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 affinity of the putative Ca²⁺ receptor(s) responsible for evoked release. Evoked, 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 [Ca²⁺](ss). Surprisingly, evoked release was also found to be highly correlated with [Ca²⁺](ss). The quantal content (m) and the rate of asynchronous release (S) showed sigmoidal dependence on [Ca²⁺](ss). The slope log m/log [Ca²⁺](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 independent of [Ca²⁺](o). These results are consistent with the assumption that evoked release is controlled by the sum of [Ca²⁺](ss) and the local elevation of Ca²⁺ concentration near the release sites resulting from Ca²⁺ influx 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 μM 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 μM. This value of K(d) is similar to that found previously for asynchronous release.