Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

Abstract

3 -induced conformational change to the open state. We also found that GFP-IP 3 R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligandinduced clustering of a ligand-gated channel protein.Many extracellular stimuli are received by trimeric G protein-or tyrosine kinase-coupled receptors and relayed to signaling molecules including phospholipase C (PLC), 1 which leads to production of a second messenger, inositol 1,4,5-trisphosphate (IP 3 ) (1). IP 3 in turn activates an internal membrane receptor/Ca 2ϩ channel, the IP 3 receptor (IP 3 R), and induces Ca 2ϩ release from internal Ca 2ϩ stores (mostly endoplasmic reticulum (ER)). This IP 3 -Ca 2ϩ pathway is particularly important because it is located at the intersection of many signaling pathways and is therefore thought to play a central role in cellular signaling.Rather recently, evidence has been accumulating that the subcellular localization of IP 3 R is tightly controlled and changes according to circumstances (2-4). In addition, IP 3 R is known to form large clusters on the ER (5). Very importantly, theoretical modeling (6 -8) predicted that IP 3 R clustering affects both elementary events (e.g. Ca 2ϩ blips and puffs) and the formation of complex cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] C ) patterns (e.g. Ca 2ϩ oscillations and waves). Particularly Shuai and Jung (6) predicted that if IP 3 concentration is relatively low (i.e. physiological situation), there is an optimal extent of clustering of IP 3 receptor for effective elevation of cytosolic calcium concentration. When the clusters of IP 3 receptor would become very large, the whole calcium signals elicited by low IP 3 concentrations would be significantly reduced. Experimental verification of this hypothesis has not been performed mainly because the molecular mechanism of IP 3 R clustering remains totally unknown, and thus it is impossible to inhibit or artificially modulate it. Wilson et al. (5) suggested that IP 3 R clustering was triggered by increased [Ca 2ϩ ] C since not only Ca 2ϩ -mobilizing agonists but a Ca 2ϩ ionophore (ionomycin) and an inhibitor of sarco/ endoplasmic Ca 2ϩ -ATPase (thapsigargin) induced it. They also showed that it is independent of ER vesiculation (9). Later it was shown that two small GTP-binding proteins, Cdc42 and Rac, are involved in agonist-induced IP 3 R clustering but that was because they augmented Ca 2ϩ signals as a whole and not because they influenced intracellular trafficking (10). In short, the detailed molecular mechanism of IP 3 R clustering remains largely unknown, and it is important to know it to understand precise roles of IP 3 R clustering. In this study we simultaneously imaged the dynamics of enhanced green fluorescent protein (GFP)-tagged IP 3 R type 1 (GFP-IP 3 R1) and [Ca 2ϩ ] C by using fluorescent Ca 2ϩ indicators. Based on the results of the mutational and pharmacological analyses, we concluded that IP 3 R clustering is initiated by a conformational c...

The Clustering of Inositol 1,4,5-Trisphosphate (IP3) Receptors Is Triggered by IP3 Binding and Facilitated by Depletion of the Ca2+ Store

“…As previous studies (11,13) have shown that the clustering of IP 3 R is not the result of ER redistribution, ]i measured using fura-2-loaded cells. In C and E, the cells were incubated with 2 μM ThG for 10 min in the presence (C) or absence (E) of extracellular Ca 2+ and then stimulated with 100 μM ATP.…”

“…Further studies are necessary to determine whether this theoretical model of the signaling capability is applicable to the clustering of GFP-IP 3 R3 observed in our experiments. It has been shown that ERp44, an ER luminal protein that inhibits the channel activity of IP 3 R1 (25), colocalizes with GFP-IP 3 R1 clusters induced by ATP in COS-7 cells (11). On the basis of this result, the authors suggested that ERp44 negatively regulates the channel activity of IP 3 R1 in the clusters.…”

“…The clustered distribution of IP 3 Rs has been predicted to be important in controlling elementary Ca 2+ release events, such as Ca 2+ puffs and blips, which act as triggers to induce the spatiotemporal patterns of global Ca 2+ signals, such as waves and oscillations (8 -10). Recently, Tateishi et al (11) reported that GFP-IP 3 R1 expressed in COS-7 cells aggregates into clusters on the ER network after agonist stimulation. They concluded that IP 3 R clustering is induced by its IP 3 -induced conformational change to the open state, not by Ca 2+ release itself, because IP 3 R1 mutants that do not undergo an IP 3 -induced conformational change failed to form clusters.…”

Derivation of Ca 2+ signals from puff properties reveals that pathway function is robust against cell variability but sensitive for control

“…Indeed, clustering in cells is a dynamic process (5,(26)(27)(28). During this control of spiking, the positive slope of the σ-T av relation and the existence of the minimal ISI guarantee that faster spiking is also more regular despite the stochastic character of spike generation.…”

Dynamic regulation of IP3 receptor clustering and activity by IP3

“…Confocal microscopy has shown that both native and tagged IP 3 Rs can form clusters. [14][15][16] But a single IP 3 R measures only ∼20 nm across, and so determining the spacings of IP 3 Rs within clusters and assessing whether IP 3 Rs are close enough to facilitate local Ca 2+ regulation lie beyond the resolution of confocal microscopy. It is generally impracticable to use patch-clamp techniques to record from ion channels within the ER, but the outer nuclear envelope is continuous with the ER membrane and amenable to patchclamp recording.…”