Alpha-latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in beta-cells.
The spider venom alpha-latrotoxin (alpha-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 beta-cells, which express endogenously the alpha-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. alpha-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, alpha-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, alpha-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.
Lajus
Sophie
S
Institut Européen de Chimie et Biologie, JE 2390 and INSERM E347, 2 rue Robert Escarpit, 33607 Pessac/Bordeaux, France.
Vacher
Pierre
P
Huber
Denise
D
Dubois
Mathilde
M
Benassy
Marie-Noëlle
MN
Ushkaryov
Yuri
Y
Lang
Jochen
J
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism*; D051381:Rats; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013111:Spider Venoms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D020746:Calcium Channels, L-Type; D010455:Peptides; D024642:Potassium Channels, Voltage-Gated; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; C020763:alpha-latrotoxin; C064719:iberiotoxin; D010738:Type C Phospholipases; D002118:Calcium