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Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins.

ABSTRACT: Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).

SUBMITTER: Kasri NN

PROVIDER: S-EPMC1271747 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins.

Kasri Nael Nadif NN Holmes Anthony M AM Bultynck Geert G Parys Jan B JB Bootman Martin D MD Rietdorf Katja K Missiaen Ludwig L McDonald Fraser F De Smedt Humbert H Conway Stuart J SJ Holmes Andrew B AB Berridge Michael J MJ Roderick H Llewelyn HL

The EMBO journal 20031218 2

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenop ...[more]

PMID: 14685260

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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+].

Project description:Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with ? and ?2-?1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal l-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ?100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal l-type channels.SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.

Dataset Information

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins.

Publications

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins.

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