Intraterminal Ca2+ and Spontaneous Transmitter Release at the Frog Neuromuscular Junction

Abstract

We investigated the relationship between intraterminal Ca2+concentration ([Ca2+]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 [Ca2+]i and MEPP frequency. Loading terminals with the fast and slow Ca2+-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 [Ca2+]i dependence of MEPP frequency determined by elevation of [Ca2+]i due to application of 0.1–10 μM ionomycin was similar to that determined when [Ca2+]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, 4α-PMA, did not. PMA application also caused an increase in [Ca2+]i. The relationship between [Ca2+]i and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca2+]i. Under all conditions tested, our data revealed a low [Ca2+]i threshold for activation of transmitter release and are consistent with a K d for [Ca2+]i on the order of 1 μM.