The rich diversity of neurons with distinct signalling capability reflects cellular differences in the expression of ion channel types, their distribution and their modulation. This biological complexity can be dissected by mutations of the genes involved. By using a collection of Drosophila mutants, the proposed experiments examine the distribution of different K+ channels and their roles in subcellular regions of the neuron. In addition, we will study the genetic control of expression, assembly and modulation of K+ channels. Electrophysiological studies comparing different cellular regions have been hampered by the small size of Drosophila neurons. We recently developed a culture system of """"""""giant"""""""" Drosophila neurons differentiated from cell division-arrested neuroblasts. The cells exhibit the various ion currents and thickened neurites, permitting patch-clamp recordings from cell bodies, neurites and terminals. It is also possible to correlate voltage-clamp and current-clamp data from the same neurons in this new system. Individual K+ currents can be eliminated to examine how they shape action potentials and firing patterns by using drugs or mutations, such as Sh (Shaker) and slo (slowpoke) that separately affect two different K+ channels. The Sh gene produces multiple messages by alternative exon splicing. The in situ expression of different Sh cDNAs will be studied in transgenic flies. The expressed Sh channels will be characterized in larval muscles, which permits precise voltage-clamp measurements. Interactions between the product of the transformed cDNA and that of the native Sh gene in the transformant will provide clues to the channel subunit assembly. Mutations of the dunce (dnc) and rutabaga (rut) genes separately affect phosphodiesterase II and adenylate cyclase, leading to altered cAMP levels and learning deficiencies. These mutants offer a unique opportunity to study cAMP-dependent channel modulation to complement the current information based on pharmacological studies. The possibility that another gene ether a go-go (eag) is involved in channel modulation will be examined in larval muscle fibers. Its unusual allele-dependent effect on different K+ currents will be correlated with recombinant DNA analysis of the structural alterations. Analysis of K+ currents in double-mutant combinations of eag, Sh and dnc will yield information about how the eag product affects Sh channels and whether it is mediated by the cAMP pathway.
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