Dataset Information

Clustering of InsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca2+.

ABSTRACT: The versatility of Ca2+ signals derives from their spatio-temporal organization. For Ca2+ signals initiated by inositol-1,4,5-trisphosphate (InsP3), this requires local interactions between InsP3 receptors (InsP3Rs) mediated by their rapid stimulation and slower inhibition\ by cytosolic Ca2+. This allows hierarchical recruitment of Ca2+ release events as the InsP3 concentration increases. Single InsP3Rs respond first, then clustered InsP3Rs open together giving a local 'Ca2+ puff', and as puffs become more frequent they ignite regenerative Ca2+ waves. Using nuclear patch-clamp recording, here we demonstrate that InsP3Rs are initially randomly distributed with an estimated separation of 1 m. Low concentrations of InsP3 cause InsP3Rs to aggregate rapidly and reversibly into small clusters of about four closely associated InsP3Rs. At resting cytosolic [Ca2+], clustered InsP3Rs open independently, but with lower open probability, shorter open time, and less InsP3 sensitivity than lone InsP3Rs. Increasing cytosolic [Ca2+] reverses the inhibition caused by clustering, InsP3R gating becomes coupled, and the duration of multiple openings is prolonged. Clustering both exposes InsP3Rs to local Ca2+ rises and increases the effects of Ca2+. Dynamic regulation of clustering by InsP3 retunes InsP3R sensitivity to InsP3 and Ca2+, facilitating hierarchical recruitment of the elementary events that underlie all InsP3-evoked Ca2+ signals.

SUBMITTER: Taufiq-Ur-Rahman

PROVIDER: S-EPMC2702691 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Clustering of InsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca2+.

Taufiq-Ur-Rahman Skupin Alexander A Falcke Martin M Taylor Colin W CW

Nature 20090401 7238

The versatility of Ca2+ signals derives from their spatio-temporal organization. For Ca2+ signals initiated by inositol-1,4,5-trisphosphate (InsP3), this requires local interactions between InsP3 receptors (InsP3Rs) mediated by their rapid stimulation and slower inhibition\ by cytosolic Ca2+. This allows hierarchical recruitment of Ca2+ release events as the InsP3 concentration increases. Single InsP3Rs respond first, then clustered InsP3Rs open together giving a local 'Ca2+ puff', and as puffs ...[more]

PMID: 19348050

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Project description:Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6 Å) and without (3.0 Å) InsP(3) bound. The arrangement of the three NT domains, SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP(3)R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore.

Project description:The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined 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 ( approximately 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+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. 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, including spontaneous InsP3-independent channel activities, 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 outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].

Dataset Information

Clustering of InsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca2+.

Publications

Clustering of InsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca2+.

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