mammalian nuclear envelope. Biophys. J. 81:117–124. Mak, D.-O.D., S. McBride, and J.K. Foskett. 2001b. ATP-dependent Cardy, T.J., and C.W. Taylor. 1998. A novel role for calmodulin: adenophostin activation of inositol 1,4,5-trisphosphate receptor Ca -independent inhibition of type-1 inositol trisphosphate re- channel gating: kinetic implications for the durations of calcium ceptors. Biochem. J. 334:447–455. puffs in cells. J. Gen. Physiol. 117:299–314. Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. Mak, D.-O.D., S. McBride, and J.K. Foskett. 2003. Spontaneous 1998. Type III InsP receptor channel stays open in the presence channel activity of the inositol 1,4,5-triphosphate (InsP ) recep- 3 3 of increased calcium. Nature. 396:81–84. tor (InsP R). Application of allosteric modeling to calcium and Hirota, J., T. Michikawa, T. Natsume, T. Furuichi, and K. Mikoshiba. InsP regulation of InsP R single-channel gating. J. Gen. Physiol. 3 3 1999. Calmodulin inhibits inositol 1,4,5-trisphosphate-induced 122:583–603. calcium release through the purified and reconstituted inositol Michikawa, T., J. Hirota, S. Kawano, M. Hiraoka, M. Yamada, T. Fu- 1,4,5- trisphosphate receptor type 1. FEBS Lett. 456:322–326. ruichi, and K. Mikoshiba. 1999. Calmodulin mediates calcium- Jiang, Q., D.-O.D. Mak, S. Devidas, E.M. Schwiebert, A. Bragin, Y. dependent inactivation of the cerebellar type 1 inositol 1,4,5-tris- Zhang, W.R. Skach, W.B. Guggino, J.K. Foskett, and J.F. Engel- phosphate receptor. Neuron. 23:799–808. hardt. 1998. Cystic fibrosis transmembrane conductance regula- Monkawa, T., A. Miyawaki, T. Sugiyama, H. Yoneshima, M. Yama- tor-associated ATP release is controlled by a chloride sensor. J. moto-Hino, T. Furuichi, T. Saruta, M. Hasegawa, and K. Miko- Cell Biol. 143:645–657. shiba. 1995. Heterotetrameric complex formation of inositol Joseph, S.K., and S. Samanta. 1993. Detergent solubility of the ino- 1,4,5-trisphosphate receptor subunits. J. Biol. Chem. 270:14700– sitol trisphosphate receptor in rat brain membranes. Evidence 14704. for association of the receptor with ankyrin. J. Biol. Chem. 268: Moraru, I.I., E.J. Kaftan, B.E. Ehrlich, and J. Watras. 1999. Regula- 6477–6486. tion of type 1 inositol 1,4,5-trisphosphate-gated calcium channels Joseph, S.K., C. Lin, S. Pierson, A.P. Thomas, and A.R. Maranto. by InsP and calcium: Simulation of single channel kinetics based 1995. Heteroligomers of type-I and type-III inositol trisphosphate on ligand binding and electrophysiological analysis. J. Gen. Phys- receptors in WB rat liver epithelial cells. J. Biol. Chem. 270:23310– iol. 113:837–849. 23316. Nosyreva, E., T. Miyakawa, Z. Wang, L. Glouchankova, A. Mizu- Joseph, S.K. 1996. The inositol trisphosphate receptor family. Cell. shima, M. Iino, and I. Bezprozvanny. 2002. The high affinity cal- Signal. 8:1–7. cium-calmodulin-binding site does not play a role in modulation Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. Inositol 1,4,5-tris- of type 1 inositol (1,4,5)-trisphosphate receptor function by cal- phosphate (InsP ) and calcium interact to increase the dynamic cium and calmodulin. Biochem. J. 365:659–667. range of InsP receptor-dependent calcium signaling. J. Gen. Nucifora, F.C., Jr., A.H. Sharp, S.L. Milgram, and C.A. Ross. 1996. Physiol. 110:529–538. Inositol 1,4,5-trisphosphate receptors in endocrine cells: localiza- Keen, J.E., R. Khawaled, D.L. Farrens, T. Neelands, A. Rivard, C.T. tion and association in hetero- and homotetramers. Mol. Biol. Bond, A. Janowsky, B. Fakler, J.P. Adelman, and J. Maylie. 1999. Cell. 7:949–960. Domains responsible for constitutive and Ca -dependent inter- Patel, S., S.K. Joseph, and A.P. Thomas. 1999. Molecular properties actions between calmodulin and small conductance Ca -acti- of inositol 1,4,5-trisphosphate receptors. Cell Calcium. 25:247– vated potassium channels. J. Neurosci. 19:8830–8838. 264. Lin, C., J. Widjaja, and S.K. Joseph. 2000. The interaction of cal- Ramos-Franco, J., S. Caenepeel, M. Fill, and G. Mignery. 1998a. Sin- modulin with alternatively spliced isoforms of the type-I inositol gle channel function of recombinant type-1 inositol 1,4,5-tris- trisphosphate receptor. J. Biol. Chem. 275:2305–2311. phosphate receptor ligand binding domain splice variants. Bio- Maeda, N., T. Kawasaki, S. Nakade, N. Yokota, T. Taguchi, M. Kasai, phys. J. 75:2783–2793. and K. Mikoshiba. 1991. Structural and functional character- Ramos-Franco, J., M. Fill, and G.A. Mignery. 1998b. Isoform-spe- ization of inositol 1,4,5- trisphosphate receptor channel from cific function of single inositol 1,4,5-trisphosphate receptor mouse cerebellum. J. Biol. Chem. 266:1109–1116. channels. Biophys. J. 75:834–839. Mak, D.-O.D., and J.K. Foskett. 1994. Single-channel inositol 1,4,5- Ramos-Franco, J., D. Bare, S. Caenepeel, A. Nani, M. Fill, and G. trisphosphate receptor currents revealed by patch clamp of iso- Mignery. 2000. Single-channel function of recombinant type 2 lated Xenopus oocyte nuclei. J. Biol. Chem. 269:29375–29378. inositol 1,4, 5- trisphosphate receptor. Biophys. J. 79:1388–1399. Mak, D.-O.D., and J.K. Foskett. 1997. Single-channel kinetics, inac- Sienaert, I., H. De Smedt, J.B. Parys, L. Missiaen, S. Vanlingen, H. tivation, and spatial distribution of inositol trisphosphate (IP ) Sipma, and R. Casteels. 1996. Characterization of a cytosolic and receptors in Xenopus oocyte nucleus. J. Gen. Physiol. 109:571– a luminal Ca binding site in the type I inositol 1,4,5-trisphos- 587. phate receptor. J. Biol. Chem. 271:27005–27012. Mak, D.-O.D., and J.K. Foskett. 1998. Effects of divalent cations on Sienaert, I., L. Missiaen, H. De Smedt, J.B. Parys, H. Sipma, and R. single-channel conduction properties of Xenopus IP receptor. Casteels. 1997. Molecular and functional evidence for multiple Am. J. Physiol. 275:C179–C188. Ca -binding domains in the type 1 inositol 1,4,5-trisphosphate 580 Regulation of Ca Inhibition of the InsP Receptor The Journal of General Physiology receptor. J. Biol. Chem. 272:25899–25906. Xia, X.M., B. Fakler, A. Rivard, G. Wayman, T. Johnson-Pais, J.E. Taylor, C.W. 1998. Inositol trisphosphate receptors: Ca -modu- Keen, T. Ishii, B. Hirschberg, C.T. Bond, S. Lutsenko, et al. 1998. lated intracellular Ca channels. Biochim. Biophys. Acta. 1436:19– Mechanism of calcium gating in small-conductance calcium-acti- 33. vated potassium channels. Nature. 395:503–507. Taylor, C.W., A.A. Genazzani, and S.A. Morris. 1999. Expression of Yamada, M., A. Miyawaki, K. Saito, T. Nakajima, M. Yamamoto- inositol trisphosphate receptors. Cell Calcium. 26:237–251. Hino, Y. Ryo, T. Furuichi, and K. Mikoshiba. 1995. The calmodu- Tu, H., T. Miyakawa, Z. Wang, L. Glouchankova, M. Iino, and I. Bez- lin-binding domain in the mouse type 1 inositol 1,4,5-trisphos- prozvanny. 2002. Functional characterization of the Type 1 inosi- phate receptor. Biochem. J. 308:83–88. tol 1,4,5-trisphosphate receptor coupling domain SII(/ ) Yang, J., S. McBride, D.O. Mak, N. Vardi, K. Palczewski, F. Haese- splice variants and the Opisthotonos mutant form. Biophys. J. 82: leer, and J.K. Foskett. 2002. Identification of a family of calcium 1995–2004. sensors as protein ligands of inositol trisphosphate receptor Ca Wojcikiewicz, R.J. 1995. Type I, II, and III inositol 1,4,5-trisphos- release channels. Proc. Natl. Acad. Sci. USA. 99:7711–7716. phate receptors are unequally susceptible to down-regulation Zhang, X., and S.K. Joseph. 2001. Effect of mutation of a calmodu- and are expressed in markedly different proportions in different lin binding site on Ca regulation of inositol trisphosphate re- cell types. J. Biol. Chem. 270:11678–11683. ceptors. Biochem. J. 360:395–400. 581 Mak et al. The Journal of General Physiology

Journal

The Journal of General Physiology – Pubmed Central

Published: Nov 1, 2003

There are no references for this article.