α-Latrotoxin induces exocytosis by inhibition of voltage-dependent K + channels and by stimulation of L-type Ca 2+ channels via latrophilin in β-cells

Abstract

The spider venom α-latrotoxin (α-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 β-cells, which express endogenously the α-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 β-cells, which lack endogenous LPH. α-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, α-LTX first induced membrane depolarization by inhibition of repolarizing K + channels followed by the appearance of Ca 2+ transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca 2+] i) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTX N4C, which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K + channels via phospholipase C, activated L-type Ca 2+ channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca 2+] i in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca 2+ channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, α-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K + and Ca 2+ channels as novel targets of its secretory activity. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.