ISOLATION OF THE GENE ENCODING THE NA-K-C1 COTRANSPORTER

点击翻译按钮获取中文摘要

The goal of this project is 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 PI that has a mutation affecting a single specific diffusional K+ pathway in the plasma membrane. It proposes to extend the functional characterization of this pathway 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 hybrids formed using the mutant reveal the mutation to be dominant and selectable. Therefore the technique of DNA-mediated gene transfer, a technique which has been successfully applied a number of times in the past to the isolation of other dominant selectable genes, is proposed to isolate this mutant gene affecting channel function. In each step of the isolation procedure, the genetically conferred altered function of the K+ channel will be tested by measuring the ability of the cells to survive in the subthreshold low K+ medium from which the mutant was isolated. This isolated gene will serve as a probe which will allow the isolation of its mRNA 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 of K+ channels from membrane vesicles of the mutant has proven feasible, a long-term objective of this proposed line of work would be the study in bilayers of pure channel proteins synthesized in vitro from isolated and specifically mutated genes, more completely defining the structure and function of these important physiological mechanisms.