Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells.

Léa Réthoré,Daniel Henrion,Michel De Waard,Harold De Pomyers,Joseph Khoury,Claire Legendre,Sébastien Nicolas,Elodie Le Seac’H,Jérôme Montnach,Ziad Fajloun,César Mattei,Christian Legros,Kamel Mabrouk,Joohee Park,Hélène Tricoire-Leignel

doi:10.3390/ijms23020827

Abstract

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Highlights

Voltage-gated Na+ (NaV) channels are key molecular components involved in the electrical-excitability properties of the so-called excitable cells, such as neurons and myocytes [1]
We demonstrated that the pharmacological activation of NaV channels induces [Ca2+]i elevation mediated by L-type CaV channel (LTCC)
In GH3b6 cells, we found that VTD-induced [Ca2+]i elevation was totally blocked by TTX in the nanomolar range, confirming the involvement of TTX-S NaV channels

Summary

Introduction

Voltage-gated Na+ (NaV) channels are key molecular components involved in the electrical-excitability properties of the so-called excitable cells, such as neurons and myocytes (i.e., they can develop action potentials in response to electrical stimulation) [1]. NaV channels constitute validated pharmacological molecular targets for a large panel of clinically used drugs, such as anti-arrhythmics, anti-convulsants, anesthetics, and analgesics [2]. Six isoforms are highly sensitive (nanomolar range) to TTX (TTX-S): NaV1.1, 1.2, 1.3, 1.4, NaV1.6, and NaV1.7 and reciprocally three isoforms are much less sensitive and called resistant to TTX (TTX-R): NaV1.5, 1.8, and NaV1.9 [2,3] These α-subunits are complex pore-forming and glycosylated membrane proteins containing all molecular determinants needed to form a rapid inactivating voltage-gated channel, highly selective to Na+ [5]. They are associated with one or two auxiliary β-subunits (NaVβ1–4), encoded by four different genes. These β-subunits play a chaperon, gating, and regulatory role for NaV channels and belong to the cell adhesion molecule family [6]

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Conclusion