Dataset Information

Selective blockade of Ca_v1.2 (α1C) versus Ca_v1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

ABSTRACT: L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Ca_v1.2 and Ca_v1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Ca_v1.2 -mediated L-type calcium currents (I_CaL) at concentrations leaving Ca_v1.3-mediated I_CaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Ca_v1.2 and Ca_v1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Ca_v1.2- and Ca_v1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Ca_v1.2 Ca²⁺ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

SUBMITTER: Mesirca P

PROVIDER: S-EPMC10762068 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Publications

Selective blockade of Cav1.2 (α1C) versus Cav1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

Mesirca Pietro P Chemin Jean J Barrère Christian C Torre Eleonora E Gallot Laura L Monteil Arnaud A Bidaud Isabelle I Diochot Sylvie S Lazdunski Michel M Soong Tuck Wah TW Barrère-Lemaire Stéphanie S Mangoni Matteo E ME Nargeot Joël J

Nature communications 20240102 1

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -media ...[more]

PMID: 38167790

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Project description:Mammalian circadian (24 h) rhythms are timed by the pattern of spontaneous action potential firing in the suprachiasmatic nucleus (SCN). This oscillation in firing is produced through circadian regulation of several membrane currents, including large-conductance Ca2+- and voltage-activated K+ (BK) and L-type Ca2+ channel (LTCC) currents. During the day steady-state BK currents depend mostly on LTCCs for activation, whereas at night they depend predominantly on ryanodine receptors (RyRs). However, the contribution of these Ca2+ channels to BK channel activation during action potential firing has not been thoroughly investigated. In this study, we used a pharmacological approach to determine that both LTCCs and RyRs contribute to the baseline membrane potential of SCN action potential waveforms, as well as action potential-evoked BK current, during the day and night, respectively. Since the baseline membrane potential is a major determinant of circadian firing rate, we focused on the LTCCs contributing to low voltage activation of BK channels during the subthreshold phase. For these experiments, two LTCC subtypes found in SCN (CaV1.2 and CaV1.3) were coexpressed with BK channels in heterologous cells, where their differential contributions could be separately measured. CaV1.3 channels produced currents that were shifted to more hyperpolarized potentials compared with CaV1.2, resulting in increased subthreshold Ca2+ and BK currents during an action potential command. These results show that although multiple Ca2+ sources in SCN can contribute to the activation of BK current during an action potential, specific BK-CaV1.3 partnerships may optimize the subthreshold BK current activation that is critical for firing rate regulation.NEW & NOTEWORTHY BK K+ channels are important regulators of firing. Although Ca2+ channels are required for their activation in excitable cells, it is not well understood how BK channels activate using these Ca2+ sources during an action potential. This study demonstrates the differences in BK current activated by CaV1.2 and CaV1.3 channels in clock neurons and heterologous cells. The results define how specific ion channel partnerships can be engaged during distinct phases of the action potential.

Project description:Clustering of L-type voltage-gated Ca2+ channels (LTCCs) in the plasma membrane is increasingly implicated in creating highly localized Ca2+ signaling nanodomains. For example, neuronal LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca2+ concentrations within a nanodomain close to the channel, without requiring bulk Ca2+ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the CaV 1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed CaV 1.3 α1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble complexes containing multiple CaV 1.3 α1 subunits under basal conditions. Moreover, CaV 1.3 LTCC complex formation was facilitated by CaV β subunits (β3 and β2a), which also interact with Shank3. Shank3 interactions with CaV 1.3 LTCCs and multimeric CaV 1.3 LTCC complex assembly were disrupted following the addition of Ca2+ to cell lysates, perhaps simulating conditions within an activated CaV 1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized CaV 1.3 LTCC clusters under basal conditions, but not after Ca2+ channel activation. Live cell imaging studies also revealed that Ca2+ influx through LTCCs disassociated Shank3 from CaV 1.3 LTCCs clusters and reduced the CaV 1.3 cluster intensity. Deletion of the Shank3 PDZ domain prevented both binding to CaV 1.3 and the changes in multimeric CaV 1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized CaV 1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions.

Dataset Information

Selective blockade of Ca_v1.2 (α1C) versus Ca_v1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

Publications

Selective blockade of Ca<sub>v</sub>1.2 (α1C) versus Ca<sub>v</sub>1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

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