Calcium channels and calcium-gated potassium channels at the frog neuromuscular junction

Two mechanisms which regulate transmitter release by regulating Ca2+ entry in the presynaptic nerve terminal were studied at the frog neuromuscular junction (nmj). First, the location of Ca2+ channels in relation to transmitter release sites and, second, the regulation of Ca2+ entry by Ca(2+)-gated potassium (gKca) channels. Ca2+ channels were disclosed using fluorescent omega-conotoxin GVIA (omega-CgTX) which blocks transmitter release and Ca2+ entry at the frog nmj. Ca2+ channels were located in bands spaced at regular intervals of 1 micron. The omega-CgTX labeling was removed following mechanical displacement of the presynaptic terminal after collagenase digestion. The bands of omega-CgTX staining matched almost perfectly the staining of cholinergic receptors with fluorescent alpha-bungarotoxin (alpha-BuTX) and therefore must be located at the active zone. The role of gKca channels in the regulation of transmitter release was assessed using charybdotoxin (ChTX) which blocks gKca channels of large and intermediate conductances. Application of ChTX (2-20 nM) induced a two-fold increase in transmitter release which was prevented when a membrane permeant Ca2+ buffer (DMBAPTA-AM) was introduced prior to the toxin application. The Ca2+ buffer by itself caused a reduction in transmitter release. Nerve-evoked Ca2+ entry in the presynaptic terminal, detected with the fluorescent indicator fluo3, was increased following gKca channel blockade by ChTX. The Ca(2+)-gated K+ channels may function to limit the duration of the presynaptic action potential and thus limit Ca2+ entry.