Ca++ is a fundamental regulator of many essential neural functions and the plasma membrane of neural cells is preeminent among cellular sites for controlling internal Ca++ levels. The studies described in ths application are a comprehensive analysis of the Ca2+ regulatory mechanism operating in the neural plasma membrane. The approach is based on preliminary studies characterizing the isolation and properties of a preparation of synaptosomal plasma membrane vesicles. This methodology provides the unique capability of analyzing biochemically the transport and regulatory mechanisms that control Ca++ fluxes across the neural plasma membrane, and relating their activity ot the control of Ca++ in the intact neural cell. The studies on Ca++ regulation utilize two different neurological systems from which to derive plasma membrane vesicles: (1) synaptosomes isolated from rat brain cerebral cortex; (2) homogeneous cloned neural cell lines, selected for their specific receptor-mediated control propeties (neuroblastoma N1E-115 and neuroblastoma-glioma NG108-15 cell lines). Initial studies concern refinement of procedures for isolation of vesicles of high plasma membrane purity and defined sidedness, from each system, and precise characterization of the Ca++ flux mechanisms within them. Major studies concern analysis of the regulatory mechanisms which critically control the activity of the Ca++ fluxes across the plasma membrane. These involve both the direct receptor-mediated modulation of Ca++ transport (in particular, via the muscarinic receptor) and the important internal regulatory mechanisms exerted by intracellular regulators (cyclic AMP and calmodulin) through phosphorylation of specific plasma membrane regulatory proteins. The plasma membrane is a primary site for pharmacological control. The studies permit a direct examination of the effects of several important classes of neuroactive pharmacological agents (including anticonvulsants, narcotic analgesics, antipsychotics, and anesthetics) which are believed to exert their neurological effects through direct modification of plasma membrane Ca++ fluxes or their regulatory components. An in depth understanding of the mechanisms which control Ca++ entry and efflux, their physiological regulation, and their pharmacological modification, is essential to determining effective counteractive therapy for diseases either affecting chemical transmission (Parkinson's disease and myasthenia gravis) or those altering neural conduction (including multiple scelorosis).
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