ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) ␣-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentrationdependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders.

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and ␥-aminobutyric acid type A receptors (GABA A Rs). To test this, we mutated Loop 2 in the ␣1 subunit of GlyRs and in the ␥ subunit of ␣1␤2␥2GABA A Rs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of ␣1GlyR subunits with Loop 2 from the ␦GABA A R (␦L2), but not the ␥GABA A R subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the ␥ subunit of GABA A Rs with ␦L2 shifted the ethanol threshold from 50 mM in WT to 1 mM in the GABA A ␥-␦L2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA A Rs. The ␦L2 mutations did not affect GlyR or GABA A R sensitivity, respectively, to Zn 2؉ or diazepam, which suggests that these ␦L2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and ␦L2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ϳ14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3-5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs) 2 have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6 -12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine 3 , ␥-aminobutyric acid type A (GABA A ), ␥-aminobutyric acid type C, and glycine receptors (GlyRs) (10,11,(15)(16)(17)(18)(19)(20) and 2) the glutamate superfamily, including N-methyl D-aspartate, ␣-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21,22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of .A series of studies that employed chim...

The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical–chemical properties at position 52 in extracellular domain Loop 2 of α1GlyRs, or the homologous α2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in α1GlyRs that contain a non‐polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position. Ethanol sensitivity in receptors with polar substitutions at position 52 was significantly lower than GlyRs with non‐polar residues at this position. The α2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTα2GlyR, thereby making it α1‐like. Collectively, these findings indicate that (i) polarity at position 52 plays a key role in determining sensitivity to ethanol and pressure antagonism of ethanol; (ii) the extracellular domain in α1‐ and α2GlyRs is a target for ethanol action and antagonism and (iii) there is structural‐functional homology across subunits in Loop 2 of GlyRs with respect to their roles in determining sensitivity to ethanol and pressure antagonism of ethanol. These findings should help in the development of pharmacological agents that antagonize ethanol.

Glycine receptors (GlyRs) are recognized as the primary mediators of neuronal inhibition in the spinal cord, brain stem and higher brain regions known to be sensitive to ethanol. Building evidence supports the notion that ethanol acting on GlyRs causes at least a subset of its behavioral effects and may be involved in modulating ethanol intake. For over two decades, GlyRs have been studied at the molecular level as targets for ethanol action. Despite the advances in understanding the effects of ethanol in vivo and in vitro, the precise molecular sites and mechanisms of action for ethanol in ligand-gated ion channels in general, and in GlyRs specifically, are just now starting to become understood. The present review focuses on advances in our knowledge produced by using molecular biology, pressure antagonism, electrophysiology and molecular modeling strategies over the last two decades to probe, identify and model the initial molecular sites and mechanisms of ethanol action in GlyRs. The molecular targets on the GlyR are covered on a global perspective, which includes the intracellular, transmembrane and extracellular domains. The latter has received increasing attention in recent years. Recent molecular models of the sites of ethanol action in GlyRs and their implications to our understanding of possible mechanism of ethanol action and novel targets for drug development in GlyRs are discussed.

The present study tested the hypothesis that several residues in Loop 2 of ␣1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50 -60 on glycine responses in ␣1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a ␤-turn structure of Loop 2, with odd-numbered residues in the ␤-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains. We also tested the hypothesis that the charge at position 53 is important for agonist activation by measuring the glycine response of wild type (WT) and E53C GlyRs exposed to methanethiosulfonate reagents. As earlier, E53C GlyRs have a significantly higher EC 50 than WT GlyRs. Exposing E53C GlyRs to the negatively charged 2-sulfonatoethyl methanethiosulfonate, but not neutral 2-hydroxyethyl methanethiosulfonate, positively charged 2-aminoethyl methanethiosulfonate, or 2-trimethylammonioethyl methanethiosulfonate, decreased the glycine EC 50 to resemble WT GlyR responses. Exposure to these reagents did not significantly alter the glycine EC 50 for WT GlyRs. The latter findings suggest that the negative charge at position 53 is important for activation of GlyRs through its interaction with positive charge(s) in other neighboring agonist activation elements. Collectively, the findings provide the basis for a refined molecular model of ␣1GlyRs based on the recent x-ray structure of a prokaryotic pentameric ligand-gated ion channel and offer insight into the structure-function relationships in GlyRs and possibly other ligand-gated ion channels.Glycine is a major inhibitory neurotransmitter in the adult mammalian central nervous system (1, 2). It reduces central nervous system excitability via activation of a ligand-gated receptor linked to an integral chloride channel, the strychninesensitive glycine receptor (GlyR).2 GlyRs are members of a superfamily of ligand-gated ion channels (LGICs) known as Cys-loop receptors (3, 4), whose members also include ␥-aminobutyric acid type A (GABA A ), nicotinic acetylcholine (nACh), and 5-hydroxytryptamine 3 , all of which assemble to form ion channels with a pentameric structure. Cys-loop receptor subunits share significant sequence homology and consist of four transmembrane (TM) ␣-helical segments, an intracellular component for cytosolic interactions, and a large, extracellular ligand-binding domain (5-8).Considerable evidence indicates that Loop 2 in the extracellular domain of Cys-loop receptors (loop terminology as defined by Sixma and co-workers (6)) is important for coupling agonist binding to channel gating in the TM domain (4, 9 -12). The importance of the ␣1GlyR Loop 2 region in agonist activation was first noted when the phenotype of the spasmodic mouse was traced to a naturally occurring alanine-to-serine ...