Intraterminal Ca(2+) and Spontaneous Transmitter Release at the Frog Neuromuscular Junction

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) 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+)](i) 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+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) 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+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.