Calcium-independent receptor for alpha-latrotoxin and neurexin 1alpha  [corrected] facilitate toxin-induced channel formation: evidence that channel formation results from tethering of toxin to membrane.
alpha-Latrotoxin binding to the calcium-independent receptor for alpha-latrotoxin (CIRL-1), a putative G-protein-coupled receptor, stimulates secretion from chromaffin and PC12 cells. Using patch clamp techniques and microspectrofluorimetry, we demonstrate that the interaction of alpha-latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca(2+). alpha-Latrotoxin interaction with CIRL-1 transiently expressed in bovine chromaffin cells produced a 400-pS channel, which rarely closed under Ca(2+)-free conditions. The major effect of overexpressing CIRL-1 was to greatly increase the sensitivity of chromaffin cells to channel formation by alpha-latrotoxin. alpha-Latrotoxin interaction with CIRL-1 transiently overexpressed in non-neuronal human embryonic kidney 293 (HEK293) cells produced channels that were nearly identical with those observed in chromaffin cells. Channel currents were reduced by millimolar Ca(2+). At alpha-latrotoxin concentrations below 500 pM, channel formation occurred many seconds after binding of toxin to CIRL-1 indicating distinct steps in channel formation. In all cases there was a rapid, sequential addition of channels once the first channel appeared. An analysis of CIRL-1 mutants indicated that channel formation in HEK293 cells is unlikely to be transduced by a G-protein-dependent mechanism. alpha-Latrotoxin interaction with a fusion construct composed of the extracellular domain of CIRL-1 anchored to the membrane by the transmembrane domain of vesicular stomatitis virus glycoprotein, and with neurexin 1alpha, an alpha-latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type CIRL-1. We propose that alpha-latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that alpha-latrotoxin itself controls the conductance properties of the channels it produces.
Hlubek
M D
MD
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Stuenkel
E L
EL
Krasnoperov
V G
VG
Petrenko
A G
AG
Holz
R W
RW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects*; D003600:Cytosol / Q000378:metabolism; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000378:metabolism; D017353:Gene Deletion; D006023:Glycoproteins; D006801:Humans; D008562:Membrane Glycoproteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D009479:Neuropeptides; D016716:PC12 Cells; D010957:Plasmids / Q000235:genetics; D051381:Rats; D018000:Receptors, Peptide / Q000378:metabolism*; D013111:Spider Venoms / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology
C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D018000:Receptors, Peptide; D013111:Spider Venoms; D014759:Viral Envelope Proteins; C034800:alpha-latrotoxin receptor; C112162:neurexophilin; C020763:alpha-latrotoxin; D019204:GTP-Binding Proteins; D002118:Calcium