CELLULAR REGULATION OF POTASSIUM CHANNELS

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Voltage-dependent potassium channels play a central role in setting the pattern of spontaneous firing and the shape of action potentials of muscle cells and neurons. Alterations in the level of expression and in the electrical characteristics of potassium channels occur in response to hormones, as well as to drugs that influence second messenger pathways linked to protein kinases. This project will investigate the mechanisms that regulated two different potassium channels whose genes have been isolated and have been termed the minK gene and the Kvl gene. Both genes are expressed in heart cells as well as in other tissues. MinK RNA, when injected into Xenopus oocytes, generates slowly activating potassium currents whose amplitude is greatly enhanced by activation of the cyclic AMP-dependent protein kinase. Electrical recordings and biochemical measurements on normal and mutant minK channels expressed in heterologous cells will then be carried out to determine whether the channel is phosphorylated directly and whether modulation of channel activity occurs through the rapid recruitment of new channels to the plasma membrane. Mammalian cells lines that normally express minK RNA and protein will be identified and the physiological role and regulation of the channel will be investigated in such cells. A second series of experiments will be carried out with Kvl, a mammalian member of the Shaker family of proteins. Although a number of channel genes belonging to this family have been identified, no gene has yet been shown to account for a specific component of potassium current in a normal cell. Hybrid arrest techniques will therefore be used to determine whether the Kvl protein contributes to the voltage-dependent potassium current in GH3 cells, a clonal pituitary cell line that expresses Kvl mRNA at high levels. Electrophysiological measurements, coupled with biochemical studies of the Kvl protein identified with Kvl-specific antisera, will then be used to study the short-term and long-term regulation of the Kvl channel by hormones and second messengers.