Alpha-latrotoxin modulates the secretory machinery via receptor-mediated activation of protein kinase C.
The hypothesis whether alpha-latrotoxin (LTX) could directly regulate the secretory machinery was tested in pancreatic beta cells using combined techniques of membrane capacitance (Cm) measurement and Ca2+ uncaging. Employing ramp increase in [Ca2+]i to stimulate exocytosis, we found that LTX lowers the Ca2+ threshold required for exocytosis without affecting the size of the readily releasable pool (RRP). The burst component of exocytosis in response to step-like [Ca2+]i increase generated by flash photolysis of caged Ca2+ was also speeded up by LTX treatment. LTX increased the maximum rate of exocytosis compared with control responses with similar postflash [Ca2+]i and shifted the Ca2+ dependence of the exocytotic machinery toward lower Ca2+ concentrations. LTXN4C, a LTX mutant which cannot form membrane pores or penetrate through the plasma membrane but has similar affinity for the receptors as the wild-type LTX, mimicked the effect of LTX. Moreover, the effects of both LTX and LTXN4C) were independent of intracellular or extracellular Ca2+ but required extracellular Mg2+. Our data propose that LTX, by binding to the membrane receptors, sensitizes the fusion machinery to Ca2+ and, hence, may permit release at low [Ca2+]i level. This sensitization is mediated by activation of protein kinase C.
Liu
Jie
J
Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074. P. R. China.
Wan
Qunfang
Q
Lin
Xianguang
X
Zhu
Hongliang
H
Volynski
Kirill
K
Ushkaryov
Yuri
Y
Xu
Tao
T
D000818:Animals; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D025802:Electric Capacitance; D004789:Enzyme Activation; D005089:Exocytosis / Q000187:drug effects; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D018408:Patch-Clamp Techniques; D010782:Photolysis; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018000:Receptors, Peptide / Q000378:metabolism*; D013111:Spider Venoms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; C020763:alpha-latrotoxin; D011493:Protein Kinase C; D002118:Calcium