Simultaneous Measurement of Evoked Release and [Ca2+]i in a Crayfish Release Bouton Reveals High Affinity of Release to Ca2+

Abstract

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.