Dissecting the Chemistry of Nicotinic Receptor-Ligand Interactions with Infrared Difference Spectroscopy

Abstract

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that inter

Lipid Composition Alters Drug Action at the Nicotinic Acetylcholine Receptor

“…The intensities and frequencies of these two bands are essentially identical to those of the two bands in Carb minus tetramethylamine difference spectra (Ryan et al, 2002), where the vibrations of the two bands can also been seen clearly because of the absence of overlapping amide I vibrations. As noted, the 1690 and 1668 cm Ϫ1 bands reflect a vibrational shift in an as yet unidentified residue that interacts directly with the ester carbonyl of Carb (Ryan et al, 2002).…”

“…The intensities and frequencies of these two bands are essentially identical to those of the two bands in Carb minus tetramethylamine difference spectra (Ryan et al, 2002), where the vibrations of the two bands can also been seen clearly because of the absence of overlapping amide I vibrations. As noted, the 1690 and 1668 cm Ϫ1 bands reflect a vibrational shift in an as yet unidentified residue that interacts directly with the ester carbonyl of Carb (Ryan et al, 2002). That procaine binding to the neurotransmitter site has no effect on the frequencies or intensities of these two bands shows that although procaine interacts with aromatic residues in the neurotransmitter sites, it does not form a hydrogen bond with the residue that typically interacts with the ester carbonyl of Carb.…”

“…Four protein side-chain vibrations centered near 1690, 1668, 1620, and 1515 cm Ϫ1 are also of interest, as they reflect vibrational shifts that result from the formation of physical interactions between Carb and neurotransmitter binding site residues. Positive and negative bands near 1690 and 1668 cm Ϫ1 , respectively, reflect a shift in the vibrational frequency from 1668 to 1690 cm Ϫ1 of an as-yet-unassigned residue upon formation of a physical interaction (likely a hydrogen bond) with the ester carbonyl functional group of Carb (Ryan et al, 2002). Note that the negative 1668 cm Ϫ1 vibration is masked by the above-discussed positive amide I difference band near 1663 cm Ϫ1 (Fig.…”

Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment

“…The higher density low lipid to protein ratio proteoliposomes are easier to pellet (particularly in higher density 2 H 2 O buffers) and thus exchange into 2 H 2 O. Low lipid to protein ratio vesicles are also ideal for structural studies using infrared difference spectroscopy, a technique that detects residue-specific changes in structure of the nAChR upon agonist-binding (27,43,44). Application of this approach to prokaryotic pLGICs would be particularly informative given the ability to express mutants and thus assign detected vibrational changes to specific amino acid side chains.…”

A Lipid-dependent Uncoupled Conformation of the Acetylcholine Receptor

“…Infrared difference spectroscopy (Fig. 2) is an effective tool for probing conformational change in these membrane environments because the technique provides a comprehensive map of the vibrational, and thus structural changes induced in the entire nAChR upon agonist binding (14). Carbamylcholine (Carb) difference spectra recorded from native-nAChR, PC/PA/Chol-nAChR, and PC-nAChR all exhibit a number of positive vibrational bands due to nAChRbound Carb (asterisk in Fig.…”