α-Latrotoxin Induces Exocytosis by Inhibition of Voltage-dependent K+ Channels and by Stimulation of L-type Ca2+ Channels via Latrophilin in β-Cells

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 Ca2+ transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca2+]i) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTXN4C, 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 Ca2+ 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 [Ca2+]i in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca2+ 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 Ca2+ channels as novel targets of its secretory activity.