Secondary Structure of the Nicotinic Acetylcholine Receptor:Implications for Structural Models of a Ligand-Gated Ion Channel

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Abstract
The secondary structure and effects of two ligands, carbamylcholine and tetracaine, on the secondary structure of affinity-purified nicotinic acetylcholine receptor (nAChR) from Torpedo has been studied using Fourier transform infrared spectroscopy (FTIR). FTIR spectra of the nAChR were acquired in both 1H2O and 2H2O buffer and exhibit spectral features indicative of a substantial alpha-helical content with lesser amounts of beta-sheet and random coil structures. The resolution enhancement techniques of Fourier self-deconvolution and Fourier derivation reveal seven component bands contributing to both the amide I band and amide I' band contours in 1H2O and 2H2O, respectively. Curve-fitting estimates of the nAChR secondary structure are consistent with the qualitative analysis of the FTIR spectra as follows: 39% alpha-helix, 35% beta-sheet, 6% turn, and 20% random coil. Of particular interest is the estimated alpha-helical content as this value places restrictions on models of the nAChR transmembrane topology and on the types of secondary structures that may contribute to functional domains, such as the ligand-binding site. The estimated alpha-helical content is sufficient to account for four transmembrane alpha-helices in each nAChR subunit as well as a substantial portion of the extracellular and/or the cytoplasmic domains. FTIR spectra were also acquired in the presence and absence of 1 mM carbamylcholine and 5 mM tetracaine to examine the effects of ligand binding on the secondary structure of the nAChR. The similarity of the spectra, even after spectral deconvolution, indicates that the secondary structure of the nAChR is essentially unaffected by desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

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