The long-term goal is to characterize the molecular basis of nicotinic acetylcholine receptor (AChR) function. The approach to be used in the proposed research involves the use of chemical modifications and site-directed mutagenesis to probe the role of free sulhydryl groups in the activation and inactivation phases of AChR-mediated ion channel function. The proposed experiments provide a unique opportunity for this laboratory to exploit the new molecular biology techniques for modifying protein structure while continuing to extend our ongoing chemical modification studies of receptor function. Specific goals of the proposed research include: 1). To identify the subunit(s) and peptide fragment(s) in purified ACHR from electric organ of the electric ray Torpedo californica that contain the sulfhydryl groups that are specifically labeled by hydrophobic maleimides, such as N-phenylmaleimide. 2). To alter the structure of the AChR by converting the DNA coding sequence for one or more of the sulfhydryl group(s) identified in (1) to a sequence for a different, non-reative amino acid (such as serine) using site-directed mutagenesis. The altered AChR will be expressed in Xenopus oocytes by injecting the oocytes with altered mRNA obtained using the SP6 system to produce the mRNA from AChR cDNA. 3). To characterize the effects of AChR sequence alterations on AChR function by measuring both the ligand binding and ion permeability control properties of the nicotinic AChR in the Xenopus oocytes. Voltage clamp techniques will be used to monitor the channel activity of both normal and altered AChR in the oocytes.
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