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Intra-membrane signaling between the voltage-gated Ca2+-channel and cysteine residues of syntaxin 1A coordinates synchronous release.

ABSTRACT: The interaction of syntaxin 1A (Sx1A) with voltage-gated calcium channels (VGCC) is required for depolarization-evoked release. However, it is unclear how the signal is transferred from the channel to the exocytotic machinery and whether assembly of Sx1A and the calcium channel is conformationally linked to triggering synchronous release. Here we demonstrate that depolarization-evoked catecholamine release was decreased in chromaffin cells infected with semliki forest viral vectors encoding Sx1A mutants, Sx1A(C271V), or Sx1A(C272V), or by direct oxidation of these Sx1A transmembrane (TM) cysteine residues. Mutating or oxidizing these highly conserved Sx1A Cys271 and Cys272 equally disrupted the Sx1A interaction with the channel. The results highlight the functional link between the VGCC and the exocytotic machinery, and attribute the redox sensitivity of the release process to the Sx1A TM C271 and C272. This unique intra-membrane signal-transduction pathway enables fast signaling, and triggers synchronous release by conformational-coupling of the channel with Sx1A.

SUBMITTER: Bachnoff N

PROVIDER: S-EPMC3621091 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Intra-membrane signaling between the voltage-gated Ca2+-channel and cysteine residues of syntaxin 1A coordinates synchronous release.

Bachnoff Niv N Cohen-Kutner Moshe M Trus Michael M Atlas Daphne D

Scientific reports 20130101

The interaction of syntaxin 1A (Sx1A) with voltage-gated calcium channels (VGCC) is required for depolarization-evoked release. However, it is unclear how the signal is transferred from the channel to the exocytotic machinery and whether assembly of Sx1A and the calcium channel is conformationally linked to triggering synchronous release. Here we demonstrate that depolarization-evoked catecholamine release was decreased in chromaffin cells infected with semliki forest viral vectors encoding Sx1A ...[more]

PMID: 23567899

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Project description:Voltage-dependent Na+ channel activation underlies action potential generation fundamental to cellular excitability. In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III-IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.

Project description:BackgroundStudies of the structure-function relationship in proteins for which no 3D structure is available are often based on inspection of multiple sequence alignments. Many functionally important residues of proteins can be identified because they are conserved during evolution. However, residues that vary can also be critically important if their variation is responsible for diversity of protein function and improved phenotypes. If too few sequences are studied, the support for hypotheses on the role of a given residue will be weak, but analysis of large multiple alignments is too complex for simple inspection. When a large body of sequence and functional data are available for a protein family, mature data mining tools, such as machine learning, can be applied to extract information more easily, sensitively and reliably. We have undertaken such an analysis of voltage-gated potassium channels, a transmembrane protein family whose members play indispensable roles in electrically excitable cells.ResultsWe applied different learning algorithms, combined in various implementations, to obtain a model that predicts the half activation voltage of a voltage-gated potassium channel based on its amino acid sequence. The best result was obtained with a k-nearest neighbor classifier combined with a wrapper algorithm for feature selection, producing a mean absolute error of prediction of 7.0 mV. The predictor was validated by permutation test and evaluation of independent experimental data. Feature selection identified a number of residues that are predicted to be involved in the voltage sensitive conformation changes; these residues are good target candidates for mutagenesis analysis.ConclusionMachine learning analysis can identify new testable hypotheses about the structure/function relationship in the voltage-gated potassium channel family. This approach should be applicable to any protein family if the number of training examples and the sequence diversity of the training set that are necessary for robust prediction are empirically validated. The predictor and datasets can be found at the VKCDB web site.

Dataset Information

Intra-membrane signaling between the voltage-gated Ca2+-channel and cysteine residues of syntaxin 1A coordinates synchronous release.

Publications

Intra-membrane signaling between the voltage-gated Ca2+-channel and cysteine residues of syntaxin 1A coordinates synchronous release.

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