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Structural determinants of CaV1.3 L-type calcium channel gating.

ABSTRACT: A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Ca(v)1.3 L-type Ca(2+) channels, in particular the voltage dependence of activation (V(0.5)) and Ca(2+) dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Ca(v)1.3 (Ca(v)1.3(L)), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCa(v)1.3(scg)). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCa(v)1.3(scg) with a rat cDNA isolated from rat pancreas (rCa(v)1.3(L)). When expressed in tsA-201 cells under identical experimental conditions rCa(v)1.3(L) exhibited Ca(2+) current properties indistinguishable from human and mouse Ca(v)1.3(L), compatible with the presence of a functional CTM. In contrast, rCa(v)1.3(scg) showed gating properties similar to human short splice variants lacking a CTM. rCa(v)1.3(scg) differs from rCa(v)1.3(L) at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCa(v)1.3(scg) explained most of the functional differences to rCa(v)1.3(L). Their mutation to the corresponding residues in rCa(v)1.3(L) (G244, V2075) revealed that both contributed to the more negative V 0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCa(v)1.3(L). We found no evidence for transcripts containing one of the single rCa(v)1.3(scg) mutations in rat superior cervical ganglion preparations. However, the rCa(v)1.3(scg) variant provided interesting insight into the structural machinery involved in Ca(v)1.3 gating.

SUBMITTER: Lieb A

PROVIDER: S-EPMC3431584 | biostudies-literature | 2012 May-Jun

REPOSITORIES: biostudies-literature

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Structural determinants of CaV1.3 L-type calcium channel gating.

Lieb Andreas A Scharinger Anja A Sartori Simone S Sinnegger-Brauns Martina J MJ Striessnig Jörg J

Channels (Austin, Tex.) 20120501 3

A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Ca(v)1.3 L-type Ca(2+) channels, in particular the voltage dependence of activation (V(0.5)) and Ca(2+) dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Ca(v)1.3 (Ca(v)1.3(L)), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCa(v)1.3(scg)). We therefore addressed the question if this represents a species-difference and compared ...[more]

PMID: 22760075

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Project description:Activation of the growth hormone (GH) secretagogue receptor (GHS-R) by synthetic GH releasing peptides (GHRP) or its endogenous ligand (Ghrelin) stimulates GH release. Though much is known about the signal transduction underlying short-term regulation, there is far less information on the mechanisms that produce long-term effects. In the current report, using an enzyme-linked immunosorbent assay for GH detection and whole-cell patch-clamp recordings, we assessed the long-term actions of such regulatory factors on voltage-activated Ca(2+) currents in bovine somatotropes (BS) separated on a Percoll gradient and detected by immunohistochemistry. After 24 h of treatment with Ghrelin (10 nM) or GHRP-6 (100 nM) enhanced BS secretory activity; GH secretion stimulated by GHS through the activation of GHS-R because treatment with the antagonist of GHS-R (D-Lys3-GHRP-6, 10 μM) blocked the GH secretion, and the effect was dose and time dependent (24, 48, and 72 h). GH secretion stimulated by GHRP-6 was abolished by nifedipine (0.5 μM), a blocker of L-type HVA Ca(2+) channels, and KN-62 (10 μM), an inhibitor of Ca(2+)/CaM-KII. After 72 h in culture, all recorded BS exhibited two main Ca(2+) currents: a low voltage-activated (LVA; T-type) and a high voltage-activated (HVA; mostly dihydropyridine-sensitive L-type) current. Interestingly, HVA and LVA channels were differentially upregulated by Ghrelin. Chronic treatment with the GHS induced a significant selective increase on the Ba(2+) current through HVA Ca(2+) channels, and caused only a small increase of currents through LVA channels. The stimulatory effect on HVA current density was accompanied by an augment in maximal conductance with no apparent changes in the kinetics and the voltage dependence of the Ca(2+) currents, suggesting an increase in the number of functional channels in the cell membrane. Lastly, in consistency with the functional data, quantitative real-time RT-PCR revealed transcripts encoding for the Cav1.2 and Cav1.3 pore-forming subunits of L-type channels. The treatment with Ghrelin significantly increased the Cav1.3 subunit expression, suggeting that the chronic stimulation of the GHS receptor with Ghrelin or GHRP-6 increases the number of voltage-gated Ca(2+) channels at the cell surface of BS.

Dataset Information

Structural determinants of CaV1.3 L-type calcium channel gating.

Publications

Structural determinants of CaV1.3 L-type calcium channel gating.

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