SECONDARY STRUCTURE OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR - IMPLICATIONS FOR STRUCTURAL MODELS OF A LIGAND-GATED ION-CHANNEL

The secondary structure and effects of two ligands, carbamylcholine an d tetracaine, on the secondary structure of affinity-purified nicotini c acetylcholine receptor (nAChR) from Torpedo has been studied using F ourier transform infrared spectroscopy (FTIR). FTIR spectra of the nAC hR were acquired in both (H2O)-H-1 and (H2O)-H-2 buffer and exhibit sp ectral features indicative of a substantial alpha-helical content with lesser amounts of beta-sheet and random coil structures. The resoluti on enhancement techniques of Fourier self-deconvolution and Fourier de rivation reveal seven component bands contributing to both the amide I band and amide I' band contours in (H2O)-H-1 and (H2O)-H-2, respectiv ely. Curve-fitting estimates of the nAChR secondary structure are cons istent with the qualitative analysis of the FTIR spectra as follows: 3 9% alpha-helix, 35% beta-sheet, 6% turn, and 20% random coil. Of parti cular interest is the estimated alpha-helical content as this value pl aces restrictions on models of the nAChR transmembrane topology and on the types of secondary structures that may contribute to functional d omains, such as the ligand-binding site. The estimated alpha-helical c ontent is sufficient to account for four transmembrane alpha-helices i n each nAChR subunit as well as a substantial portion of the extracell ular and/or the cytoplasmic domains. FTIR spectra were also acquired i n the presence and absence of 1 mM carbamylcholine and 5 mM tetracaine to examine the effects of ligand binding on the secondary structure o f the nAChR. The similarity of the spectra, even after spectral deconv olution, indicates that the secondary structure of the nAChR is essent ially unaffected by desensitization. The FTIR data are discussed in te rms of structural models of the nAChR and of nAChR desensitization.