Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction.

We investigated the relationship between intraterminal Ca(2+) concentration (Ca(2+)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in Ca(2+) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The Ca(2+) dependence of MEPP frequency determined by elevation of Ca(2+) due to application of 0.1-10 microM ionomycin was similar to that determined when Ca(2+) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in Ca(2+). The relationship between Ca(2+) and MEPP frequency in PMA was the same as was determined by the other methods of raising Ca(2+). Under all conditions tested, our data revealed a low Ca(2+) threshold for activation of transmitter release and are consistent with a K(d) for Ca(2+) on the order of 1 microM.