The long-term goal of this project is to understand how voltage-gated Ca2+ channels sense and decode electrical and molecular signals that regulate neurotransmitter release. Ca2+ channels are large (about 370 kDa) heteromultimeric proteins composed of alpha1, alpha2delta, and beta subunits that work in concert to control the amount of Ca2+ that enters a neuron in response to a given stimulus. The subunits form the Ca2+ channel pore (4 large transmembrane homology domains) and are encoded by at least ten genes that, based on structural, electrophysiological, and pharmacological differences, can be divided into three major sub-groups, Cav1, Cav2 and Cav3. Cav1 and Cav2 are high-voltage activating channels, whereas Cav3 channels activate at more negative membrane potentials. Cav1 genes express channels with L-type (long lasting) electrophysiological characteristics, Cav2 genes express P/Q, N, and R-type (intermediate lasting) channels, and Cav3 genes express T-type (transient) channels. The alpha1 subunits serve as targets for several classes of therapeutic agents, including antiarrhythmics (diltiazem, L-type antagonist) and analgesics (ziconotide, N-type antagonist from a marine snail), and for a host of peptide spider toxins (e.g., Aga IVA, P-type antagonist). Cav2 genes, which will be studied in this proposal, are expressed principally at synapses.The intracellular beta subunits, encoded by 4 distinct genes, interact with the alpha1 subunit at specific binding sites on between-homology-domain linker sequences. The beta subunits modulate Ca2+ channel expression levels, as well as the voltage dependence and kinetics of Ca2+ channel activation and inactivation. Our preliminary studies show that alternative splicing of the N-terminus of the beta4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating. They also show that splicing affects channel pharmacology (altered sensitivity to omegaCgTx GVIA) and responsiveness of alpha1 subunits to repetitive stimuli. Thus, understanding the molecular details of the events brought about by beta4 alternative splicing is essential for the development of analgesic drugs, and for furthering our understanding of the role that voltage-gated Ca2+ channels play in synaptic plasticity. To this end, our most remarkable preliminary result, obtained by using simple methods in structural genomics, is the discovery that the beta4 subunit and the synaptic scaffolding (MAGUK) protein, PSD-95, have evolved from a common ancestor. The two proteins share very similar predicted secondary structure, and with the crystal structure of PSD-95 now available, a number of beta4 subunit tertiary structure predictions can now be made. The objectives of this application are to confirm, using advanced NMR techniques, our tertiary structure predictions and to determine whether the well-characterized inter- and intramolecular interactions of PSD-95 have been conserved in beta4 subunits. Our hypothesis is that the beta4 subunit acts as a multi-modular docking site for a myriad of proteins, including calmodulin, kinase anchoring proteins, and PDZ domains, and serves as a director, transmitting molecular signals from inside the cell to the gating machinery of alpha1 subunits
