Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1

Abstract

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive α1E subunit is functionally less well characterized than the structurally related α1A (ω-agatoxin-IVA sensitive, P- /Q-type) and α1B (ω-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of α1E (α1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of α1Ee. In α1E-transfected human embryonic kidney (HEK-293) cells the α1E-selective peptide antagonist SNX-482 (100 nM) reduces α1Ed- and α1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits β3 and α2δ-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits α1G, α1H, and α1I showing that the toxin from the venom of Hysterocrates gigas is useful as an α1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 μM (+/-)-isradipine plus 0.5 μM ω-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 ± 1.2 pA/pF to -7.6 ± 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that α1E, in addition to L-type and non-L-type (α1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.