Abstract
The effects of both neutral and anionic lipids on the structure of the nicotinic acetylcholine receptor (nAChR) have been probed using infrared difference spectroscopy. The difference between infrared spectra of the nAChR recorded using the attenuated total reflectance technique in the presence and absence of the neurotransmitter analog, carbamylcholine, exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon ligand binding and subsequent desensitization. This spectral map is essentially identical in difference spectra recorded from native, native alkaline-extracted, and affinity-purified nAChR reconstituted into either soybean asolectin or egg phosphatidylcholine membranes containing both neutral and anionic lipids. This result suggests both a similar structure of the nAChR and a similar resting to desensitized conformational change in each membrane environment. In contrast, difference spectra recorded from the nAChR reconstituted into egg phosphatidylcholine membranes lacking neutral and/or anionic lipids all exhibit an essentially identical pattern of band intensity variations, which is similar to the pattern of variations observed in difference spectra recorded in the continuous presence of the desensitizing local anesthetic, dibucaine. The difference spectra suggest that the main effect of both neutral and anionic lipids in a reconstituted egg phosphatidylcholine membrane is to help stabilize the nAChR in a resting conformation. In the absence of neutral and/or anionic lipids, the nAChR is converted into an alternate conformation that appears to be analogous to the local anesthetic-induced desensitized state. Significantly, the proportion of receptors found in the resting versus the putative desensitized state appears to be dependent upon the final lipid composition of the reconstituted membrane. A lipid-dependent modulation of the equilibrium between a channel-active resting and channel-inactive desensitized state may account for the modulations of nAChR activity that are observed in different lipid membranes.
Highlights
The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ATR, attenuated total reflectance; Carb, carbamylcholine; Chol, cholesterol; Dib, dibucaine; DOPA, dioleoylphosphatidic acid; DOPS, is a large integral membrane protein that has been studied extensively as a model of both neurotransmitter-gated ion channel structure/function and protein-lipid interactions [1, 2]
Relative to the difference between spectra recorded in the presence and absence of Carb from the nAChR in native membranes, those recorded from the nAChR reconstituted into EPC membranes either with or without a variety of neutral and/or anionic lipids all exhibit subtle difference band intensity variations that reflect lipid-induced structural changes in those regions of the nAChR that are involved in ligand binding and desensitization
The positive and negative bands observed in the difference between Fourier transform infrared (FTIR) spectra of the nAChR recorded in the presence and absence of Carb should reflect the structural changes induced in the nAChR upon the binding of Carb
Summary
The difference between infrared spectra of the nAChR recorded using the attenuated total reflectance technique in the presence and absence of the neurotransmitter analog, carbamylcholine, exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon ligand binding and subsequent desensitization This spectral map is essentially identical in difference spectra recorded from native, native alkaline-extracted, and affinity-purified nAChR reconstituted into either soybean asolectin or egg phosphatidylcholine membranes containing both neutral and anionic lipids. Relative to the difference between spectra recorded in the presence and absence of Carb from the nAChR in native membranes, those recorded from the nAChR reconstituted into EPC membranes either with or without a variety of neutral and/or anionic lipids all exhibit subtle difference band intensity variations that reflect lipid-induced structural changes in those regions of the nAChR that are involved in ligand binding and desensitization. A lipid-dependent modulation of the equilibrium between the channel competent resting and a putative channel-inactive desensitized state may account for the effects of a variety of different lipids on the ion flux activity of the nAChR
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