This project concerns the membrane proteins -- receptors and ion channels -- that underlie cellular excitability. In the next project period, VV will be exploited as a new system for the heterologous expression of these proteins in the membrane of mammalian and avian cells. 1. Vaccinia affords a new system for studying the 7-helix receptor-G protein interactions and the ion channel pathway, which employs direct coupling rather than soluble second messengers. Experiments will therefore work toward a complete reconstitution of this pathway. As a first step, the serotonin 5-HT-1A receptor will be expressed in primary cultures of cardiac atrial cells (chicks or guinea-pigs). It is expected that it will couple to the existing G protein(s), which in turn will activate K+ channels. Quantitative studies will follow. 2. It is of interest to understand the molecular basis for the diversity of voltage-dependent Ca++ channels from rat brain. Efforts will be continued to construct full-length clones for four classes of putative voltage-dependent Ca++ channels from rat brain. These clones will then be expressed in mammalian cell lines using VV and electrophysiological testing. Site-directed mutagenesis will follow. 3. Milligram quantities of channel proteins will be expressed and purified for structural studies.
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