Voltage-dependent Ca2+ channels are vital for regulated Ca2+ entry and Ca2+ signaling in neurons. Functional studies have uncovered several types of Ca2+ channels, including L-, N-, T- and P- type, distinguished by pharmacology, electrophysiology and neuronal distribution. More recently, molecular cloning has revealed an even greater diversity arising from multiple genes and alternative splicing. The availability of cloned cDNAs for Ca2+ channel subunits opens up new approaches to molecular determinants of key Ca2+ channel features. Ca2+ channel selectivity for permanent and blocking ions. Mutagenesis experiments will probe the contributions of a hypothesized ring of glutamate residues within the pore, a putative outer ring of negatively charged residues, and other structural features. Changes in selectivity for Ca2+ and blocking cations, pore size, and ion-ion interactions will be tested. High-affinity binding of peptide toxins. omega-CTx-GVIA blocks N-type channels and omega-CTx-MVIIC is a potent blocker of BI channels highly expressed in cerebellum. What is the structural basis for the potent block? Is toxin specificity determined by residues near the outer mouth of the pore? Such questions will be addressed with chimeric constructs and point mutants. This may help further development of type-selective agents. G-proteins, gating modes and modulation of N-type channels. N-type Ca2+ channels display multiple patterns of gating (modes). Norepinephrine down-modulates Ca2+ channel entry by biasing against a mode with high open probability (p) and in favor of a low-p mode or no activity at all, modulation thought to involve G-protein. Experiments will explore what region(s) of the Ca2+ channel are required for G-protein signaling, whether the interaction is direct, and how neurotransmitter modulation, prepulse disinhibition, and spontaneous modal switching are altered by functional ablation of specific G-proteins. Interactions between Ca2+ channels and presynaptic membrane and vesicle membrane proteins. Antibodies to synaptotagmin (p65) or syntaxin (p35) are known to immunoprecipitate omega-CTx-GVIA binding sites (N-type Ca2+ channels). Do such interactions exist for other channel types? What are the molecular determinants of such interactions? What are the functional consequences of such interactions for transmitter release?