This project is a continuation of studies using a combination of molecular biology and electrophysiology in order to understand how ion channel structure determines function. It will focus on two different ion channels, the nicotinic acetylcholine receptor (AChR), which is the """"""""prototypical"""""""" ligand-gated channel, and the Shaker (SH) K+ channel of Drosophila, which is likely to serve a similar role for voltage gated channels. In vitro transcription of cloned cDNAs for the various channel subunits is used to produce large quantities of subunit-specific mRNAs, and the appropriate mixture of RNAs is injected into Xenopus oocytes, which then produce functional channels coded for by those RNAs. Factors that govern channel assembly such as transcript availability and subunit RNA stoichiometry can be manipulated by varying the composition of the RNA mixture that is injected into the oocyte. Site-directed mutagenesis can be used to introduce specific, predefined amino acid changes in the channel, and the structural domains involved in various functions can be mapped out through comparison of the electrophysiological and biochemical properties of the mutant channel with the wild-type. Regions of particular interest in the AChR are the putative ACh-binding domain on the alpha subunit, the sites of phosphorylation by second messenger-activated protein kinases (which are thought to influence the rate of receptor desensitization), and the sites of N-linked protein glycosylation (which may influence receptor assembly through effects on protein targeting and subunit stability, as well as receptor function). The experiments on Sh K+ channels deal with the subunit composition of the channel (monomer vs multimer; homo- vs hetero- oligomer). In addition, site-directed mutagenesis will be used to alter the charge distribution located at the putative """"""""mouth"""""""" of the channel to determine the environment that a K+ ion encounters as it enters and exits the channel. In addition, we will alter the charge distribution of the putative voltage sensor of the Sh channel to study the nature of the voltage-dependent gating.. It is expected that these studies on representatives of both classes of ion channels will help us to map out the structural features of ion channels involved in gating, ion channels will help us to map out the structural features of ion channels involved in gating, ion transport, and assembly. It is further expected that some of the structural features relevant to the assembly and function of these two channels will be common to other ion channels, and thus provide insight on the molecular basis of normal and abnormal cellular electrical activity.
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