STRUCTURE/FUNCTION ANALYSIS OF VOLTAGE-GATED CA CHANNELS

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Voltage grated Ca2+ channels control entry of Ca2+ into cells under physiologic and pathologic conditions. They are targets of a major class of hypertension controlling drugs, they are targets of autoimmune reactions that lead to cell death as may happen in rare diseases such as amyotrophic lateral sclerosis as well as in more frequent diseases such as type I diabetes. An understanding of their diversity, their complexity and their functioning is of central importance. The aim of the research is to relate structural elements of the alpha1 and beta subunits of Ca2+ channels to their functional properties. The basic tools to be used are the construction via recombinant DNA techniques of mutant and chimeric molecules and the measurement of their interaction and properties upon expression in Xenopus oocytes. The expectation that positive information will be obtained through these manipulations is based on 1. the acquisition through molecular cloning on our part of two homologous alpha1 cDNA's, type C and type E, that have distinguishing electrophysiological and pharmacological properties and can be expressed well in Xenopus oocytes; 2. the acquisition through molecular cloning on our part of several beta subunit cDNA's that can also be expressed in Xenopus oocytes and of which some impart distinguishing properties to the alpha1 subunits when co-expressed in Xenopus oocytes; and 3. the association through collaboration of the PI with Dr. Enrico Stefani, who will carry out a thorough and insightful analysis of the electrophysiological properties of the constructed molecules. Specific aims center on the elucidation through construction of chimeras of the major structure-function relations for alpha1 in an attempt to determine those structures required for Ca2+ inactivation of some but not others, for voltage inactivation, for regulation by phosphorylation and for key pharmacologic properties. The application is submitted as part of an Investigator-initiated Research Project Grant (IRPG). The companion grant is Dr. Stefani's type II renewal application AR-38970. Results from these studies may contribute to the design of better therapeutic agents.