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 art ificial lipid bilayers or in the plasma membrane of non-secretory cells. Ho wever, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. S ignaling-deficient (truncated) mutants of latrophilin and latrophilin-neure xin hybrids also facilitate pore induction, which correlates with toxin bin ding 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 isothiocya nate and norepinephrine. Bound alpha -latrotoxin remains on the cell surfac e without penetrating completely into the cytosol. Higher temperatures faci litate insertion of the toxin into the plasma membrane, where it co-localiz es with latrophilin (under all conditions) and with neurexin tin the presen ce of Ca2+). Interestingly, on subsequent removal of Ca2+, alpha -latrotoxi n dissociates from neurexin but remains in the membrane and continues to fo rm pores. These receptor-independent pores are inhibited by anti-alpha -lat rotoxin antibodies. Our results indicate that (i) c alpha -latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha -latrotoxin facilitate i ts insertion into the membrane, (iii) the receptors are not physically invo lved in the pore structure, (iv) alpha -latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha -latrotoxi n interaction with other neuronal proteins.