Sites of Excitatory and Inhibitory Actions of Alcohols on Neuronal  2 4 Nicotinic Acetylcholine Receptors

Abstract

To define potential alcohol binding sites in the neuronal nicotinic acetylcholine receptor (nAChR) we used cysteine mutagenesis and sulfhydryl-specific labeling. The basis of this strategy is that covalent addition of an alkylthiol group to a cysteine in an alcohol binding site will mimic the action of an irreversibly bound alcohol. Each amino acid in the extracellular region of the second transmembrane segment of the nAChR subunit alpha2 was mutated to cysteine. The resulting alpha2 subunits were coexpressed with wild-type beta4in Xenopus laevis oocytes, and the responses were studied using two-electrode voltage clamp. Of the 11 mutants tested, 2 fulfilled criteria for participation in an alcohol binding site: alpha2(L262C)beta4 and alpha2(L263C)beta4. Covalent binding of propanethiol to these cysteines did not change acetylcholine (ACh) affinity, but modified ACh maximal response in both cases: it increased for alpha2(L263C)beta4 and decreased for alpha2(L262C)beta4. The same modifications on ACh responses were obtained with ethanol on alpha2(L263C)beta4 and octanol on alpha2(L262C)beta4. This suggested that alcohol binding at L263 enhances receptor function, whereas binding at L262 inhibits function. We studied different n-alcohols (ethanol, butanol, pentanol, and octanol), as well as isoflurane and urethane, on these two mutants. Covalent binding of propanethiol to the cysteines revealed changes in the alcohol modulation consistent with an excitatory site (L263) or an inhibitory site (L262) being no longer accessible to alcohol. Thus, n-alcohols appear to act on both sites and their ability to enhance (short-chain), inhibit (long-chain), or produce no effect (intermediate-chain) depends upon their relative action at these two sites.

Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors

“…In addition, these results suggest that there are structural and functional differences between the different sites upon which ethanol acts. The concept of multiple sites of ethanol action with different properties is similar to findings in other LGICs [38][39][40][41][42][43]. IVM is a hydrophobic molecule that is known to bind at the lipid-protein interfaces and to act on multiple sites within the TM segments of P2X4Rs [25,26].…”

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

“…But despite decades of investigation, the evidence is inconclusive for any specific molecular target for ethanol on any protein (for reviews see (6,7)). Many potential protein targets have been identified (4,8) including voltage-gated Na þ channels (9), adenylyl cyclase (10,11), guanine nucleotide binding protein G s (12), the GLT1 glutamate transporter (13), gap junctions (14), g-aminobutyric acid (GABA) A receptors (6), n-methyl-daspartate (NMDA) receptors (15), nicotinic acetylcholine receptor (nAChR) channels (16,17), and the cannabinoid receptor 1 (18). One system within the brain that appears to be sensitive to alcohol dependence is the endocannabinoid system (for review see (19)).…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…The packing of helical bundles within the TMD supports distinct water pockets, referred to as the intrasubunit and intersubunit cavities. These water vestibules form the binding site for several allosteric ligands and therapeutic agents (11,26,27) that modulate channel gating. Although these independent pieces of information point toward the indisputable role of the ECD-TMD region in allosteric mechanisms of gating and modulation, the mechanistic details of the interplay between each of these components are unknown.…”