The goals of this project are: (1) to elucidate the structure of the site(s) on neuronal nicotinic acetylcholine receptors (nAChRs) where zinc binds and potentiates receptor function, and (2) to elucidate the mechanism through which zinc potentiates receptor function. ? Nicotinic ligands are potentially useful as anxiolytics and analgesics, in the treatment of neurological disorders such as schizophrenia, Parkinson's disease, and Alzheimer's disease, and are also the sites at which nicotine exerts its psychoactive and addictive effects. Effective pharmacological intervention at neuronal nAChRs requires development of subtype selective ligands. Pursuit of this goal has traditionally been directed toward development of ligands that act at the acetylcholine (ACh) binding sites. We propose to pursue a new class of site on neuronal nAChRs, the site at which zinc potentiates these receptors. The ACh binding sites on neuronal nAChRs are at the interfaces between the extracellular domains of subunits. In many neuronal nAChRs, two agonist-binding sites are formed at interfaces between two pairs of dissimilar subunits, leaving three alternate interfaces with no involvement in ACh binding. We hypothesize that one or more of the alternate interfaces bind zinc, a modulator of neuronal nAChR function.
In aim 1, we will use site-directed mutagenesis and functional analysis to identify amino acid residues on neuronal nAChRs that coordinate zinc at the potentiation site.
In aim 2, we will explore the larger structure of the potentiation site using Substituted Cysteine Accessibility Mutagenesis. Information obtained in aims 1 and 2 will be used to refine a homology model of the extracellular domains of neuronal nAChRs.
In aim 3, we will determine whether zinc potentiates neuronal nAChRs through changes in the single channel open probability, the single channel current, and/or the number of channels. Changes in open probability will be characterized in terms of the underlying gating mechanism. ? ? ?
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