Our long term aim is to elucidate the molecular mechanisms of neural signaling by characterization of mutations in Drosophila that affect ion channels. We discovered a new, evolutionarily conserved family of voltage-activated K+ channels consisting of three subtypes (Eag, Elk, and Erg) encoded by the genes eag, elk, and sei. A primary goal of the present application is to characterize the physiological properties of these channels and to elucidate their in vivo functions, using a combination of genetic, molecular, and electrophysiological techniques. We will relate the physiological roles of these channels with the molecular structure and function of the corresponding polypeptides by in vivo electrophysiological studies of eag and sei mutants, immunohistochemical analyses of the distribution of the channels, and biophysical characterization of wildtype and mutant channels expressed in Xenopus oocytes. We will extend these analyses to Elk channels as well by isolation and characterization of elk mutations. New genes that regulate the expression, localization, or function of Erg channels will be sought by screening for enhancers of sei. Such mutations can pave the way for molecular dissection of these regulatory mechanisms, as our work on Hk has shown. We discovered that the Hk locus encodes a K+ channel Beta subunit that coassembles with, and affects the functional properties of, pore-forming subunits. We also found that Hk is a member of a family of NADPH-utilizing oxidoreductases. To further elucidate the role of Beta subunits in regulating the diversity and function of K+ channels in vivo, we will perform molecular and electrophysiological experiments to describe the expression, localization, and properties of Hk splice variants. The possibility that Hk retains an essential enzymatic activity will be tested by characterizing the electrophysiological effects of site-directed mutations in vivo and in Xenopus oocytes. Because Drosophila is the only organism in which in vivo studies of K+ channel mutations can be readily combined with in vitro manipulations and functional analyses in heterologous systems, these studies will yield novel insights on K+ channel structure, function, expression, regulation, diversity, and evolution. Since K+ channels are essential for neural function in all higher organisms and mutations of K+ channels (including members of the Eag family) cause genetic disease in humans, results from our studies should continue to have broad biological and medical significance.
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