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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.37 n.4 Santiago  2004

http://dx.doi.org/10.4067/S0716-97602004000400004 

 

 

Biol Res 37: 513-519, 2004

ARTICLE

Novel model of calcium and inositol 1,4,5-trisphosphate regulation of InsP3 receptor channel gating in native endoplasmic reticulum

J. Kevin Foskett and D.-O. Daniel Mak

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA

Dirección para Correspondencia


ABSTRACT

The InsP3R Ca2+-release channel has biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing high [Ca2+]i inhibition. To determine whether relieving Ca2+ inhibition is sufficient for activation, we examined single-channels in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent activities with low open probability Po (~ 0.03) were observed in [Ca2+]i < 5 nM, whereas none were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i in the absence of InsP3 and demonstrate that the channel can be active when all of its ligand-binding sites are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies, the tetrameric channel can adopt six conformations, the equilibria among which are controlled by two inhibitory, one activating Ca2+-binding, and one InsP3-binding sites in a manner similar to the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site.

Key words: InsP3 receptor; calcium release channel; calcium; ion channel; model.


 

INTRODUCTION

In many cell types, the second messenger inositol 1,4,5-trisphosphate (InsP3) is generated in the cytoplasm in response to the binding of extracellular ligands to plasma membrane receptors. InsP3 binds to its receptor, the InsP3R, in the endoplasmic reticulum (ER) and activates it as a Ca2+ channel to liberate stored Ca2+ from the ER lumen into the cytoplasm. This rapid release of Ca2+ modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), which serves as a ubiquitous cellular signal that can be manifested temporally as repetitive spikes or oscillations and spatially as propagating waves or highly localized events. The temporal and spatial complexity of this signaling system involves sophisticated regulation of the activity of the InsP3R by various mechanisms, including cooperative activation by InsP3 (2) and biphasic feedback from the permeant Ca2+ ion (2). A family of three InsP3 receptor isoforms has been identified: types 1, 2, and 3, with different primary sequences derived from different genes. The channel open probability (Po) of types 1 and 3 InsP3R isoforms is modulated with biphasic dependencies on [Ca2+]i, suggesting that the channels have two distinct types of functional Ca2+ -binding sites: activating and inhibitory (2,4). InsP3 activates the InsP3R by tuning the sensitivity of the channel to Ca2+ inhibition, with increases in the cytoplasmic concentration of InsP3 ([InsP3]) causing a decrease in the apparent Ca2+ affinity of the inhibitory binding sites of the channel. Nevertheless, the fully InsP3-liganded channel can still be inhibited by Ca2+, albeit at sufficiently high concentrations (2,4). Importantly, InsP3 has little apparent effect on activation parameters (half-maximal activation [Ca2+]i, Kact; and activation Hill coefficient, Hact) of the biphasic Hill equation that describes the Ca2+ response of the channel, nor does it affect the robust maximum Po exhibited by either InsP3R isoform under optimal activating conditions.

REGULATION OF InsP3R BY CALCIUM AND InsP3: A MODEL

Because the InsP3R Ca2+-release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration (Fig. 1), and InsP3 appears to activate gating primarily by reducing the ability of high [Ca2+]i to inhibit the channel (Fig. 1) (2), we determined whether relieving Ca2+ inhibition was sufficient for channel activation by examining single-channel activities in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po (~ 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+ (5). These results establish the half-maximal inhibitory [Ca2+]i of the channel in the absence of InsP3 (~1.2_4.0 nM) and demonstrate that the channel can be active when none (including InsP3) of its ligand-binding sites are occupied. Spontaneous InsP3R channel activity was observed because the inhibitory Ca2+-binding sites of the channel have a finite affinity, even in the absence of InsP3, so that in [Ca2+]i < 5 nM, the inhibitory Ca2+-binding sites are not occupied and there is no Ca2+ inhibition of the channel. The observation of spontaneous, ligand-independent activity suggests that Ca2+ and InsP3 regulation of the InsP3R channel should be described by an allosteric molecular model in which a channel that is not bound to Ca2+ or InsP3 nevertheless has a finite, non-zero, probability of adopting an open conformation. In contrast, all previous models have assumed that channel opening has a strict requirement for InsP3 binding.

 

Figure 1. Top (A-D): Typical single-channel current traces of the rat type 3 InsP3 at various [Ca2+]i in the presence of 10 mM InsP3. Arrows indicate closed channel current level in all current traces. Bottom: Ca2+ dependence of r-InsP3R-3 channel open probability under various [InsP3]. Different symbols denote data for various [InsP3] as tabulated. The curves are theoretical fits using the biphasic Hill equation:
Po = Pmax {1 + (Kact / [Ca2 +]i )Hact } -1 {1 + ([Ca2+]i / Kinh)Hinh }-1
with Kinh varying with [InsP3] as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of [InsP3] with values tabulated in the graph. Taken from (4).

In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms (1,2,3,5), including spontaneous InsP3-independent channel activities (5) and abrogation of high [Ca2+]i inhibition after exposure of InsP3R to a low (< 5 nM) Ca2+ bath (6), the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner depicted by the Monod-Wyman-Changeux model (Fig. 2). InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site. The model also suggests that in addition to the mechanism controlling ligand-dependent conformation transitions, there is a ligand-independent gating mechanism that accounts for maximum channel Po of less than unity (5).

This model postulates that the InsP3R monomers, and therefore the InsP3R tetrameric channel as a whole, can adopt six different conformations (Fig. 2). The channel is open when it is in the A* and C* conformations. The B, D, A' and C' conformations are closed. The closed B and D conformations are completely inter-convertible with the open A* and C* conformations (Fig. 2). The equilibria between A*, B, C* and D conformations are dependent on InsP3 and Ca2+ binding to the channel, so that channel activity is regulated by [InsP3] and [Ca2+]i. In contrast, the equilibrium A* ↔ A' is not affected by [InsP3] or [Ca2+]i, i.e., the affinities of the InsP3 and Ca2+ sites of the InsP3R channel are the same in A* and A'. The ratio of the total durations that an InsP3R channel spends in the A* conformation and in the A' conformation is the same regardless of [InsP3] and [Ca2+]i. Thus, the A* and A' conformations can be grouped together as the 'active' A conformation (a conformation in which the channel can open) when we consider the effects of InsP3 and Ca2+ on channel conformations. Similarly, C' and C* are grouped together as the active C conformation because the equilibrium C* ↔ C' is likewise not affected by [InsP3] or [Ca2+]i. In this model, even in the presence of optimal [InsP3] and [Ca2+]i, when the InsP3R channel hardly exists in the closed B and D conformations, the maximum observed channel Po is < 1 because the channel exists a fraction of the time in the closed A' and C' conformations. This accounts for the observation in both regular and ultra-low bath [Ca2+] (2,6) that the maximum InsP3R channel Po in saturating [InsP3] (10 µM) and optimal [Ca2+]i is only ~ 0.8 (< 1).

Figure 2. The MWC-based four+two-conformation model for InsP3R channel gating. Only conformation transitions are represented in the schemes. Reactions involving binding of InsP3 and Ca2+ to the InsP3R channel and the state of occupation of the various ligand-binding sites of the channel are omitted from the schemes for clarity. The dotted rectangles represent the grouping of the open A* and closed A' conformations into the active A conformation, and the grouping of the C* and C' conformations into the active C conformation. Taken from (5).

 

The model further postulates that each of the four InsP3R monomers has one InsP3-binding site (Q) and three different functional Ca2+-binding sites (F, G and H) on the cytoplasmic side of the channel. Because of its tetrameric structure, an InsP3R channel can bind a maximum of four InsP3 in its Q sites and four Ca2+ in each of the three types (F, G and H) of Ca2+ sites. The affinities of these ligand-binding sites are different in the different channel conformations (A, B, C and D). Both InsP3 and Ca2+ regulate channel activity because differential binding of InsP3 or Ca2+ to these sites will stabilize those conformations in which the sites have high affinities, thereby affecting the equilibria among the A, B, C and D conformations, as laid out in (7).

Regulation by [Ca2+]i: In our model, the F sites in the A and C conformations of the channel have higher Ca2+ affinity than in the B and D conformations. Thus, Ca2+ binding to F sites is activating because binding preferentially stabilizes the active A and C conformations. The H sites, on the other hand, have higher Ca2+ affinity in the B and D channel conformations than in the A and C conformations in the presence of regular, 300-500 nM bath [Ca2+]. Therefore, Ca2+ binding to the H sites is inhibitory, since binding stabilizes the closed B and D conformations. The Ca2+ affinities of neither the activating F sites nor the inhibitory H sites are InsP3 dependent. The experimentally-observed abrogation of Ca2+ inhibition in InsP3R channels exposed to ultra-low bath [Ca2+] (6) is caused by the loss of functionality of the inhibitory H sites. As a result of exposure of the channel to ultra-low bath [Ca2+], the H sites in all the channel conformations adopt the same affinity. Thus, Ca2+ binding to these sites (or the lack thereof) has no effect on the conformation equilibria. Parenthetically, in addition to the three types of Ca2+-binding sites (F, G and H) that are sensitive to the instantaneous [Ca2+]i, the InsP3R channel must also have another type of Ca2+-binding site that is sensitive to the bath [Ca2+] and affects channel activity by modifying the affinity of the H sites and thereby the Ca2+ inhibition of the channel, as described in (6). This type of bath [Ca2+] `sensing' site is not included in the present molecular model because under physiological [Ca2+]i it is always Ca2+-liganded and the H site is therefore presumably always functional.

Regulation by InsP3: A description of the InsP3 regulation of the channel is more complicated than that of the biphasic regulation by [Ca2+]i. In the absence of InsP3, the InsP3R channel exists predominantly in the A and B conformations. InsP3 binding to the Q sites preferentially stabilizes the C and D channel conformations, because the Q sites in those conformations have higher InsP3 affinity. Importantly, this stabilization of the C and D channel conformations in turn affects Ca2+ inhibition of the channel. The modeling reveals that the Ca2+ affinity of the G sites in the closed B conformation is higher than that in the active A conformation. Therefore, when [InsP3] is low and the InsP3R channel exists mostly in the A and B conformations, Ca2+ binding to the G sites is inhibitory, because G-site binding stabilizes the closed B conformation. In contrast, the Ca2+ affinity of the G sites in the closed D channel conformation is lower than that in the active C conformation. Therefore, when the channel exists mostly in the C and D conformations in the presence of high [InsP3], Ca2+ binding to the G sites is activating because this stabilizes the active C conformation. Thus, InsP3 activates InsP3R channel gating by changing the affinity of the G sites in the InsP3R, which in turn changes the nature of the G sites from inhibitory to activating.

Our modeling effort is the first to incorporate spontaneous channel activity into an allosteric model to describe the InsP3R channel. It is also the first model that takes into consideration the tetrameric structure of the InsP3R channel and thus fully and quantitatively addresses the cooperative nature of the activation and inhibition of InsP3R channel gating by [Ca2+]i and the cooperative nature of InsP3R channel regulation by InsP3. We examined various allosteric models to find a molecular model that could describe channel gating characteristics observed in extensive electrophysiological studies of the InsP3R in native endoplasmic reticulum membrane. We explicitly defined 12 distinct observations that such a model should be able to explain for both the types 1 and 3 InsP3R over a wide observed range of [Ca2+]i (~ 3 nM to 200 µM) and [InsP3] (0 to 10 µM), including the lack of Ca2+ inhibition (up to 1.5 mM) of channel activity of XInsP3R1 exposed to ultra-low bath [Ca2+] (< 5 nM) (6) and spontaneous activities (5). Although more than one molecular model can describe adequately the observed channel behaviors, the MWC-based four+two-conformation model with one InsP3- and three Ca2+-binding sites can do so with the minimum number of free parameters and therefore is considered most likely (5).

The model provides insights into the possible molecular mechanisms that enable the InsP3R channel to be regulated exquisitely, with the apparent affinity of a Ca2+ binding inhibitory site (Kinh) changing over 2 orders of magnitude (from 160 nM to 60 µM) by changes in [InsP3] over a narrow range (10 to 100 nM) (2,4) even though there are only four InsP3-binding sites in each InsP3R tetrameric channel. The model also provides a possible explanation for the rapid and total saturation of its response to [InsP3] once [InsP3] goes beyond 100 nM (2,4). Both of these properties of the InsP3R regulation are highly relevant to the generation of rapid and well-controlled Ca2+ signals in the cell. Accordingly, the high sensitivity of the InsP3R to low [InsP3] and the rapidity of saturation of the channel response to InsP3 can be accounted for by the InsP3R monomer having three different functional Ca2+-binding sites that directly affect the equilibria among the conformations of the channel. One of these sites is activating (F), whereas another is inhibitory (H), but both are independent of InsP3. In contrast, a third Ca2+-binding site (G) is affected by InsP3, being inhibitory in the absence of InsP3 but becoming activating as [InsP3] increases. All previous models of Ca2+ regulation of InsP3R function have assumed that each channel monomer possessed a single inhibitory Ca2+binding site. Our previous analyses of the effects of InsP3 on channel gating could also be empirically described by assuming a single inhibitory Ca2+binding site, that was allosterically modulated by InsP3 binding (2,4). Thus, InsP3 binding reduced the apparent affinity of the one inhibitory site for Ca2+, enabling the channel to be active at [Ca2+]i that are inhibitory in the absence of InsP3. The molecular model derived here now suggests that two inhibitory Ca2+ binding sites are present in each monomer. InsP3 changes the apparent affinity of one of them, and in so doing transforms it into an activating site. This InsP3-mediated transformation of the nature of this Ca2+ binding site contributes substantially to the extremely high sensitivity of InsP3R channel gating to [InsP3]. The second inhibitory Ca2+ binding site (H) has lower Ca2+ affinity (~ µM range) that is not modulated by InsP3 binding. Binding to this H site accounts for Ca2+ inhibition of the fully-InsP3-liganded channel. We previously described the Ca2+ dependence of inhibition of the InsP3 liganded channel as reflecting the finite affinity of the InsP3-sensitive Ca2+ inhibition site (2,4). However, that was a simple empirical description, which our molecular model now indicates was incorrect. Whereas the properties of the low-affinity Ca2+ inhibition site (H) are insensitive to InsP3 binding, they are sensitive to exposure to an ultra-low bath [Ca2+]. As discussed in (6), it is possible that this Ca2+ inhibition site could be a target of as yet undiscovered physiological regulation. In the four+two-conformation model, the abrogation of Ca2+ inhibition of InsP3R channel exposed to ultra-low bath [Ca2+] is caused by the loss of functionality of the InsP3-independent Ca2+ inhibitory H sites. Nevertheless, the channel in ultra-low bath [Ca2+] remains dependent on InsP3 because the InsP3-dependent Ca2+-binding sites (G sites) are not affected by the low bath [Ca2+] and inhibit InsP3R activity in the absence of InsP3.

Our molecular model suggests that not all conformation transitions of the InsP3R that affect the channel opening are regulated by InsP3 and [Ca2+]i. In our model, Pmax of the InsP3R channel is limited to ~0.8 ( 1) by conformation transitions that affect channel opening but are independent of [Ca2+]i and [InsP3]. Such conformation transitions probably arise from a channel gating mechanism different from the one regulated by ligands (InsP3 and Ca2+). This ligand-independent channel activity can account for the observed constancy of the mean InsP3R channel open durations over a wide range of [Ca2+]i and [InsP3].

This model developed here will be useful in future experimental research. First, it will be important for providing a quantitative framework for understanding the roles of other channel regulators. Second, as mutagenesis is applied to this channel in attempts to discover the molecular bases for ligand-binding, for example, this model may aid in discriminating sites that are truly binding sites from those that are allosterically coupled to them. Third, when transient kinetic responses of channels are measured in response to these changes, the model may help to constrain the set of possible schemes that need to be considered. Fourth, application of the model to data sets obtained from distinct InsP3R isoforms may provide useful in identifying the properties that distinguish them and account for any observed distinct behaviors.

ACKNOWLEDGEMENTS

We thank Sean McBride for technical assistance and the National Institutes of Health for support.

REFERENCES

1. MAK DOD, FOSKETT JK (1997) Single-channel kinetics, inactivation and spatial distribution of inositol trisphosphate (IP3) receptor in Xenopus oocyte nucleus. J Gen Physiol 109:571-587         [ Links ]

2. MAK DOD, MCBRIDE S, FOSKETT JK (1998) Inositol 1,4,5-trisphosphate activation of inositol trisphosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition. Proc Nat Acad Sci USA 95:15812-15825         [ Links ]

3. MAK DOD, MCBRIDE S, RAGHURAM V, YUE Y, JOSEPH SK, FOSKETT JK (2000) Single channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor. J Gen Physiol 115:241-255         [ Links ]

4. MAK DOD, MCBRIDE S, FOSKETT JK (2001) Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 InsP3 receptors. J Gen Physiol 117:435-446         [ Links ]

5. MAK DOD, MCBRIDE S, FOSKETT JK (2003a) Spontaneous channel gating of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating. J Gen Physiol 122:583-603         [ Links ]

6. MAK DOD, MCBRIDE S, PETRENKO NB, FOSKETT JK (2003b) Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium release channel. J Gen Physiol 122:569-581         [ Links ]

7. MONOD J, WYMAN J, CHANGEUX P (1965) On the nature of allosteric transitions: a plausible model. J Mol Biol 12:88-118         [ Links ]

 

Corresponding author. Dr. J. Kevin Foskett, Department of Physiology, B39 Anatomy-Chemistry Bldg., University of Pennsylvania, Philadelphia, PA 19104-6085, USA, Tel: 215-898-1354, Fax: 215-573-6808, e-mail: foskett@mail.med.upenn.edu

Received: June 16, 2004. Accepted: November 7, 2004.

 

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