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CELLULAR |
Departments of
1 Physiology and
2 Cell and Developmental Biology, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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(Received 13 March 2006;
accepted after revision 24 April 2006;
first published online 27 April 2006)
Corresponding author J. K. Foskett: Department of Physiology, B39 Anatomy-Chemistry Building, 414 Guardian Drive, University of Pennsylvania, Philadelphia, PA 19104-6085, USA. Email: foskett{at}mail.med.upenn.edu
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A fundamental aspect of InsP3R-mediated intracellular signalling is the phenomenon of ‘quantal release’, defined here as the ability of cells to have graded release of Ca2+ from intracellular stores in response to incremental levels of extracellular agonist or cytoplasmic InsP3 concentrations ([InsP3]) (Muallem et al. 1989; Meyer & Stryer, 1990) (reviewed in Bootman, 1994; Missiaen et al. 1994; Parys et al. 1996; Taylor, 1998). Ca2+ release in response to InsP3 has a transient fast phase, whose rate is proportional to [InsP3], followed by a much slower one even in constant [InsP3]. Consequently, sustained exposure to submaximal levels of agonists, even over extensive periods, only mobilizes a fraction of total releasable Ca2+ in a cell. Quantal Ca2+ release has been observed in many cell types under a variety of experimental conditions, including cells with the plasma membrane permeabilized, or with ATP depleted, or with [Ca2+]i kept constant, and in response to poorly metabolizable InsP3 analogues (Bootman, 1994; Missiaen et al. 1994; Parys et al. 1996; Taylor, 1998). Quantal Ca2+ release is surprising because it might have been expected that all InsP3R channels should become activated in response to any agonist concentration, releasing all of the InsP3-sensitive Ca2+ stores, albeit at different rates depending on the agonist concentration. Despite considerable investigation, there is no consensus regarding the mechanisms that either tune the initial rate of release of Ca2+ to [InsP3], or subsequently significantly slow or terminate release. Several mechanisms involving either InsP3R or Ca2+ store heterogeneity have been invoked to account for graded transient Ca2+ release, including the presence of discrete Ca2+ stores with different densities of InsP3R or sensitivities to [InsP3], or the presence of heterogeneous InsP3R channels in a continuous store (different channel isoforms with alternatively spliced variants and variable post-translational modifications have been proposed) with different proposed mechanisms of release termination, including regulation of InsP3R activity by ER luminal [Ca2+] or by desensitization or inactivation processes (Bootman, 1994; Missiaen et al. 1994; Parys et al. 1996; Taylor, 1998). Nevertheless, the mechanisms involved in graded transient Ca2+ release have remained controversial because experimental evidence has been obtained that has disputed every proposed scheme (Bootman, 1994; Missiaen et al. 1994; Parys et al. 1996; Taylor, 1998).
High-resolution electrophysiological and imaging studies have provided more recent insights into the mechanisms of graded release. First, optical imaging of [Ca2+] indicator dyes has revealed the presence of highly localized Ca2+ release events whose numbers and frequency are graded with stimulus intensity (Parker & Ivorra, 1990b; Lechleiter et al. 1991; Bootman & Berridge, 1996; Horne & Meyer, 1997; Sun et al. 1998; Thomas et al. 1998). Second, recordings of single InsP3R channels have suggested that feedback inhibition of channel gating by high [Ca2+]i, tuned by [InsP3], can grade channel activity with [InsP3] (Mak et al. 1998, 2001). Together, these observations provide a mechanism to grade Ca2+ release that is based on ligand-dependent activity of independent release sites. Thus, in the presence of low [InsP3] during weak agonist stimulation, sporadic random openings of InsP3R channels (Mak et al. 1998, 2001, 2003a) generate small localized [Ca2+]i elevations (termed ‘blips’) (Parker & Yao, 1996; Bootman et al. 1997). As [InsP3] increases, sensitivity of the InsP3R to Ca2+ inhibition is reduced (Mak et al. 1998, 2001), enabling Ca2+-induced Ca2+ release (CICR) (Iino, 1990) to coordinate openings of neighbouring InsP3Rs in a local cluster (Yao et al. 1995; Horne & Meyer, 1997; Mak & Foskett, 1997; Thomas et al. 1998) to generate larger Ca2+ ‘puffs’ (Parker & Yao, 1995; Yao et al. 1995; Sun et al. 1998). Puffs remain localized when the level of released Ca2+ is insufficient to allow Ca2+ diffusing from puffs to activate InsP3Rs beyond the local cluster (Parker & Yao, 1996), whereas even greater Ca2+ release at higher [InsP3] can enable Ca2+ to diffuse far enough to activate channels in other clusters. This progressive recruitment of release sites by even higher [InsP3] may then account for transitions from local to global Ca2+ signals (Bootman & Berridge, 1995; Berridge, 1997). Although this model can account for graded Ca2+ release, it is not known if InsP3-tuning of high [Ca2+] inhibition is the primary mechanism used, or whether additional mechanisms also contribute to InsP3R channel recruitment. Furthermore, this model does not account for observed apparent heterogeneity of InsP3 sensitivity among release sites throughout different regions of the cytoplasm (Bootman & Berridge, 1996; Parker et al. 1996; Thorn et al. 1996), nor does it provide insights into the mechanisms that underlie time-dependent termination of Ca2+ release.
Here, we demonstrate that mechanisms intrinsic to the single InsP3R channel itself can account for quantal Ca2+ release. Patch-clamp electrophysiology of nuclei isolated from insect Sf9 cells yielded a consistent and high probability of detecting apparently homogenous endogenous InsP3R channels, which has enabled two novel observations. First, by accurate determinations of channel activity durations, we have established InsP3-induced InsP3R inactivation as an inevitable process that terminates single channel activity, with the rate of inactivation regulated by [InsP3] and [Ca2+]i. We established this ligand-dependent process as true inactivation by demonstrating its reversibility. Second, we have been able to accurately quantify the mean number of activated channels in a typical membrane patch under precisely controlled ligand conditions. Whereas it was anticipated that all channels in a homogeneous population would always become activated, even in suboptimal ligand conditions, albeit to lower levels of activity, we found instead that the number of channels activated is a graded function of both [Ca2+]i and [InsP3]. A qualitative model can account for graded channel recruitment by suggesting that apparent heterogeneous ligand sensitivities can be generated in a homogeneous population of InsP3R channels. This model also accounts for the ligand concentration dependence of the rate of fateful InsP3-induced channel inactivation. Thus, our results suggest that quantal Ca2+ release can be generated by mechanisms that are intrinsic to the InsP3R Ca2+-release channel itself.
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Methods |
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300 nM) in an experimental chamber on the stage of an inverted microscope. Isolated nuclei are 10 µm in diameter, and were distinguished from intact cells based on their unique morphology (Fig. 1B). Fresh homogenizations were performed every 2 h (Mak et al. 2005). The standard pipette solution contained (mM): 140 KCl, 0.5 Na2ATP, 10 Hepes, pH 7.3, and various [Ca2+] and [InsP3], as indicated. All solutions were carefully buffered to desired free [Ca2+] (Mak et al. 1998), confirmed by fluorometry.
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Particular consideration was given to the accurate determination of the number of active channels in nuclear membrane patches (NA) from the experimental current records. For simplicity, we consider a membrane patch containing n active, identical and independent channels exhibiting Markovian stochastic kinetics with open probability Po and a single open channel kinetic state with mean open duration of to. With these assumptions, events when all n channels open simultaneously follow Markovian kinetics with a probability (Po)n and mean duration of to/n. If T is the minimum duration of an open event that is detectable after filtering in the experimental system, then average interval 
n between two successive detectable events when all n channels were open is 
. Therefore, the number of active channels in a membrane patch (N) can be assumed, with high level of confidence (P < 0.01), to be the maximum number of open channel current levels observed if the channel record lasted longer than 5(N+1). In our patch-clamp set-up, T was empirically determined to be 0.2 ms using test pulses of variable duration (Mak et al. 2001). The to value of the Sf9 InsP3R channels is 30 ms over the range of experimental conditions used. In experiments performed using conditions that generated the lowest channel Po (0.09 at [InsP3]
= 33 nM and [Ca2+]i
= 7.5 µM), at most one active channel was involved, and the current records analysed all lasted longer than 5(2). In experiments using conditions that produced higher channel Po, multi-channel current records were more common, but the duration of each current record analysed exceeded 5(N+1), where N is the number of maximum open channel current levels detected in that record. Thus, the uncertainty in the determination of the number of active channels in a membrane patch was insignificant in the studies described here.
We observe that gating activities of all InsP3R channels inevitably terminate, i.e. InsP3R channel activities observed in nuclear patch-clamp experiments have finite durations. To quantify the activity duration for an InsP3R channel in an experiment, we assume that all channels observed in one experiment inactivated identically, independently, following simple Markovian kinetics with a single time constant (Ta). Then, for an experiment with N active channels and channel activity lasting TN from the beginning of the experiment to the last channel closing observed in the channel current record, 
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Besides the durations of observation of channel activities, another factor affecting the accurate determination of the number of active channels in nuclear membrane patches is the finite time ts between the initial exposure of the InsP3R channels to the ligand conditions in the pipette solution as the tip of the patch-clamp pipette made contact with the outer nuclear envelope, and the quality of seal between the isolated membrane patch and the patch pipette tip becoming good enough (seal resistance >200 M
) for single-channel current recording. Because of the inactivation of the InsP3R channels in every experiment, a fraction of all InsP3R channels stimulated by ligand conditions in the patch pipette were not observed because they underwent inactivation during ts. For isolated Sf9 nuclei, mean ts was 6.0 s (standard deviation 2.8 s). With the same assumptions used in Ta evaluation, the actual number of InsP3R channels activated in an experiment NA was estimated as Nobs exp(ts/Ta), where Nobs is the number of InsP3R channels observed in the current record. For most ligand conditions ts
<<
Ta so that the values of NA are not significantly different from those of Nobs, except in 10 µM InsP3 and 89 µM
Ca2+i, when Ta is so short (9.1 s) that NA cannot be confidently estimated from Nobs.
All data points shown in all graphs are means of at least four experiments performed at the same [InsP3] and [Ca2+]i. Error bars indicate S.E.M. The S.E.M. for NA under one set of ligand conditions was calculated by taking into account the effects of Nobs, Ta and ts.
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Results |
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Single InsP3R channels have been recorded in native ER membranes by patch-clamping nuclei isolated from Xenopus oocytes (Mak & Foskett, 1997) and COS-7 cells (Boehning et al. 2001a). This technique is rationalized by the continuity of the ER with the outer membrane of the nuclear envelope and the role of the nuclear envelope as a Ca2+ store similar to ER. Shortcomings of the previously used Xenopus and COS-7 cell systems include lack of consistency in detecting active InsP3R channels and relatively rapid (t
<30 s) apparent channel inactivation following InsP3-induced activation (Mak & Foskett, 1997; Mak et al. 2000; Boehning et al. 2001a). To discover a better system for studying single InsP3R channels, we examined insect Sf9 cells. Gigaohm seals were readily achieved (>80% success rate) on carefully selected isolated nuclei (Fig. 1B). With 10 µM InsP3 and 1 µM Ca2+ in the pipette solution, conditions that maximize activity of rat and Xenopus InsP3R channels in nuclear membrane patches (Mak et al. 1998, 2001; Boehning et al. 2001a), Sf9 InsP3R channels were consistently detected in 60–80% of patches obtained from nuclei isolated from different batches of cells. Channel activity was evident upon gigaseal formation, but the number of active channels decreased during continuous recording (Fig. 1A) due to abrupt activity termination. The channels had linear current–voltage relationship with slope conductance of 477 ± 3 pS (n
= 10) (Fig. 1C), larger than the 360 pS of vertebrate channels (Mak & Foskett, 1998). Channels varied somewhat in their conductance, which sometimes fluctuated during a continuous recording (Mak & Foskett, 1998) (S.D. 12 pS), with occasional openings to subconductance levels that accounted for <2% of all open durations (Fig. 1A). The permeability sequence, determined from reversal potential measurements of currents in asymmetrical solutions, PCa:PMg:PK:PCl was 10:6.8:1:0.22. No channels were observed in the absence of InsP3 (0/13 patches) or presence of 10 µM InsP3 and the competitive inhibitor heparin (100 µg ml–1) (0/14 patches), whereas with 10 µM InsP3 they were detected in 5/6 patches obtained from the same batch of cells. Thus, the channels observed were confirmed to be endogenous Sf9 InsP3R channels.
Distribution of InsP3R channels on Sf9 nuclei
A significant proportion (41/53) of active patches in optimal conditions (10 µM InsP3, 1 µM Ca2+) contained multiple InsP3R channels, as identified by two or more equally spaced current levels, each corresponding to the predominant 480 pS state (Fig. 1A). Comparing the number of channels observed in 75 patches with the theoretical Poisson distribution revealed a significant disparity between the two (Fig. 1D), suggesting that the channels are not evenly distributed in the outer nuclear membrane. Thus, Sf9-InsP3R channels may be organized in clusters, a conclusion similar to that reached in an analogous analysis of Xenopus oocyte InsP3R in outer nuclear membrane (Mak & Foskett, 1997) and in Ca2+-imaging studies of several cell types (Yao et al. 1995; Horne & Meyer, 1997; Thomas et al. 1998; Bootman et al. 2001).
[Ca2+] and [InsP3] dependencies of Sf9-InsP3R channel gating kinetics
InsP3R channel gating is under complex allosteric regulation by both InsP3 and [Ca2+]i. Activity of Sf9-InsP3R is biphasically regulated by [Ca2+]i with maximum channel Po exhibited over a broad range of [Ca2+]i in the presence of saturating [InsP3] (Fig. 2A–C). The [Ca2+]i dependence of Po in the presence of 10 µM InsP3 can be fitted with a biphasic Hill equation (Fig. 2B) (Mak et al. 1998):
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| (1) |
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In all ligand conditions, channel activity invariably terminated in the continuous presence of InsP3 and absence of local change in [Ca2+]i (Figs 1 and 3A). In optimal ligand conditions, the mean channel activity duration (Ta) was 120 s. Similar apparent inactivation has been observed for nuclear membrane-patched InsP3R in Xenopus oocytes (Mak & Foskett, 1994, 1997) and COS-7 cells (Boehning et al. 2001a) but with significantly shorter Ta. In 10 µM InsP3, Ta of the Sf9-InsP3R was reduced in [Ca2+]i >1 µM, with reduction by over 10-fold at 89 µM Ca2+ (P < 0.05) (Fig. 3A). In subsaturating (33 nM) InsP3, Ta began to decrease in [Ca2+]i
300 nM, substantially lower than that observed in saturating InsP3 (P < 0.05) (Fig. 3A). The [InsP3] and [Ca2+]i dependencies of Ta suggest that the observed inevitable termination of channel activity represents the InsP3R channels entering a true inactivated state instead of an experimental artefact.
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The conductance, activation and inactivation gating properties and their regulation by InsP3 and [Ca2+]i, and spatial distribution of the observed Sf9 InsP3R channels are all highly reminiscent of other InsP3R channels observed in nuclear patch clamp studies. In those previous studies, the probability of detecting an active InsP3R channel in the patched membrane (Pd) was generally low, i.e. most patches contained no active channels; and there was significant variability in Pd (Mak & Foskett, 1997; Mak et al. 2000; and unpublished observations in COS-7 cells). In contrast, active Sf9-InsP3R channels were detected in optimal ligand conditions in up to 80% of patches obtained from nuclei from different batches of cells. This consistency allowed us to examine the possible ligand dependence of Pd (Fig. 3B). In 10 µM InsP3 in low [Ca2+]i (50 nM), Pd was 0.47. That is, nearly half of all patches contained at least one active InsP3R channel. Pd increased to 0.81 when [Ca2+]i was raised to 500 nM. Between 0.5 and 7.5 µM, Pd remained high (0.6–0.8). When [Ca2+]i was increased beyond 10 µM, Pd gradually decreased. Thus, Pd varied as a biphasic function of [Ca2+]i. With 33 nM InsP3, Pd increased from 0.5 at [Ca2+]i
= 100 nM to 0.67 at [Ca2+]i
= 500 nM, and then decreased to 0.21 at [Ca2+]i
= 10 µM (Fig. 3B). Pd was sensitive to [InsP3] as well as [Ca2+]i, with decreasing [InsP3] within the subsaturating range enhancing high [Ca2+]i reduction of Pd, reminiscent of the effect of suboptimal [InsP3] on high [Ca2+]i inhibition of channel gating (Fig. 2B).
To calculate Pd, a membrane patch containing multiple channels was given the same weight as one containing a single channel. However, it was noted that patches obtained under optimal ligand conditions usually contained several active channels, whereas those obtained under suboptimal conditions often contained only one. The relatively long channel lifetimes allowed the number of channels to be determined accurately under most conditions (see Methods). To more accurately characterize the number of channels activated, we defined NA as the total number of channels observed in all patches divided by the total number of patches obtained under that set of experimental conditions. In [Ca2+]i = 50 nM, in the presence of 10 µM InsP3, NA was 1.3 ± 0.3. That is, on average each patch contained 1.3 active InsP3R channels. In higher [Ca2+]i, more channels were detected in each patch, with maximum NA of 3.0 ± 0.4 at [Ca2+]i = 500 nM (P < 0.01). In 1 µM < [Ca2+]i < 8 µM, NA ranged between 2.2 and 2.8 (Fig. 3C). Further increases in [Ca2+]i were associated with reduced NA (P < 0.01). Compared with NA observed in 10 µM InsP3, consistently lower NA was observed in 33 nM InsP3 over all [Ca2+]i (P < 0.01). In [Ca2+]i = 110 nM, NA was 0.7 ± 0.2. It increased to 1.6 ± 0.3 at [Ca2+]i = 500 nM. Higher [Ca2+]i then decreased NA (P < 0.01), such that at [Ca2+]i = 7.5 µM, only 0.26 ± 0.10 channels per patch were detected.
The observation that NA was a function of stimulus strength was unexpected since it was anticipated that the entire channel population in a membrane patch would always become activated, albeit to different levels of activity (Po) depending on the strength of ligand activation. These differences in NA cannot be explained by under-estimation of the number of channels when Po was low, since Sf9-InsP3R channels were observed long enough for NA to be accurately determined (see Methods). Furthermore, since the observation period following exposure of the membrane patch to ligands was many tens of seconds, the inability of suboptimal ligand concentrations to engage all channels cannot be accounted for by stochastic probabilities of ligand binding to the channels. These results indicate that suboptimal ligand concentrations are insufficient to activate all InsP3R channels in a membrane patch that can be activated by optimal ligand concentrations. Therefore, ligand activation of InsP3R is associated with recruitment of channels to an activated state, as well as with increasing the Po of those channels that are recruited.
The total ion flux (J) associated with activation of the entire population of InsP3R in the ER membrane is defined by:
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is the single-channel conductance, N is the number of activated channels, and Po is the average single-channel open probability. The patch-clamp experiments were able to measure all three parameters independently, enabling the effects of ligand stimulation on the magnitude of InsP3R-mediated Ca2+ release from the ER store to be estimated. With being ligand independent, NAPo is a measure of the ligand-induced InsP3R-mediated Ca2+ release. As both parameters were sensitive to ligand concentrations, NAPo is a more accurate assessment of the consequences of ligand activation of the channel than either parameter alone. In saturating [InsP3], the dependence of NAPo on [Ca2+]i was biphasic: NAPo increased by over 10-fold as [Ca2+]i was increased from 50 to 500 nM (Fig. 3D), and then gradually decreased as [Ca2+]i was further increased. NAPo was also strongly dependent on [InsP3]. With [InsP3] reduced to 33 nM, the dependence of NAPo on [Ca2+]i was again biphasic, with maximal NAPo observed in [Ca2+]i
0.5–1 µM, but peak NAPo was an order of magnitude lower than that observed in saturating [InsP3] (Fig. 3D). |
Discussion |
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Functional properties of the endogenous Sf9 InsP3R
We utilized a nuclear isolation protocol that avoids purification and reconstitution procedures, enabling the InsP3R channels to remain in their native membrane environment, preserving specific lipid interactions during our experiments. Previously, microsomes obtained from transduced Sf9 cells were used as a source of recombinant InsP3R in biochemical studies (Cardy et al. 1997) and lipid bilayer reconstitution studies (Tu et al. 2002, 2004; Srikanth et al. 2004). Whereas it was reported in those studies that endogenous Sf9 InsP3R were not detected biochemically or functionally, the InsP3 signalling system is present and functional in Sf9 cells (Ross et al. 1994; Knight et al. 2003; Knight & Grigliatti, 2004), and our studies here have revealed that the Sf9-InsP3R is functionally expressed at a high levels in the outer nuclear membrane of freshly isolated nuclei. The apparent discrepancy may reflect a substantially higher sensitivity of detection of InsP3R function in patch clamp electrophysiology, enrichment of InsP3R in the nuclear envelope, or use of antibodies in biochemical studies that only poorly recognized the Sf9 InsP3R.
Under our experimental conditions, with symmetrical K+ as the charge carrier (Mak et al. 1998), the Sf9-InsP3R channel exhibited a linear current–voltage relation, similar to that observed for nuclear-patched endogenous Xenopus InsP3R-1, rat cerebellar InsP3-R (Marchenko et al. 2005) and recombinant rat types 1 and 3 InsP3R (Mak & Foskett, 1998; Boehning et al. 2001a; Mak et al. 2001); however, the single-channel conductance of 480 pS of the Sf9 InsP3R channel is higher than the 360 pS conductance of the vertebrate channels. This may be due to the membrane environment of the Sf9 cell. Alternatively, it may be due to differences at the molecular level between the insect and mammalian InsP3R pore sequences. Although the amino acid sequence of the Sf9-InsP3R is not known, all known invertebrate InsP3R pore selectivity filter sequences (BLAST search: Drosophila, Caenorhabditis elegans, Panulirus, Anopheles, Asterina, Apis, Strongylocentrotus, Aplysia and Tetrahymena) contain a GGIGD motif, whereas the vertebrate sequence is GGVGD. By site-directed mutagenesis of rat InsP3R-1, it was previously demonstrated that this sequence is involved in ion conductance and selectivity (Boehning et al. 2001b). A mutant mammalian InsP3R channel with the pore Val replaced with Ile to resemble the invertebrate InsP3R, had a higher conductance (490 ± 13 pS) (Boehning et al. 2001b), close to that (480 pS) of the Sf9-InsP3R measured here. Thus, other invertebrate InsP3R channels may also have higher single-channel conductance than their mammalian counterparts. In contrast, the ion permeability selectivity of the Sf9 channel is very similar to that of its mammalian homologs, with the channel cation selective (PK:PCl
5) with 10-fold higher selectivity for Ca2+ than K+ (Mak & Foskett, 1994, 1998; Mak et al. 2000; Boehning et al. 2001a; Marchenko et al. 2005).
We examined the gating properties of the Sf9-InsP3R under a wide range of ligand concentrations using experimental conditions similar to those used to study other InsP3R channels in native ER membranes (Mak et al. 1998; Boehning et al. 2001a). The most fundamental conclusion is that the ligand regulation properties of the insect Sf9-InsP3R have remarkable similarities to those of the Xenopus type 1 and rat types 1 and 3 channels studied previously (Mak et al. 1998, 2001; Boehning et al. 2001a). Like the vertebrate channels, the Sf9-InsP3R has a high maximum Po of 0.7–0.8, and the Sf9 InsP3R channel Po has a biphasic dependence on [Ca2+]i, with the ranges of [Ca2+]i over which the channel was activated or inhibited similar to those of the vertebrate channels recorded under similar conditions of [InsP3] and [ATP]. Also like the vertebrate channels, the Sf9-InsP3R channel Po remained at high levels over a broad range of [Ca2+]i, with inhibition by high [Ca2+]i not pronounced until [Ca2+]i > 10 µM in the presence of saturating [InsP3]. However, inhibition of the Sf9-InsP3R channel by high [Ca2+]i is less cooperative than the vertebrate InsP3R isoforms (Mak et al. 2001). Consequently, Po is already <0.1 in the vertebrate channels at [Ca2+]i = 90 µM, whereas Sf9-InsP3R Po is still about 0.2 at [Ca2+]i = 90 µM (Fig. 2B).
The main effect of reducing [InsP3] to subsaturating levels (100 nM) on the Sf9-InsP3R channel activity was to increase the sensitivity of the channel Po to inhibition by high [Ca2+]i. In contrast, Ca2+ activation was not significantly affected by [InsP3]. This is highly reminiscent of InsP3 regulation of endogenous Xenopus InsP3R-1 (Mak et al. 1998) and recombinant rat InsP3R-3 (Mak et al. 2001). Nevertheless, the Sf9-InsP3R has a lower effective sensitivity to InsP3 and the relief of Ca2+ inhibition by InsP3 has less apparent cooperativity in Sf9-InsP3R compared with the vertebrate channels (Fig. 2B). Both Sf9-InsP3R and Xenopus InsP3R-1 have low channel activity in 10 nM InsP3, but whereas Xenopus InsP3R-1 is already fully activated by 100 nM InsP3, Sf9-InsP3R responds to InsP3 over a significantly broader range of concentrations: increasing [InsP3] from 100 nM to 1 µM gave rise to further relief of Ca2+ inhibition of Po in Sf9-InsP3R (Fig. 2B).
Finally, the kinetic basis for Sf9-InsP3R channel activation by InsP3 and Ca2+ involves primarily modulation of tc. The to value remained within a relatively narrow range, whereas tc varied over an order of magnitude over a range of [Ca2+]i from 50 nM to 90 µM. Thus, the [Ca2+]i dependence of channel tc is mostly responsible for the channel Po versus [Ca2+]i relation. This is similar to the effect of [Ca2+]i on the Xenopus InsP3R-1 and rat InsP3R-3 channels (Mak et al. 1998, 2001).
The broad similarities of ligand regulation among the Sf9-InsP3R channel and two types of vertebrate InsP3R channels suggest that common conserved mechanisms underlie ligand regulation of all InsP3Rs. A previous study of the Drosophila InsP3R also concluded that its functional properties were similar to those of mammalian InsP3Rs (Swatton et al. 2001). The [Ca2+]i and [InsP3] dependencies of the Sf9-InsP3R channel activity can be adequately described by an allosteric MWC-based model (Fig. 2C) that accounted for the ligand regulation of the Xenopus InsP3R-1 and rat InsP3R-3 channels observed in previous nuclear patch-clamp studies (Mak et al. 2003a). The remarkable functional similarity of Sf9-InsP3R with the endogenous Xenopus and recombinant rat InsP3Rs may be a consequence of the high sequence homology among different InsP3R isoforms from various species.
InsP3-induced InsP3R channel inactivation
In single-channel studies, gating activities of Sf9-InsP3R (the present study) and vertebrate InsP3R (Mak & Foskett, 1997; Mak et al. 2000; Boehning et al. 2001a) recorded in native ER membranes inevitably abruptly terminate after InsP3 activation. Whereas the previous vertebrate single-channel studies suggested that the abrupt termination was likely to be due to channel inactivation, it was impossible to rule out non-physiological artefacts associated with patching, for example collisions of the channels with the walls of the glass pipette. However, the present results, by demonstrating both InsP3 as well as Ca2+ dependencies of the channel activity duration, and the reversibility of the activity termination, clearly establish this process as true channel inactivation. Previously, the presence of InsP3-induced InsP3R inactivation has been controversial. Pre-exposure of permeabilized hepatocytes to InsP3 under conditions of constant [Ca2+]i was followed by a time-dependent reduction of InsP3R-mediated Ca2+ release (Hajnóczky & Thomas, 1994, 1997; Dufour et al. 1997; Marchant & Taylor, 1998) as well as transformation of the receptor from a low-affinity active state to a high-affinity, desensitized state (Coquil et al. 1996; Marchant & Taylor, 1998), consistent with InsP3-mediated InsP3R inactivation. Biphasic kinetics of Ca2+ release rates in intact cells have been observed in many studies (reviewed in Bootman, 1994; Missiaen et al. 1994; Parys et al. 1996; Taylor, 1998), but either inactivation has been ruled out as a mechanism for release termination (Taylor & Potter, 1990; Oldershaw et al. 1992; Hirose & Iino, 1994; Parys et al. 1995; Combettes et al. 1996; Beecroft & Taylor, 1997) or other types of mechanisms have been invoked (reviewed in Bootman, 1994; Missiaen et al. 1994; Parys et al. 1996; Taylor, 1998). In rapid perfusion protocols using isolated microsomes or permeabilized cells that attempted to maintain [InsP3] and [Ca2+]i constant, transient Ca2+ release kinetics indicated that channel inactivation or partial inactivation played a role in the decay of release rates (Champeil et al. 1989; Finch et al. 1991a; Combettes et al. 1994; Wilcox et al. 1996; Dufour et al. 1997; Marchant & Taylor, 1998; Adkins et al. 2000), but questions have been raised regarding the efficacy of maintaining constant conditions in these protocols (Taylor, 1998). Observations of refractory periods following either global (Khodakhah & Ogden, 1995; Carter & Ogden, 1997; Ogden & Capiod, 1997) or more focal (Parker & Ivorra, 1990a; McCarron et al. 2004) InsP3-mediated Ca2+ release in intact cells are also consistent with channel inactivation in intact cells. However, in rapid perfusion and cell studies, it has remained unclear whether release termination or decay is caused by a true intrinsic InsP3-induced inactivation process, possibly accelerated by high [Ca2+]i, or whether it is due to Ca2+-feedback inhibition of channel gating. Furthermore, it has not been established if apparent inactivation as a consequence of pre-exposure to Ca2+ before InsP3 exposure (Parker & Ivorra, 1990a; Payne et al. 1990; Finch et al. 1991b; Combettes et al. 1994; Oancea & Meyer, 1996; Ogden & Capiod, 1997; Swatton & Taylor, 2002; McCarron et al. 2004) is related to apparent inactivation as a result of InsP3 binding.
The single-channel studies reported here, performed under conditions of constant ligand concentrations and ER luminal conditions, indicate that InsP3-induced inactivation is an intrinsic property of the single InsP3R channel. Although the kinetics of apparent inactivation observed in superfusion experiments (Finch et al. 1991b; Combettes et al. 1994; Dufour et al. 1997) and in intact cells in response to photo-release of InsP3 (Khodakhah & Ogden, 1995) were rapid (<1 s), the inactivation kinetics reported here are slower. However, the kinetics of inactivation for InsP3R in nuclear patch-clamp studies observed here (Ta
10–100 s) and previously (Ta
20–30 s; Mak & Foskett, 1997), and here in the InsP3-pre-exposure protocols, are similar to those estimated by ER permeability measurements in permeabilized hepatocytes (Hajnóczky & Thomas, 1994), as well as to the kinetics of InsP3-induced increases in InsP3 affinity of an apparently desensitized InsP3R in cerebellar microsomes (Coquil et al. 1996) (t
15–45 s) and of the transient fast phase of Ca2+ release in response to initial exposure to InsP3 in permeabilized and intact cells (e.g. see Muallem et al. 1989; Meyer & Stryer, 1990; Taylor & Potter, 1990). Of note, InsP3R inactivation observed in permeabilized hepatocytes (Hajnóczky & Thomas, 1994), which had kinetics similar to those observed in our single channel studies, was shown to account for release termination associated with [Ca2+]i oscillations (Hajnóczky & Thomas, 1997). Together these results suggest that the kinetics of inactivation observed in single-channel studies are of physiological relevance for [Ca2+]i signalling observed in cells. However, it remains unclear whether distinct inactivation kinetics observed in different studies reflects methodological differences, distinct types of inactivation or inhibition, or a physiologically relevant range of kinetics of a common inactivation mechanism.
Our results here demonstrate that the kinetics of single channel inactivation are sensitive to both [InsP3] and [Ca2+]i. Inactivation was observed for every activated channel, even under conditions of saturating [InsP3] and very low [Ca2+]i (50 nM). Thus, inactivation appears to be fatefully linked to activation, but the rate of channel inactivation was accelerated at lower [InsP3] and higher [Ca2+]i, with channel activity duration reduced by over an order of magnitude between 1 and 89 µM Ca2+, with average channel duration in 89 µM Ca2+ reduced to under 10 s. In addition to ligand concentrations, other factors may also be important in determining channel inactivation kinetics. For example, the rate of channel inactivation of the Xenopus InsP3R (Mak & Foskett, 1997) was faster than that the Sf9-InsP3R studied here under identical ligand conditions. Furthermore, the rate of the Sf9-InsP3R inactivation was faster in the patched membranes than in the InsP3-pre-exposure protocols. Thus, factors such as membrane tension, membrane lipid composition and associated proteins may also contribute to the rate of InsP3R channel inactivation. It is interesting that inactivation has not been reported for reconstituted InsP3R channels recorded in artificial planar bilayer membranes. Our results suggest that inactivation rates of single InsP3R channels are regulated by ligand concentrations and possibly by additional, including cell-type-specific, factors as well, consistent with a common regulated mechanism of inactivation. Importantly, however, additional studies will be required to determine whether inactivation observed in single channel studies contributes to Ca2+ release termination in cells.
Inactivation versus inhibition
Because the curves that describe the transient kinetics of Ca2+ release observed in cells and rapid perfusion experiments are reminiscent of the biphasic curves that describe the [Ca2+]i dependencies of steady-state channel Po, there has been a tendency in the literature to equate the two and to account for release termination by effects of high [Ca2+]i on single-channel gating activity. Consequently, the terms ‘inhibition’ and ‘inactivation’ and ‘partial inactivation’ have sometimes been used to refer to similar phenomena. However, as pointed out (Sneyd et al. 2004), the bell-shaped or otherwise biphasic shape of the steady state Po versus [Ca2+]i curve has ‘very little, if anything’, to do with the fact that the InsP3R exhibits complex rapid kinetic behaviours. Our single-channel studies of Sf9-InsP3R demonstrate that channel inactivation occurs over all [Ca2+]i studied, with the rate of inactivation not necessarily correlated with channel Po. Of note, it was previously observed that Xenopus oocyte InsP3R channels that lack high [Ca2+]i inhibition of channel Po (as a result of an experimental manoeuvre) nevertheless still inactivated (Mak et al. 2003b). Thus, InsP3R inhibition by high [Ca2+]i, and InsP3-induced inactivation of the InsP3R, are distinct processes. The [Ca2+]i dependencies of channel Po inhibition and inactivation are likely to be mediated by distinct Ca2+-binding sites, as discussed in more detail below. Consequently, we propose that the term ‘inhibition’ be used to refer to the effects of high [Ca2+]i on steady-state channel Po, whereas ‘inactivation’ be used to refer to the time-dependent loss of the capacity of the channel to open even in the presence of constant [InsP3] and [Ca2+]i.
Ligand stimulation by both InsP3R channel recruitment and activation
We found that the average number of channels activated in a membrane patch (NA) depends on the strength of ligand activation. NA had a biphasic dependence on [Ca2+]i, and was smaller when InsP3 was reduced to suboptimal concentrations over the entire range of [Ca2+] examined (Fig. 3C). These differences in NA cannot be explained by underestimation of the number of channels when Po was low, since the channel current records were long enough for NA to be accurately determined. Notably, the ligand concentration dependencies of Po and NA are similar. Thus, the product NAPo is a sensitive function of both [Ca2+]i and [InsP3] (Fig. 3D), and NAPo quantitatively accounts for the magnitude of the Ca2+ flux associated with InsP3-mediated [Ca2+]i signals in cells (eqn (2)) because channel conductance is constant. Our single-channel results therefore suggest that the magnitudes of InsP3R-mediated Ca2+ signals in cells are determined by both channel recruitment as well as single-channel activity.
The observed ligand dependence of the number of channels that become recruited into the activated state is unexpected. Because the channels were observed for tens of seconds during their exposure to the ligands, graded channel recruitment cannot be accounted for by stochastic probabilities of ligand binding to the channels. Thus, it was anticipated that the entire channel population in a membrane patch would always become engaged, albeit to different levels of activity (Po) depending on the strength of ligand stimulation. The graded recruitment of the number of activated channels seemingly implies that the InsP3R channels in Sf9 cell nuclei have heterogeneous sensitivities to both [InsP3] and [Ca2+]i. Nevertheless, it seems unlikely that heterogeneous ligand sensitivity exists among the channels. InsP3R isoform diversity cannot be involved since no invertebrate has been demonstrated to express more than one InsP3R isoform. Furthermore, it is unlikely that heterogeneous covalent modifications could account for the observed graded responsiveness to such a wide range of both ligands in nuclei from different batches of cells. In addition, possible local determinants of channel activity such as luminal [Ca2+] or channel density can be ruled out in the system studied here.
A qualitative model that accounts for ligand dependencies of channel recruitment and inactivation
A simple qualitative kinetic scheme (Fig. 5) involving channel sequestration can account for the observed ligand-dependent channel recruitment without invoking intrinsic channel heterogeneity. The model assumes that InsP3R channels can become sequestered in a non-active S state by the binding of Ca2+ to a sequestration site. In the absence of InsP3, the regular closed C state of the channel is in equilibrium with the S state, with the C state being favoured (Fig. 5A). When the channel is exposed to InsP3, InsP3 binding to the channel enables the channel to enter activated A states (including the A* state in which the channel is open to conduct ion flux, and the closed A' state). A competition ensues in which the probability of the channel becoming activated, which depends on [Ca2+]i according to the previously developed MWC-based allosteric model (Mak et al. 2003a), is countered by the probability of the channel becoming sequestered into the non-active S state (Fig. 5B). Ca2+ concentrations that favour activation of the InsP3R channel (optimal [Ca2+]i) decrease the chance of the channel being sequestered before activation, increasing NA; in contrast, suboptimal [Ca2+]i (subactivating [Ca2+]i or high inhibitory [Ca2+]i) that are less favourable for channel activation increase the chance that the channel is sequestered into the S state and therefore reduce NA. Consequently, the [Ca2+]i dependence of NA is biphasic and similar to the [Ca2+]i dependence of channel Po under all [InsP3]. Once an InsP3-bound channel enters the S state, it is stabilized there because the affinities of its InsP3-binding sites and the sequestration Ca2+-binding sites are so high that it cannot escape from that state in the continuous presence of InsP3. For any particular [Ca2+]i, NA is lower in subsaturating [InsP3] than saturating [InsP3] because lower [InsP3] allows more channels to be siphoned off into the S state by Ca2+ binding to the sequestration site before InsP3 can bind to the channel. Subsequent binding of InsP3 to the channel stabilizes it in the S state.
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There are obvious similarities in the properties of channel sequestration, which results in a certain fraction of available InsP3R channels not being activated despite the continuous presence of InsP3, and channel inactivation, which results in InsP3-induced termination of channel activity. In both processes, the InsP3R channel ends up in a state from which it cannot escape in the continuous presence of InsP3. Both processes are regulated by [InsP3] and [Ca2+]i. And, in our scheme, both processes involve Ca2+ binding to the InsP3R channel. However, the time scales of the two processes are very different. Channel sequestration must occur on a time scale shorter than the activation of the InsP3R channel after it is exposed to InsP3, within 1–5 s (from the time the patch pipette makes contact with the membrane and the channels in the isolated membrane patch are exposed to InsP3, to the time when the gigaohm seal forms to allow recording of channel currents). In contrast, channel inactivation occurs at least an order of magnitude slower, 20–100 s. Therefore, it is likely that sequestration and inactivation are distinct processes, involving two distinct Ca2+-binding sites.
This simple qualitative modelling of our single-channel results indicates that InsP3-induced channel inactivation (I states in Fig. 5) from active conformations may play a role in terminating Ca2+ release. Here, we now propose that channel sequestration before it releases Ca2+, plays a role in tuning the number of release channels involved in responses in cells. Interestingly, this model is reminiscent of a kinetic model developed by Sneyd & Dufour (2002), that closely followed the ideas of Taylor (Marchant & Taylor, 1997; Adkins & Taylor, 1999), to account for Ca2+ release kinetics from hepatic microsomes, and of another kinetic model developed by Dawson et al. (2003) that was adapted from a kinetic scheme of ryanodine receptor adaptation (Sachs et al. 1995). In both kinetic models, inactivated InsP3R states were also accessible from either open or closed channel states. Although our simple
, Ca2+ binding to sequestration site; 
, Ca2+ binding to inactivation site. A bigger arrowhead indicates the side of the reaction or transition that the equilibrium favours. Reactions or transitions with thicker arrow shafts have higher rates. The activated InsP3-bound A state can gate between channel open (A*) and closed (A') conformations through ligand-independent transitions represented by arrows with chevron arrowheads. Equilibrium between InsP3R channels in C and A states is represented by the open arrow marked with MWC, discussed in detail in (