Localized Ca2+ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release.

Abstract

The release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes) was found to be tightly coupled to the entry of Ca2+ through voltage-dependent Ca2+ channels, but is relatively unresponsive to "bulk" increases in cytosolic Ca2+ concentrations ([Ca2+]c) effected by Ca2+ ionophore. Under the same conditions, this dependence on Ca2+ influx, specifically through Ca2+ channels, was also seen for the dephosphorylation of a 96-kDa protein, (P96), present in the nerve terminals, as well as the phosphorylation of proteins migrating at 75 kDa (P75), corresponding to the synapsins, a group of well characterized synaptic vesicle-associated proteins. P96 dephosphorylation, following Ca2+ influx, was persistent and insensitive to the phosphatase inhibitor okadaic acid, suggesting a phosphatase other than protein phosphatase 1 and 2A as being responsible. Perhaps through the same phosphatase activity the increase in P75 phosphorylation was rapidly reversed with a time course similar to P96 dephosphorylation. When release, P96 dephosphorylation, and P75 phosphorylation were considered as functions of the [Ca2+]c increases achieved by depolarization and Ca2+ ionophore, there was no correlation of any of these with the overall concentration of Ca2+ in the cytosol. Since the fura-2 method used to measure [Ca2+] gives an averaged [Ca2+]c, these results imply that the release and protein dephosphorylation events are functionally coupled to local [Ca2+]c, in the immediate vicinity of Ca2+ channels. The reported clustering of the latter at the active zone area of the synapse and the parallelism between synaptic vesicle exocytosis and the phosphorylation of synaptic vesicle-associated proteins (p75:synapsins Ia/Ib), suggests that P96 may be similarly localized at the active zone area and, therefore, may be of significance in a modulatory role in glutamate release.