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The CaV2?1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels.

ABSTRACT: The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming ?1 subunit (CaV2?1), and auxiliary CaV?, and CaV?2? subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2?1, CaV?, and CaV?2? and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2?1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.

SUBMITTER: Dunn TW

PROVIDER: S-EPMC5818475 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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The CaV2α1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels.

Dunn Tyler W TW Fan Xiaotang X Ase Ariel R AR Séguéla Philippe P Sossin Wayne S WS

Scientific reports 20180219 1

The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVβ, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plastici ...[more]

PMID: 29459734

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Project description:Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mg(i)) inhibits L-type Ca(2+) currents through Ca(V)1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mg(i) acts through the COOH-terminal EF-hand of Ca(V)1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg(2+) affinity. In whole-cell patch clamp experiments, increased Mg(i) reduced both Ba(2+) and Ca(2+) currents conducted by wild type (WT) Ca(V)1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT Ca(V)1.2 to lower Mg(i) (0.26 mM) increased the amplitudes of Ba(2+) currents 2.6 +/- 0.4-fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mg(i) to 2.4 or 7.2 mM reduced current amplitude to 0.5 +/- 0.1 and 0.26 +/- 0.05 of the control level at 0.8 mM Mg(i). The effects of Mg(i) on peak Ba(2+) currents were approximately fit by a single binding site model with an apparent K(d) of 0.65 mM. The apparent K(d) for this effect of Mg(i) was shifted approximately 3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mg(i) up to 7.2 mM. In contrast to these results, peak Ba(2+) currents through the K1543D mutant were inhibited by lower concentrations of Mg(i) compared with WT, consistent with approximately fourfold reduction in apparent K(d) for Mg(i), and inhibition of mutant K1539D by Mg(i) was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mg(i) was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of Ca(V)1.2 channels by Mg(i). Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of Ca(V)1.2 channels, and reveal a potentially important role of Mg(i) binding to the COOH-terminal EF-hand in regulating Ca(2+) influx in physiological and pathophysiological states.

Dataset Information

The CaV2?1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels.

Publications

The Ca<sub>V</sub>2α1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of Ca<sub>V</sub>2 channels.

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