The regulatory peptide called calcitonin gene-related peptide (CGRP) was detected by immunofluorescence in frog motor neurons and motor nerve terminals. In motor nerve terminals, CGRP-like immunoreactivity was found to be segregated within large dense-core vesicles. To determine whether exocytosis from acetylcholine-containing small synaptic vesicles and from CGRP-containing large dense-core vesicles can be independently stimulated, nerve-muscle preparations were exposed to alpha-latrotoxin. This toxin induced complete depletion of acetylcholine-containing small synaptic vesicles but did not induce a parallel depletion of CGRP-like immunoreactivity and of large dense-core vesicles. These effects were independent of the presence of extracellular Ca2+ and occurred both at room temperature and at low temperature (1-3 degrees C). These findings suggest that exocytosis from the two vesicle populations is mediated by distinct biochemical mechanisms, which might be differentially regulated by physiological stimuli.

The changes in ionic permeability induced by the application of alpha-latrotoxin to NG108-15 neuroblastoma x glioma cells were examined using the nystatin perforated-patch technique for whole-cell recording. Complex single channel activity appeared in the plasmalemmas after delays that ranged from 1-20 min in Krebs' solution. The conductance of a channel fluctuated among at least three broad, approximately equispaced bands, the maximum conductance being about 300 pS, and the reversal potential approximately 0 mV. The channels were permeable to Na+, K+, Ca2+ and Mg2+, poorly permeable to glucosamineH+ and Cl-, and were blocked by La3+. The channels stayed fully open in Ca(2+)-free solutions with 4 mM Mg2+, in solutions with no divalent cations and in solutions with 2 mM Ca2+ and 96 mM Mg2+. They opened infrequently if both internal and external Cl- were replaced by glutamate-. If alpha-latrotoxin opened similar channels in nerve terminals, the flux of ions through them could account for the massive release of neurotransmitter induced by the toxin.

Whole-cell voltage clamp and singie-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of Ina and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 tzM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KN~ channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the K~ channel was 50 pS. This conductance increased to 174 pS when the intra-and extracellular potassium concentrations were 75 and 150 mM, respectively.