The goal of this project is renewal remains to isolate and define a membrane protein responsible for K+ channel activity in the plasma membrane of a mammalian cell. K+ channels are found widely in animal cells and are central participants in cellular excitability, allowing transmission of the action potential in nerve and muscle and the response of many cell types to a variety of neurohormonal agonists. The molecular basis and mechanism of K+ channel activity is unknown. This grant proposes the use of a cell line isolated and characterized by the P.I. that has a mutation affecting a single specific K+ channel and diffusional K+ pathway in the plasma membrane. It proposes to extend the functional characterization of this channel through the electrophysiological techniques of patch electrode recording and bilayer reconstitution. In addition, it proposes a method to allow the isolation of the channel protein mediating this transport activity. This relies upon recently developed innovations in recombinant DNA technology. Somatic cell hybridization and DNA-mediated gene transfer experiments have demonstrated the mutation to be dominant, selectable, and transferable. A cosmid genomic library has been formed from the mutant DNA and the mutant channel gene has been localized to a specific volume in this library through the iterative process of sib selection. In each step of the isolation procedure, the genetically conferred altered function of the K+ channel is tested by measuring the ability of the cells to survive in the subthreshold low K+ medium, by determining K+ fluxes, and with single channel recording. This isolated gene will serve as a probe which will allow the isolation of its message's cDNA and, from its sequence, the determination of the amino acid sequence of the mutant and parent transport proteins. This project is a necessary step in the isolation and characterization of the protein responsible for this important transport mechanism which has thus far eluded molecular definition. Since bilayer reconstitution and patch electrode records demonstrate the alteration of the K channel, an ultimate objective of this proposed line of work would be the study in bilayers, oocuytes, and transfected cells of channel proteins synthesized from isolated and specifically mutated genes, more completely defining the structure and function of these important physiological mechanisms and, it is hoped, further extending a characterization of their roles in physiological processes.
