The various types of neurons display vast diversity in signalling capability, reflecting differences in the expression, distribution and modulation of different ion channels. In Drosophila, a number of genes encoding channel subunits and components in the second messenger pathways have been well characterized molecularly, providing opportunities for in vivo studies of the genetic bases and functional significance of ion channel diversity and modulation. Each of the genes Sh, eag and slo produce multiple splicing products and code for variants of particular types of K+ channel subunits. A collection of mutations and molecular probes will be employed to study how the splicing variants of the Sh, eag, slo and Hk genes correspond to the various K+ currents, and how K+ currents of different properties contribute to the diverse excitability patterns. We have developed a culture system of """"""""giant"""""""" neurons that makes it possible to correlate voltage- and current-clamp data within individual mutant neurons, enabling direct demonstration of how perturbation of particular K+ channels leads to alterations of firing pattern. In addition expression of Sh splicing variants will be examined in neuronal cultures and larval muscle fibers from transgenic lines carrying different Sh cDNAs. K+ currents in larval muscle fibers have been well characterized and allow precise voltage-clamp measurements to detect interaction between the transgenic subunits, as well as those between the transgenic and the host. Such data will provide clues to the channel subunit assembly in different cell types. The mutants dnc, rut and ala, each affect a specific step in the cAMP and CaM kinase second messenger systems. They will be used in the study of channel modulation in both neurons and muscle fibers. The long-term effects of chronic mutational perturbation will be compared to the short- term effects of acute pharmacological actions on the second messenger systems to reveal regulatory mechanisms of channel function. Our previous mutational analysis suggests co-assembly of the eag subunits with different K+ channel subunits to mediate channel modulation. We will further examine its interactions with second messenger systems in double- mutant combinations of eag with dnc and ala and its interaction with specific Sh splicing variants in Sh transformants in the eag host background. Parallel studies in the Xenopus oocyte expression system will be made to seek independent lines of evidence.
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