Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.
Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.
Volynski
K E
KE
Biochemistry Department, Imperial College, London, SW7 2AY, United Kingdom.
Meunier
F A
FA
Lelianova
V G
VG
Dudina
E E
EE
Volkova
T M
TM
Rahman
M A
MA
Manser
C
C
Grishin
E V
EV
Dolly
J O
JO
Ashley
R H
RH
Ushkaryov
Y A
YA
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal; D002462:Cell Membrane / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000502:physiology*; D018000:Receptors, Peptide / Q000502:physiology*; D013111:Spider Venoms / Q000378:metabolism*
D015816:Cell Adhesion Molecules, Neuronal; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; C020763:alpha-latrotoxin; D002118:Calcium