The role of protein dephosphorylation as mediated by Ca2+/calmodulin-dependent protein phosphatase (calcineurin), in the regulation of neurotransmitter release from nerve terminals in mammalian brain will be studied. The proposed studies will offer insight into Ca2+-regulated processes at the synapse, contributing to the long-term objective of describing the molecular mechanisms involved in neurosecretion. Specifically, the physiological and pharmacological regulation of calcineurin in isolated brain nerve terminals will be examined. These studies will examine the Ca2+- dependent dephosphorylation of exogenous and endogenous protein phosphosubstrates in isolated brain nerve terminal fractions. Exogenous substrate dephosphorylation will be measured to assess the extent to which calcineurin in nerve terminals can be activated by Ca2+ and calmodulin.Endogenous substrates for Ca2+-dependent dephosphorylation will be studied in intact terminals after standard radiolabeling of endogenous ATP, focusing on two phosphoproteins termed P96 and P139, proteins known to dramatically dephosphorylate upon depolarization-induced Ca2+ entry. Physiological regulation of these dephosphorylation events will be explored by comparing the kinetics of depolarization-induced changes in intraterminal Ca2+ levels with the kinetics of calcineurin activation, and by determining the nature and source of the Ca2+ changes essential for dephosphorylation (e.g., Ca channels, endoplasmic reticulum, Na/Ca antiporter). Pharmacological regulation will be studied using a variety of agents known to change intraterminal Ca2+ levels (e.g. carbachol). The function of calcineurin in neurotransmitter uptake and release will be investigated by separately introducing activated calcineurin, inhibitory synthetic peptides (derived from the regulatory region of calcineurin), and antibodies to calcineurin into isolated nerve terminals, using transient freeze/thaw permeabilization, and examining subsequent effects of neurotransmitter uptake and release. Purification and characterization of the endogenous protein substrates P96 and P139 will be undertaken. Utilizing antibodies raised against the purified proteins and/or partial knowledge of their amino acid sequences, isolation of the cDNAs encoding P96 and P139 from rat brain cDNA libraries will be attempted. The roles that P96 and P139 play in nerve terminal function will be addressed by separately introducing these protein substrates and antibodies against these proteins into isolated terminals, and assessing subsequent effects on neurotransmitter uptake and release.Because calcineurin is differentially distributed throughout brain, being predominantly in corpus striatum and hippocampus, determining the role of calcineurin in nerve terminals may provide new insights into altered function in neuropathological disease states involving these brain regions.
| Nichols, R A; Suplick, G R; Brown, J M (1994) Calcineurin-mediated protein dephosphorylation in brain nerve terminals regulates the release of glutamate. J Biol Chem 269:23817-23 |
