Address correspondence to J. Kevin Foskett, Department of Physiology, B39 Anatomy-Chemistry Bldg/6085, University of Pennsylvania, Philadelphia, PA 19104-6085. Fax: (215) 573-6808; email: foskett{at}mail.med.upenn.edu
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ABSTRACT |
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Key Words: single-channel electrophysiology patch clamp calcium Xenopus oocyte nucleus
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INTRODUCTION |
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Three isoforms of the InsP3R, with spliced variants, have been identified (Joseph, 1996). Most mammalian cell types express multiple InsP3R isoforms in distinct and overlapping intracellular locations with their absolute and relative expression levels regulated by gene transcription, alternative splicing and receptor degradation that differ during different stages of cell development and in response to extracellular stimuli (Taylor et al., 1999
). Furthermore, formation of hetero-tetrameric channels is possible in cell types expressing more than one InsP3R isoform (Joseph et al., 1995
; Monkawa et al., 1995
; Wojcikiewicz, 1995
; Nucifora et al., 1996
). Although this diversity of InsP3R expression is impressive, its functional correlates and physiological implications remain unclear. Studies of the single-channel properties of the various InsP3R isoforms have revealed that whereas their permeation and conductance properties are very similar (Mak et al., 2000
; Ramos-Franco et al., 2000
), their gating may be differentially inhibited by high [Ca2+]i (Bezprozvanny et al., 1991
; Hagar et al., 1998
; Mak et al., 1998
; Ramos-Franco et al., 1998a
,b
, 2000
; Boehning et al., 2001
; Mak et al., 2001a
). Because high [Ca2+]i inhibition of InsP3R channel gating may be a pivotal feedback mechanism for the regulation of intracellular Ca2+ signaling (Taylor, 1998
), it has been suggested that differential inhibition by high [Ca2+]i of the different InsP3R isoforms may generate distinct Ca2+ signals in different cell types with different patterns of InsP3R isoform expression, and that this may be a reason for the diversity of InsP3R expression (Hagar et al., 1998
).
It has been suggested that high [Ca2+]i inhibition of the InsP3R is mediated by calmodulin (CaM), a ubiquitous Ca2+-binding protein that binds to and regulates the functions of many proteins. CaM was found to bind to the InsP3R-1 in the presence of free Ca2+ to a single site in the regulatory domain (Maeda et al., 1991; Yamada et al., 1995
; Hirota et al., 1999
). Purified InsP3R-1 channels lacking bound CaM were not inhibited by high [Ca2+]i, whereas addition of CaM restored inhibition of channel gating by high [Ca2+]i (Hirota et al., 1999
; Michikawa et al., 1999
). The notion that high Ca2+ inhibition of channel gating was mediated by CaM was reinforced by observations that the type 3 InsP3R (InsP3R-3) did not bind CaM (Yamada et al., 1995
; Cardy and Taylor, 1998
; Lin et al., 2000
) and was not inhibited by high [Ca2+]i (Hagar et al., 1998
). Nevertheless, other data suggest that the role of CaM in high [Ca2+]i inhibition of InsP3R channel gating is far from unequivocal. Despite the absence of detectable interaction between CaM and a mutant InsP3R-1 in which the putative CaM binding site was eliminated (Yamada et al., 1995
), more recent studies have demonstrated that this mutant channel is nevertheless still inhibited by high [Ca2+]i (Zhang and Joseph, 2001
; Nosyreva et al., 2002
). Furthermore, whereas the InsP3R-3 lacks the CaM binding site present in the InsP3R-1 and no interaction between InsP3R-3 and CaM has been detected (Yamada et al., 1995
; Cardy and Taylor, 1998
; Lin et al., 2000
), electrophysiological studies of the recombinant rat InsP3R-3 in its native membrane environment demonstrated that it is nevertheless inhibited by high [Ca2+]i (Mak et al., 2001a
) with quantitative features similar to those of inhibition of the InsP3R-1 in the same membrane (Mak et al., 1998
).
Here, we investigated the possible effects of CaM on high [Ca2+]i inhibition of the gating of single endogenous InsP3R-1 channels in their native membrane environment using nuclear membrane patch clamp electrophysiology (Mak and Foskett, 1994). Our experiments do not provide evidence supporting any role for CaM in this process. However, we discovered a novel regulation of high [Ca2+]i inhibition of InsP3R-1 channel gating. Inhibition of InsP3R-1 gating by high [Ca2+]i can be reversibly abrogated by exposure of the channel to a bathing solution containing ultra-low [Ca2+] (<5 nM). Our observations indicate that inhibition of InsP3R-1 channel gating by high [Ca2+]i can be disrupted by environmental conditions experienced by the channel, and therefore may not be an invariant property of a specific InsP3R isoform. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.
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MATERIALS AND METHODS |
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Western Analysis and Immunoprecipitation
Western analysis was performed on oocyte extracts (cRNA-injected and uninjected), as described in Mak et al. (2000), to ascertain the levels of endogenous and heterologously expressed CaM in the oocytes using a specific antibody (Upstate Biotechnology). Immunoprecipitation of InsP3R (type 1) and CaM was performed using oocyte lysates, as described in (Mak et al., 2000
), with a specific type 1 InsP3R antibody (Joseph and Samanta, 1993
; Joseph et al., 1995
) and protein A agarose (GIBCO BRL), and an antibody to CaM and protein G agarose (GIBCO BRL), respectively.
Solutions for Patch Clamp Experiments
All patch clamp experiments were performed with solutions containing 140 mM KCl and 10 mM HEPES with pH adjusted to 7.1 with KOH. The free Ca2+ concentration ([Ca2+]i) of the pipette solutions (to which the cytoplasmic side of the InsP3R is exposed in patch-clamp experiments) was tightly controlled by buffering various amounts of added CaCl2 (40400 µM) with 500 µM of the high-affinity Ca2+ chelator, BAPTA (1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid; Molecular Probes) and 0.5 mM Na2ATP (100 nM < [Ca2+]i < 2.5 µM); or 500 µM of the low-affinity Ca2+ chelator, 5,5'-dibromo BAPTA (Molecular Probes) and 0.5 mM Na2ATP (5 µM < [Ca2+]i < 15 µM); or 0.5 mM Na2ATP alone (15 µM < [Ca2+]i < 300 µM). Solutions with [Ca2+]i > 300 µM contained no Ca2+ chelator for buffering. The normal Ca2+ bath solution (NCaS) contained 500 µM BAPTA and 250 µM CaCl2 (free [Ca2+] 400500 nM), and the physiological Ca2+ bath solution (PCaS) contained 500 µM BAPTA and 70 µM CaCl2 (free [Ca2+] = 48 ± 5 nM). The free [Ca2+] of these solutions was directly measured using Ca2+-selective mini-electrodes (Baudet et al., 1994
). The ultra-low Ca2+ bath solution (ULCaS) contained 1 mM BAPTA and no added CaCl2. The contaminating [Ca2+] in the solution was determined by induction-coupled plasma mass spectrometry (Mayo Medical Laboratory) to be
610 µM. Ca2+-selective minielectrodes were unable to determine accurately the free [Ca2+] in the ULCaS because of the nonlinear response of the electrode in free [Ca2+] < 5 nM. Free [Ca2+] was calculated using the Maxchelator software (C. Patton, Stanford University, Stanford, CA) to be
0.91.5 nM.
Unless specified otherwise, all pipette solutions contained a saturating concentration (10 µM) of InsP3 (Mak and Foskett, 1994) from Molecular Probes. When specified, the pipette solutions also contained 500 µM W-7 (a CaM binding antagonist; N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride; Calbiochem), or 10 µM purified bovine CaM (Calbiochem). All reagents were used with no further purification.
Oocyte Nucleus Isolation Protocols
A stage V or VI oocyte was gently teased open mechanically in the isolation bathing solution, enabling the translucent nucleus to be isolated from the cytoplasmic material. The isolated nucleus was either directly transferred to the experimental bathing solution (protocol Nd, Ld, and Pd, Fig. 1), or it was transferred through a series of culture dishes containing 45 ml of incubation bath solutions (protocol L, LN, and LNL, in Fig. 1) before it was ultimately transferred to the experimental bath. The nucleus remained in each incubation bath for at least 20 min before the next transfer, to ensure that the solution in the perinuclear lumen between the outer and inner nuclear envelope had attained ionic equilibrium with the bath solution (Mak and Foskett, 1994). Approximately 20 µl of the previous bath solution accompanied the nucleus to the new bath in a transfer. The culture dish containing the nucleus in the experimental bath solution was finally moved onto the stage of the inverted microscope where patch clamp experiments were performed.
Acquisition and Analysis of Single-Channel Patch-clamp Current Records
The isolated nucleus was gently immobilized as described previously (Mak and Foskett, 1994) so that membrane patches could be repeatedly obtained from the same region (±2 µm) of the outer nuclear membrane (Mak and Foskett, 1997
). Due to abrupt termination of channel activity (Mak and Foskett, 1994
, 1997
), patch clamp experiments were performed in "on-nucleus" configuration to maximize the duration of channel activities recorded. To prevent contamination of the pipette solution by the bath solution (especially the Ca2+ chelator in the bath solution) by diffusion through the pipette tip during the time when the pipette was immersed in the bath and before giga-Ohm seal formation, a positive pressure (
10 mmHg) was maintained inside the pipette until the pipette tip was properly positioned on the nuclear membrane. Then suction was applied in the pipette to obtain the giga-Ohm seal. All experiments were performed at room temperature with the pipette electrode at +20 mV relative to the reference bath electrode unless specifically stated otherwise. Each experiment recorded the InsP3R channel activity at a specific [Ca2+]i and [InsP3], with no change of the pipette or bath solutions during the experiment. Data acquisition was performed as previously described (Mak et al., 1998
), with currents recorded with a filtering frequency of 1 kHz and a digitizing frequency 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 InsP3R channel, or two InsP3R channels determined to be identical and independently gated (Mak and Foskett, 1997), were used for channel open probability (Po) evaluation. The number of channels in the membrane patch was assumed to be the maximum number of open channel current levels observed throughout the current record. In experimental conditions with Po > 0.1, only current records with longer than 10 s of InsP3R channel activities were used for determination of Po, so there is little uncertainty in the number of channels in the current traces used. In experimental conditions with Po < 0.1, only current records exhibiting one open channel current level with InsP3R channel activities lasting longer than 30 s were used, to ensure that they were truly single-channel records (Mak et al., 2001a
). The Po data shown for each set of experimental conditions are the means of results from at least four separate patch-clamp experiments performed under the same conditions. Error bars indicate the SEM.
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RESULTS |
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The q.m. CaM, which has all EF hands mutated and therefore is Ca2+ insensitive, was overexpressed in Xenopus oocytes by cytoplasmic microinjection of cRNA. Western analysis (n = 5) indicated that the exogenous q.m. CaM was expressed to a level that was at least an order of magnitude higher than the endogenous wild-type CaM (Xia et al., 1998; Fig. 2). Patch-clamp experiments using nuclei isolated by protocol Nd (Fig. 1) from q.m. CaM-expressing oocytes revealed that InsP3R channel gating was still inhibited by high [Ca2+]i: InsP3R channel activities were detected in 11 out of 11 patches with pipette solutions containing [Ca2+]i = 2.1 µM (Fig. 3 C), but no channel activity was detected in any of 9 patches with pipette solutions containing 290 µM [Ca2+]i (Fig. 3 D). These results therefore also did not support the hypothesis that Ca2+ inhibition of InsP3R channel gating is mediated by CaM.
The lack of effect of overexpression of the q.m. CaM on Ca2+ inhibition of gating may suggest that endogenous CaM is not normally associated with the InsP3R. We examined the biochemical association between the InsP3R and CaM by coimmunoprecipitation. Using lysates prepared from cRNA-injected oocytes overexpressing either w.t. or q.m. CaM (Fig. 2), immunoprecipitation of the endogenous type 1 InsP3R with a specific antibody did not coimmunoprecipitate either w.t. or q.m. CaM (n = 4; unpublished data). In the converse experiments, immunoprecipitation of CaM with an antibody that binds to both w.t. and q.m. forms did not coimmunoprecipitate the InsP3R (n = 4; unpublished data). These results therefore do not provide evidence of an association between CaM and the InsP3R.
In summary, our single-channel patch clamp experiments revealed that neither high concentrations of a CaM antagonist, nor overexpression of a Ca2+-insensitive q.m. CaM had any effect on [Ca2+]i inhibition of InsP3R channel gating. In addition, coimmunoprecipitation failed to demonstrate an association between CaM and the InsP3R. Thus, our investigations did not provide any evidence supporting the hypothesis that high [Ca2+]i inhibition of InsP3R gating observed in in vitro patch clamp studies is mediated by CaM. These conclusions are therefore in agreement with those reached in some other studies (Zhang and Joseph, 2001; Nosyreva et al., 2002
).
Abrogation of Ca2+-dependent Inhibition of InsP3R Channel Gating
Our experimental results suggested that CaM is not involved in the inhibition of InsP3R channel gating by high [Ca2+]i. However, it remained possible that a different molecule may be involved, and that conditions could be identified which would strip such a putative effector from the InsP3R in the isolated nucleus, thereby rendering the InsP3R insensitive to Ca2+ inhibition. We reasoned that the putative effector, as a sensor of [Ca2+]i, might be dependent on normal [Ca2+]i for its association with the InsP3R. We therefore incubated the isolated nuclei in an ultra-low Ca2+ bath solution (ULCaS) before using them for nuclear patch clamp experiments to determine the Ca2+ dependence of the InsP3R gating.
In the first set of experiments, nuclei were isolated by protocol L (Fig. 1) into a bath of ULCaS ([Ca2+] < 5 nM). In the presence of 10 µM cytoplasmic (pipette) [InsP3] and [Ca2+]i < 20 µM, gating of the InsP3R exposed to the ULCaS was very similar to that of InsP3R in nuclei isolated directly into NCaS by protocol Nd (Fig. 4; Mak et al., 1998). In both cases, channel Po was low (<0.2) in [Ca2+]i < 150 nM, it increased dramatically to 0.8 as [Ca2+]i was increased from 150 nM to 1µM, and then Po remained at the maximum level of 0.8 when [Ca2+]i was further increased from 1 to 20 µM (Fig. 5). The InsP3R in nuclei isolated by protocol Nd were inhibited by [Ca2+]i > 20 µM (Mak et al., 1998
) but, remarkably, InsP3R in nuclei isolated into ULCaS by protocol L exhibited robust channel activities in [Ca2+]i as high as 1.5 mM (Fig. 4) with no decrease in channel Po (Fig. 5). Thus, a 20-min exposure to the ULCaS containing <5 nM Ca2+ caused the gating of InsP3R channel to be no longer inhibited by high [Ca2+]i. All of the InsP3R channel activities observed in the ultra-low [Ca2+] bath solution also terminated abruptly after
30 s, like those previously observed in the regular bath solution (Mak and Foskett, 1994
, 1997
).
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We previously demonstrated that the Ca2+ dependence of channel Po in nuclei isolated by protocol Nd into NCaS was well fitted by a biphasic Hill equation
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Nuclei isolated directly into a ULCaS bath by protocol Ld were used to determine the minimum duration of exposure to ULCaS bath required to relieve high [Ca2+]i inhibition of InsP3R gating. We found that channel activities could be detected with a pipette solution containing 10 µM InsP3 and 290 µM [Ca2+]i no earlier than 5 min after the nucleus was isolated into the ULCaS bath. Thus, the process involved in the relief of Ca2+ inhibition of InsP3R channel gating by exposure of the isolated nuclei to ULCaS is a slow one, requiring a few minutes.
To determine if normal cytoplasmic [Ca2+] (50 nM) is low enough to cause the relief of high [Ca2+]i inhibition of InsP3R gating, we isolated oocyte nuclei directly in PCaS bath (protocol Pd, Fig. 1). In a series of experiments performed in areas of the nuclear membrane identified with very high Pd, using pipette solutions with 10 µM InsP3 and 0.5 mM ATP, containing alternately 630 nM or 221 µM [Ca2+]i, InsP3R channels were observed in seven out of seven patches with 630 nM [Ca2+]i, but no InsP3R channel activity was observed in any of 11 patches with 221 µM [Ca2+]i, even when the nucleus was exposed to the PCaS bath for over 160 min. Thus, the normal resting [Ca2+] of the cytoplasm (
50 nM) is not sufficiently low to induce the relief of Ca2+ inhibition observed in the ultra-low Ca2+ condition.
InsP3 Dependence of the InsP3R in ULCaS Bath
Our previous studies (Mak et al., 1998, 2001a
) revealed that InsP3 activates gating by relieving the Ca2+ inhibition of the channel. InsP3 increases Kinh, the inhibitory half-maximal [Ca2+]i, with no effect on the values of the channel Ca2+ activation parameters (Kact, Hact) or Pmax in Eq. 1. It seemed likely that this mode of InsP3 activation cannot operate if the channel is not inhibited by high [Ca2+]i as observed after the channel had been exposed to the ULCaS bath for a few minutes. We therefore examined whether InsP3 was still required to gate the InsP3R under conditions that abrogated Ca2+ inhibition of the channel.
A series of experiments was performed using nuclei isolated by protocol L into ULCaS bath, patching in regions of the nuclei identified to exhibit high Pd, with pipette solutions alternately containing either 10 µM InsP3 and [Ca2+]i = 755 nM, or no InsP3 and [Ca2+]i between 60 nM and 290 µM. Again, the former solution was used to ascertain the presence of functional InsP3R channels in the regions of the isolated nuclei selected for our experiments for the entire duration of the series. InsP3R channel activities were observed in 27 out of 30 membrane patches in the presence of InsP3, but no channel activity was detected in any of the 10 patches without InsP3 (Fig. 6 A). Therefore, even though the InsP3R was no longer inhibited by high [Ca2+]i when the nucleus was isolated into ULCaS, InsP3 was nonetheless still necessary for channel gating.
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Reversibility of the Regulation by Bath [Ca2+] of Ca2+ Inhibition of the InsP3R Channel
It is possible, as we stated before, that the inhibition of InsP3R gating by high [Ca2+]i is mediated by some molecule that is tightly bound to the InsP3R in the NCaS bath, and that dissociates from the channel in the presence of extremely low [Ca2+] in the ULCaS bath. Dissociation of this putative effector from the InsP3R channel can then render the channel insensitive to inhibition by high [Ca2+]i. Accordingly, after dissociation, the putative effector molecule could possibly diffuse away into the essentially infinitely large volume of the bath. If this model is correct, the loss of Ca2+ inhibition should be irreversible. To explore the reversibility of the loss of Ca2+ inhibition, we performed patch-clamp experiments on nuclei isolated from the same batch of oocytes using different isolation/incubation protocols. As described above, Ca2+ inhibition was abrogated when the nuclei were isolated into ULCaS bath by protocol L (Fig. 7 A). However, when the nuclei were returned to the NCaS bath for 20 min before patch clamping (protocol LN, Fig. 1), no InsP3R channel activities were detected at [Ca2+]i = 290 µM (Fig. 7 B) in any of the 11 patches obtained, even though channel gating was observed in 4 out of 5 patches using pipette solutions with [Ca2+]i = 5.5 µM (Fig. 7 C). Thus, despite prior exposure to ULCaS, normal Ca2+ inhibition of InsP3R channel gating was restored when the nuclei were transferred back into NCaS. This restoration of normal Ca2+ inhibition was in turn reversible. Reexposure of the nuclei to ULCaS (protocol LNL, Fig. 1) again eliminated normal Ca2+ inhibition of gating (Fig. 7 D). The InsP3R channels in nuclei isolated by protocol LNL exhibited the same Po (Fig. 5, filled square) as those in nuclei isolated into ULCaS by protocol L without ever being exposed to NCaS (Fig. 5, open circles). These experiments indicated, first, that abolition of Ca2+ inhibition of channel gating by exposure of nuclei to ultra-low bath [Ca2+] was fully reversible, and second, that it was affected only by the [Ca2+] of the bathing solution in which the patch-clamp experiments were performed, independent of the history of bath [Ca2+] to which the nuclei were previously exposed. These results suggest either that the sensitivity of Ca2+ inhibition of the InsP3R to the bath [Ca2+] is an intrinsic property of the InsP3R channel, or that it is mediated by some molecule that remains in a stable complex with the channel throughout the multiple transfers of the nucleus into various baths containing different [Ca2+].
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DISCUSSION |
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What then could be the mechanism of Ca2+ inhibition? The simplest hypothesis is that the Ca2+ binding sites responsible for Ca2+ inhibition of channel gating are contained within the structure of the InsP3R protein itself. Many regions of the protein have been shown to bind Ca2+ in in vitro studies (Sienaert et al., 1996, 1997
). One or more of these or as yet unidentified sites may play a role, although there are no data available that address this issue. Alternately, another molecule could perhaps be involved. The InsP3R interacts with other proteins (Patel et al., 1999
; Yang et al., 2002
). Of interest, a calmodulin-like protein, CaBP1, interacts with high affinity with the ligand-binding region of the channel (Yang et al., 2002
). Whereas it is highly unlikely that CaBP1 and its isoforms mediate Ca2+ inhibition, since they are likely neurally restricted and have been shown to stimulate channel gating (Yang et al., 2002
), the identification of noncalmodulin Ca2+-binding protein interactions with the receptor lends credence to the notion that a Ca2+-binding protein could possibly be involved in mediating Ca2+ responses of the channel. Because Ca2+ inhibition of channel activity has been observed in a number of distinct experimental systems from different species, such a putative effector would need to be ubiquitously expressed and tightly associated with the channel.
A Novel Regulation of [Ca2+]i Inhibition of InsP3R-1 Channel Gating
Our investigations have revealed a novel CaM-independent regulation of the InsP3R-1 channel: abrogation of high [Ca2+]i inhibition of InsP3R-1 channel gating by exposure of the channel to ultra-low bath [Ca2+] (<5 nM). The physical location of the low [Ca2+]bath-sensing mechanism on the InsP3R protein is unknown. The abrogation could possibly be caused by low [Ca2+] in the perinuclear space between the inner and outer nuclear envelope, to which the lumenal side of the InsP3R-1 channel is exposed. In this case, exposure to the ultra-low bath [Ca2+] causes the lumenal [Ca2+] to fall to low levels due to uncompensated Ca2+ leak; and the [Ca2+] sensing mechanism responsible for switching high [Ca2+]i inhibition of InsP3R-1 channel on and off is located on the lumenal side of the InsP3R channel. The existence of a Ca2+-binding site on the lumenal side of the InsP3R-1 channel has been reported (Sienaert et al., 1996). Our previous studies indicated that the ionic composition of the solution in the perinuclear space of the isolated oocyte nucleus is likely to be similar to that of the bath solution (Mak and Foskett, 1997
). The long lag time (
300 s) between the isolation of the nucleus into the ultra-low [Ca2+] bath solution and the earliest detection of InsP3R channel activities that could not be inhibited by high [Ca2+]i may reflect the time required for the solution in the perinuclear space to become fully equilibrated with the bath solution, or the time taken for Ca2+ bound to the lumenal Ca2+-binding sites of the InsP3R channel to dissociate from the sites after the drop in lumenal [Ca2+], or a combination of the two.
Alternately, the [Ca2+]-sensing mechanism could possibly be located on the cytoplasmic side of the channel. In this case, the long lag time (300 s) between exposure of the channel to ultra-low [Ca2+] and the abrogation of high [Ca2+]i inhibition would imply that dissociation of Ca2+ from the sensing mechanism is slow (rate
0.003 s-1). Although such a sensing mechanism would be exposed to high [Ca2+] in the pipette solution as soon as the giga-ohm seal was formed, InsP3R channel activities were nevertheless observed for typically >10 s when the channel was exposed to [Ca2+]i
290 µM before the activities abruptly terminated (Mak and Foskett, 1997
; Boehning et al., 2001
). Thus, binding of Ca2+ to the sensing mechanism to restore normal high [Ca2+]i inhibition must also be a very slow process (rate <0.1 s-1). If the [Ca2+] sensing mechanism is in equilibrium with the cytoplasmic solution, the forward rate constant (kf) for Ca2+ dissociation from the [Ca2+]-sensing mechanism is
0.003 s-1 and the reverse rate constant (kr) is such that 0.1 s-1
kr x 290 µM. If the [Ca2+]-sensing mechanism is a simple Ca2+ binding site, then the equilibrium constant K (kf/kr) for Ca2+ dissociation from the site should then be
10 µM. However, abrogation of channel inhibition was not observed in our normal bath solutions that contain 300500 nM Ca2+ (Mak et al., 1998
), or in our physiological Ca2+ bath solution containing 50 nM free Ca2+. It could only be observed when bath [Ca2+] was reduced to very low levels. Thus, if the [Ca2+]bath-sensing mechanism is located on the cytoplasmic side of the channel, it is likely to be a set of cooperative Ca2+-binding sites. Further studies are necessary to distinguish whether cytoplasmic or lumenal [Ca2+] is being sensed in the disruption of high [Ca2+]i inhibition of the InsP3R, and to determine the molecular mechanisms involved in that process.
Mechanism of Regulation of High [Ca2+]i Inhibition of InsP3R-1 Channel Gating by Exposure to Low [Ca2+] Bath
A novel allosteric model, developed in the accompanying manuscript, can account for the effect of ultra-low [Ca2+] bath exposure on the abrogation of high [Ca2+]i inhibition as well as the effect of InsP3 to modulate maximum channel Po, rather than Kinh, under these conditions. In brief, this model accounts for our results by postulating the existence of two functional inhibitory Ca2+ binding sites associated with each monomer of the tetrameric channel. One site is only inhibitory when the channel is not liganded with InsP3, because InsP3 binding relieves the Ca2+ inhibition imposed by this site. In contrast, the properties of the other inhibitory site are not affected by InsP3 binding. In normal physiological [Ca2+]i conditions, Ca2+ binding to this InsP3-insensitive site provides the observed high [Ca2+]i inhibition (Kinh 50 µM) of the fully InsP3-liganded channel. The ability of this InsP3-insensitive site to be inhibitory is reversibly lost after exposure of the channel for >5 min to an ultra-low bath [Ca2+] (<5 nM). Thus, the observed abrogation of high [Ca2+]i inhibition of channel activity in saturating [InsP3] can be accounted for by the fact that there is no longer any functional inhibitory Ca2+-binding site. On the other hand, in the absence of InsP3, the InsP3-sensitive Ca2+ inhibition site is functional and keeps the channel closed. Thus, the channel still requires InsP3 to gate open even when the InsP3-insensitive site has been disrupted by exposure to ultra-low bath [Ca2+]. A detailed description of this model, which can account for these and many other features of ligand regulation of the channel observed in nuclear patch clamp experiments, is developed in the accompanying manuscript (Mak et al., 2003
, in this issue).
Are Different Sensitivities to Inhibition by High [Ca2+]i a Fundamental Distinguishing Feature among the InsP3R Isoforms?
The three isoforms of InsP3R have complicated patterns of expression in various tissues with complex regulation by various mechanisms (Taylor et al., 1999). Because the permeation and conductance properties of the InsP3R isoforms are very similar (Mak et al., 2000
; Ramos-Franco et al., 2000
), differences among the isoforms in localization and channel gating and its regulation are likely to be reasons for the existence of InsP3R diversity. A review of published single-channel studies of various InsP3R isoforms suggests that different sensitivities to inhibition by high [Ca2+]i may be one distinguishing functional feature among the various InsP3R isoforms. Nevertheless, it is not clear whether such differences are intrinsic to the channels, or whether they are perhaps artificially generated by the different experimental protocols used for studying InsP3R channel activity.
In the presence of 1 µM InsP3, native and recombinant InsP3R-1 channels (including various splice variants) reconstituted into lipid bilayers exhibited similar strong inhibition by [Ca2+]i with half-maximal inhibitory [Ca2+]i of 0.12 µM (Bezprozvanny et al., 1991
; Ramos-Franco et al., 1998a
,b
; Tu et al., 2002
), whereas native Xenopus and recombinant rat InsP3R-1 channels studied in their native membrane environment using nuclear patch clamp techniques exhibited inhibition by high [Ca2+]i, but with a significantly higher half-maximal inhibitory [Ca2+]i of
50 µM (Mak et al., 1998
; Boehning et al., 2001
). When reconstituted into planar bilayers, Ca2+ inhibition of InsP3R-1 could be alleviated by very high [InsP3] (180 µM) (Kaftan et al., 1997
; Moraru et al., 1999
), whereas Ca2+ inhibition of InsP3R-1 studied in the native membrane environment was not further affected by [InsP3] once the channel was saturated with [InsP3] >100 nM (Mak et al., 1998
).
InsP3R-2 channels reconstituted in lipid bilayers exhibited variable but low sensitivity to inhibition by high [Ca2+]i, with a half-maximal [Ca2+]i of 400 µM for recombinant InsP3R-2 channels (Ramos-Franco et al., 2000
) and >1 mM for native channels (Ramos-Franco et al., 1998b
, 2000
) in 1 µM InsP3.
Native type 3 InsP3R channels reconstituted into lipid bilayers exhibited no detectable inhibition by high [Ca2+]i and its Po remained at its maximum value (0.05) in [Ca2+]i between 1 and 100 µM in the presence of 2 µM InsP3 (Hagar et al., 1998
). In marked contrast, recombinant r-InsP3R-3 in the nuclear membrane of oocytes is inhibited by high [Ca2+]i in an InsP3-dependent manner very similar to that for X-InsP3R-1 under identical experimental conditions (Mak et al., 2001a
).
How can we account for such divergent results? Our studies here demonstrate that Ca2+ inhibition of the X-InsP3R-1 channel in its native membrane environment can be completely, specifically and reversibly abrogated under certain experimental conditions (after exposure to a nominally Ca2+-free bath). Associated with this effect, the InsP3 dependence of the channel Po was also changednormally, InsP3 affects the apparent affinity of the inhibitory Ca2+-binding sites of the channel (Mak et al., 1998), whereas after ULCaS bath exposure, InsP3 affects the maximum Po observed (Fig. 6 C). Of note, this InsP3 dependence of maximum Po is very similar to the observed effect of InsP3 on the Po of InsP3R-2 channels reconstituted into lipid bilayers (Ramos-Franco et al., 1998b
). These observations raise the intriguing possibility that the observed differences in the sensitivities to Ca2+ inhibition of various InsP3R isoforms may be a consequence of the different environment and/or isolation conditions to which the channels were exposed, rather than the result of differences in fundamental intrinsic characteristics of the individual isoforms. For example, the InsP3R-1 and InsP3R-3 channel isoforms exhibited very similar inhibition by high [Ca2+]i when they are studied in a native ER membrane environment (Mak et al., 1998
, 2001a
), but they behaved differently in reconstitution systems. We suggest that it is worth considering the possibility that procedures employed in the isolation and reconstitution and recording of the InsP3R-3 used in (Hagar et al., 1998
) disrupted the normal high [Ca2+]i inhibition of the InsP3R-3, causing the observed lack of Ca2+ inhibition, in very much the same way that exposure to a ULCaS bath abrogated the high [Ca2+]i inhibition of InsP3R-1 observed in this study. Whereas the procedures used in the isolation and reconstitution and recording of InsP3R-1 by themselves did not eliminate high [Ca2+]i inhibition of the channel (Bezprozvanny et al., 1991
; Ramos-Franco et al., 1998a
,b
; Tu et al., 2002
), they may account for the ability, observed only in the reconstituted systems, of extremely high [InsP3] to abrogate high [Ca2+]i inhibition (Kaftan et al., 1997
; Moraru et al., 1999
). By the same token, it is possible that the very low sensitivity to high [Ca2+]i inhibition of the InsP3R-2 channel isoform reconstituted in lipid bilayers (Ramos-Franco et al., 1998b
, 2000
) was induced by the isolation and reconstitution and recording protocols. Obviously, these issues will need to be resolved in future studies, for example, of the Ca2+ responses of type 2 InsP3R channels in the native ER membrane environment, under the same experimental conditions as those used for the types 1 and 3 InsP3R isoforms; and of the sensitivities of Ca2+ inhibition of the other InsP3R isoforms to exposure to ultra-low bath [Ca2+]. Nevertheless, our identification in this study of conditions that can radically alter the [Ca2+]i inhibition properties of the channel suggests that careful consideration of the isolation protocols and other conditions to which InsP3R channels are exposed before they are examined will be warranted in future studies.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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Olaf S. Andersen served as editor.
Submitted: 27 January 2003
Accepted: 2 September 2003
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REFERENCES |
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