NEUROGENETICS OF MEMBRANE EXCITABILITY

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Ion channels play essential roles in cellular communication and signal transduction in most cell types. K+ channels comprise a large and diverse group of proteins that largely determine action potential waveform, firing pattern, and amount and duration of transmitter release in excitable cells. They are also central to cellular mechanisms of learning and memory. Despite important recent advances in the molecular analysis of K+ channels in the Shaker (Sh) family, many unanswered questions remain concerning the structure, function and regulation of other K+ channels. The long term aim of this work is to address these questions by isolating and characterizing Drosophila mutations affecting genes encoding K+ channels or that otherwise affect their function. Our recent molecular analysis of the eag locus showed that it encodes a novel K+ channel polypeptide with significant similarity both to K+ channels in the Sh family,and to cyclic nucleotide-gated cation channels. We identified a second member of the eag family, elk, whose encoded polypeptide is 45% identical with eag. To elucidate the in vivo functions of these and related genes and to characterize the functional properties of the channels they encode we propose to: determine when and where these genes are expressed; identify the relevant upstream sequences for use in germline transformation and in vivo functional assays of wildtype and site-directed mutant constructs; map the mutant sites in a collection of in vivo-generated eag point mutations; screen for additional family members by use of PCR; screen for and characterize mutations of elk; and characterize the basic properties of mutant and wildtype eag and elk channels expressed in oocytes alone or in combination with Sh family members or each other. We found that Hk mutations also affect the function or regulation of K+ channels, cloned genomic DNA from this locus, and identified two sets of incomplete candidate cDNAs. To identify with certainty the polypeptide encoded by Hk and understand its function in vivo we propose to: define the limits of the gene by transformation rescue experiments; isolate, map, and sequence additional cDNAs to determine the complete Hk open reading frame; and infer possible functions from sequence data, expression experiments in oocytes and immunolocalization studies. Finally, we found that a particular lethal allele of a Na+ channel-structural gene is rescued in double mutant combinations with known K+ channel mutations. We propose to take advantage of this to develop a selective screen to identify new mutations affecting K+ channels. Because Drosophila is the only organism in which in situ mutational analysis of K+ channels can be combined with site-directed mutagenesis and functional expression in heterologous systems, these studies will enable us to derive novel information about K+ channel structure, function, and regulation. Various human disorders are known to be caused by perturbations in ion channels. Further, K+ channels are highly conserved between mammals and flies and are involved in a wide array of cellular functions in all organisms. Thus, these studies will have broad biological and medical significance.