The activity of voltage-sensitive calcium channels is vital to the normal function nerve cells; Ca entry through these channels underlies bursting patterns, neurotransmitter release, neurite growth, and changes in protein phosphorylation. The functional properties several types of neuronal Ca channels have been described, but our understanding of their structures and of the molecular interactions responsible for channel activation, inactivation and modulation remains primitive' This proposal describes studies of neuronal Ca channnels isolated from their complex cellular environment. First, Ca channels from synaptosomes will reconstituted into planar bilayers and other simple membrane systems where their functional properties can be examined. Second, neuronal Ca channels will be solubilized and partially purified in order to study their protein structures. Reconstitution experiments will help answer some fundamental questions. Do the several types of neuronal Ca channels share key elements of ion selectivity and permeation cardiac Ca channels? Are the ion permeation pathways functionally symmetric like cardiac channels? What is the molecular mechanism of Ca-dependent inactivation of L-type channels? Can direct evidence for the role of cAMP-dependent phosphorylation in maintaining active Ca channels be obtained in the cell-free system? Can Ca channels in artificial membranes be modulated by phosphorylation or by direct interactions with purified G-proteins? These questions can be answered with reconstituted systems because of the easy experimental access to the internal side of the channel. The solubilization and isolation of Ca channels (as identified by the binding of a specific peptide neurotoxin w-conotoxin VIA) will provide information about the physical structure neuronal Ca channels. What is the peptide composition? How are neuronal w-conotoxin sensitive Ca channels different from skeletal muscle dihydropyridine-sensitive Ca channels? Are there immunological similarities? Are there important differences in carbohydrate composition? Is there evidence for direct interactions of modulatory proteins with the channels molecules. Are interactions with cell matrix proteins important for the localization and clustering of channels in the presynaptic membrane? Are w-conotoxin binding proteins functional, channels when they are reincorporated into membranes?
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