Mechanism of alpha-latrotoxin action as revealed by patch-clamp experiments on Xenopus oocytes injected with rat brain messenger RNA.

ABSTRACT

Single-channel currents produced by alpha-latrotoxin from the black widow spider venom were recorded on Xenopus oocytes injected with rat brain messenger RNA fraction of 7-8 kb. Single-channel conductance varied from 3 pS to 200 pS and sublevels of similar conductance were observed at both normal and high external concentration of Ca2+. Currents reversed at 0 mV, and the channels were permeable to Ca2+, Na+ and K+ indicating non-selective cation channel produced by the toxin. Ca2+ stabilized the channel mainly at one conducting sublevel. Studies of channel kinetics indicated that openings co-operated into groups of bursts. Within these groups the histograms of closed and open times showed two exponentials with mean times near 1.5 ms and 20 ms for the closed time histogram and 85 ms and 300 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 with hyperpolarization. Elevation of external Ca2+ or toxin concentration promoted the appearance of groups of burst openings while within these 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 action has been proposed. The data strongly suggest that alpha-latrotoxin molecules are cation channels associated into clusters that insert into the membrane after binding to the receptor located at active zones of synaptic transmission. Binding and synchronization of channel openings in a cluster are promoted by Ca2+. Influx of Ca2+ through this near permanently open cation channel seems to induce intensive synaptic vesicle fusion and massive neurotransmitter release.