PROBING THE STRUCTURE-FUNCTION RELATIONSHIP OF ALPHA-LATROTOXIN-FORMED CHANNELS WITH ANTIBODIES AND PRONASE

The major toxic component of black widow spider (Latrodectus mactans t redecimguttatus) venom, alpha-latrotoxin, is known to form ionic chann els in different membranes, In order to probe the extramembrane domain s of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar li pid membrane were treated with antibodies to latrotoxin or with pronas e added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conduc tance only at positive potentials across the membrane. In contrast, tr ans side addition of antibodies changed the channel conductance at bot h positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the ini tial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and a trans applicati on of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, L i+ ions did not change significantly but Cd2+ block was decreased. Tra ns pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings a s evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depend s on membrane potential. Copyright (C) 1996 Elsevier Science Ltd