MECHANISM OF ALPHA-LATROTOXIN ACTION AS REVEALED BY PATCH-CLAMP EXPERIMENTS ON XENOPUS-OOCYTES INJECTED WITH RAT-BRAIN MESSENGER-RNA

Single-channel currents produced by alpha-latrotoxin from the black wi dow spider venom were recorded on Xenopus oocytes injected with rat br ain messenger RNA fraction of 7-8 kb. Single-channel conductance varie d from 3 pS to 200 pS and sublevels of similar conductance were observ ed at both normal and high external concentration of Ca2+. Currents re versed at 0 mV, and the channels were permeable to Ca2+, Na+ and K+ in dicating non-selective cation channel produced by the toxin. Ca2+ stab ilized the channel mainly at one conducting sublevel. Studies of chann el kinetics indicated that openings co-operated into groups of bursts. Within these groups the histograms of closed and open times showed tw o exponentials with mean times near 1.5 ms and 20 ms for the closed ti me histogram and 85 ms and 3000 ms for the open time histogram at -40 mV. Open times increased with membrane hyperpolarization while closed times did not. Open probability was near 0.8 and slightly increased wi th hyperpolarization. Elevation of external Ca2+ or toxin concentratio n promoted the appearance of groups of burst openings while within the se groups, the single-channel conductance, the reversal potential and channel kinetics did not depend on Ca2+ or toxin concentration. On the basis of the experimental results, the kinetic mechanism of toxin act ion has been proposed. The data strongly suggest that alpha-latrotoxin molecules are cation channels associated into clusters that insert in to the membrane after binding to the receptor located at active zones of synaptic transmission. Binding and synchronization of channel openi ngs in a cluster are promoted by Ca2+. Influx of Ca2+ through this nea r permanently open cation channel seems to induce intensive synaptic v esicle fusion and massive neurotransmitter release.