Latrophilin, neurexin, and their signaling-deficient mutants facilitate α-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 Ca2+ 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 tin the presence of Ca2+). Interestingly, on subsequent removal of Ca2+, 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) c 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.