Simultaneous measurement of evoked release and [Ca2+](i) in a crayfish release bouton reveals high affinity of release to Ca2+

The opener neuromuscular junction of crayfish was used to determine the aff inity of the putative Ca2+ receptor(s) responsible for evoked release. Evok ed, asynchronous release, and steady-state intracellular Ca2+ concentration , [Ca2+](ss), were measured concomitantly in single release boutons. It was found that, as expected, asynchronous release is highly correlated with [C a2+](ss). Surprisingly, evoked release was also found to be highly correlat ed with [Ca2+](ss). The quantal content (m) and the rate of asynchronous re lease (S) showed sigmoidal dependence on [Ca2+](ss). The slope log m/log [C a2+](ss) varied between 1.6 and 3.3; the higher slope observed at the lower [Ca2+](o). The slope log S/log [Ca2+](ss) varied between 3 and 4 and was i ndependent of [Ca2+](o). These results are consistent with the assumption t hat evoked release is controlled by the sum of [Ca2+](ss) and the local ele vation of Ca2+ concentration near the release sites resulting from Ca2+ inf lux through voltage-gated Ca2+ channels (Y). On the basis of the above, we were able to estimate Y. We found Y to be significantly <10 mu M even for [ Ca2+](o) = 13.5 mM. The dissociation constant (K-d) of the Ca2+ receptor(s) associated with evoked release was calculated to be in the range of 4-5 mu M. This value of K-d is similar to that found previously for asynchronous release.