title: Latrophilin, neurexin, and their signaling-deficient mutants facilitate alpha -latrotoxin insertion into membranes but are not involved in pore formation.
abstract: 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.
authors.affiliation: Biochemistry Department, Imperial College, London, SW7 2AY, United Kingdom.
authors.forename: K E
authors.initials: KE
authors.lastname: Volynski
authors.forename: F A
authors.initials: FA
authors.lastname: Meunier
authors.forename: V G
authors.initials: VG
authors.lastname: Lelianova
authors.forename: E E
authors.initials: EE
authors.lastname: Dudina
authors.forename: T M
authors.initials: TM
authors.lastname: Volkova
authors.forename: M A
authors.initials: MA
authors.lastname: Rahman
authors.forename: C
authors.initials: C
authors.lastname: Manser
authors.forename: E V
authors.initials: EV
authors.lastname: Grishin
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
authors.forename: R H
authors.initials: RH
authors.lastname: Ashley
authors.forename: Y A
authors.initials: YA
authors.lastname: Ushkaryov
mesh_terms: 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*
chemical_list: 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