Lipid Composition Alters Drug Action at the Nicotinic Acetylcholine Receptor

Abstract

We tested the hypothesis that membrane lipid composition influences drug action at membrane proteins by studying local anesthetic action at the nicotinic acetylcholine receptor (nAChR). Infrared difference spectra show that concentrations of tetracaine consistent with binding to the ion channel (Ͻ50 M) stabilize a resting-like state when the nAChR is reconstituted into phosphatidylcholine membranes containing the anionic lipid, phosphatidic acid, but have no effect on the nAChR reconstituted into membranes lacking phosphatidic acid, either in the presence or absence of cholesterol. Concentrations of tetracaine above 200 M lead to neurotransmitter site binding in all membranes. In the presence of phosphatidic acid, cholesterol, or both, neurotransmitter site binding leads to the formation of quaternary amine-aromatic interactions between tetracaine and binding site tyrosine/tryptophan residues and the stabilization of a desensitized state. One interpretation suggested by lipid partitioning studies is that phosphatidic acid enhances tetracaine action at the channel pore by increasing the partitioning of tetracaine into the lipid bilayer, thereby enhancing access to the transmembrane pore. However, subtle membrane-dependent variations in the vibrations of tyrosine and tryptophan residues, and agonist analog binding studies indicate that the structures of the agonist-bound neurotransmitter sites of the nAChR in membranes lacking both phosphatidic acid and cholesterol differ from the structures of the agonist-desensitized neurotransmitter sites in the presence of both lipids. Lipid action at the nAChR thus involves more than a simple modulation of the equilibrium between resting and desensitized states.

A Lipid-dependent Uncoupled Conformation of the Acetylcholine Receptor

“…This is consistent with the finding that another non-competitive inhibitor, tetracaine, also has no effect on the conformation of PC-nAChR (13). These data confirm that the agonist-binding sites and transmembrane pore in PC-nAChR are allosterically uncoupled.…”

“…the nAChR is desensitized prior to Carb binding; Fig. 2B), we conclude that these positive vibrations result from the Carb-induced resting-to-desensitized conformational transition (10,13,16,17). Positive intensity is also absent at these frequencies (i.e.…”

“…As the chemical labeling pattern of the nonactivatable PC-nAChR is similar to that of the desensitized nAChR, it has been suggested that lipids modulate the natural equilibrium between resting and desensitized receptors (8,9,11). Other studies, however, hint that PC-nAChR is not desensitized, suggesting a more complex mechanism of nAChR-lipid interactions (7,12,13). The lack of detailed characterization of the inactive PC-nAChR conformation has prevented the development of descriptive models of nAChR-lipid interactions.…”

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.…”

Gating-induced Conformational Rearrangement of the γ-Aminobutyric Acid Type A Receptor β-α Subunit Interface in the Membrane-spanning Domain

“…Furthermore, crystals are obtained in detergent-solubilized protein, usually in the absence of lipids. How the absence of lipids affects the protein structure is uncertain, but interactions with the lipids certainly affect channel function (45). A further issue is that cryopreservation of crystals induces tighter packing of the protein (46).…”