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
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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