Other articles by Daryl L. Davies on PubMed

• Ethanol Sensitivity in ATP-gated P2X Receptors is Subunit Dependent Alcoholism, Clinical and Experimental Research. Jun, 2002  |   Pubmed ID: 12068244 P2X receptors are ligand-gated cation channels that are gated by synaptically released extracellular adenosine 5'-triphosphate (ATP). P2X receptors are inhibited by ethanol; however, few investigations have focused on ethanol's effects in P2X receptors. Recently, recombinant homomeric P2X4 receptors were reported to be sensitive to ethanol's inhibitory action, whereas recombinant P2X3 receptors were insensitive to ethanol. The two recombinant studies were conducted in different expression systems by using different techniques; therefore, questions remain. The present study tests the hypothesis that ethanol sensitivity in P2X receptors is subunit dependent.
• Biophysical Evidence for His57 As a Proton-binding Site in the Mammalian Intestinal Transporter HPepT1 Pharmaceutical Research. Dec, 2003  |   Pubmed ID: 14725353 The objective of this study was to provide direct evidence of the relative importance of the His57 residue present in transmembrane domain 2 (TMD 2) and the His121 residue in TMD 4 as proton-binding sites in human PepT1 (hPepT1) by using a novel mutagenesis approach.
• Benzodiazepine Agonist and Inverse Agonist Coupling in GABAA Receptors Antagonized by Increased Atmospheric Pressure European Journal of Pharmacology. May, 2003  |   Pubmed ID: 12782183 Past work found that exposure to 12 times normal atmospheric pressure (ATA) of helium-oxygen gas (heliox) selectively antagonizes (uncouples) and differentiates allosteric coupling in GABA(A) receptors initiated by benzodiazepines versus neurosteroids. The present study tested the hypothesis that pressure can differentiate coupling initiated by a spectrum of benzodiazepine receptor ligands by measuring the effects of pressure on benzodiazepine ligand modulation of GABA-activated 36Cl(-) uptake in mouse brain membranes. 12 ATA completely antagonized allosteric modulation by: benzodiazepine receptor agonists diazepam and flunitrazepam; Type-1 selective benzodiazepine receptor agonist zolpidem and the benzodiazepine receptor partial inverse agonist ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-alpha][1,4]benzodiazepine-3-carboxylate (Ro15-4513). The similar, non-competitive-like characteristics of pressure antagonism of these ligands suggest common structural/functional elements underlying their coupling. Pressure also antagonized allosteric modulation by the benzodiazepine receptor inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), but the antagonism was not complete and appeared to be surmountable (competitive-like) suggesting unexpected differences in coupling for DMCM versus Ro15-4513. These studies represent the first attempt to use pressure as a tool to dissect benzodiazepine receptor coupling. The results suggest that there is a common, pressure antagonism sensitive structural/functional element underlying coupling for benzodiazepine receptor ligands and that coupling for the full inverse benzodiazepine receptor agonist DMCM differs from coupling for benzodiazepine receptor agonists and benzodiazepine receptor partial inverse agonists.
• Ethanol Potentiation of Glycine Receptors Expressed in Xenopus Oocytes Antagonized by Increased Atmospheric Pressure Alcoholism, Clinical and Experimental Research. May, 2003  |   Pubmed ID: 12766618 Behavioral and biochemical studies indicate that exposure to 12 times normal atmospheric pressure (12 ATA) of helium-oxygen gas (heliox) is a direct, selective ethanol antagonist. The current study begins to test the hypothesis that ethanol acts by a common mechanism on ligand-gated ion channels by expanding previous hyperbaric investigations on gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs) at the biochemical level to alpha(1)glycine (GlyRs) expressed in Xenopus oocytes.
• Effects of the Abused Solvent Toluene on Recombinant P2X Receptors Expressed in HEK293 Cells Brain Research. Molecular Brain Research. Jun, 2004  |   Pubmed ID: 15193425 ATP acts as a neurotransmitter in both the peripheral and central nervous systems by activating receptors in the P2Y and P2X families. P2Y receptors are coupled to intracellular signaling pathways, while P2X receptors contain an integral membrane-spanning pore and act as ion channels. Previous studies have established that certain abused drugs such as alcohol inhibit P2X receptors. In this study, we have examined the sensitivity of both homomeric and heteromeric P2X receptors to toluene, a commercial solvent widely used as a drug of abuse. P2X receptors were transiently expressed in HEK293 cells, and agonist-gated currents were measured using whole-cell patch clamp electrophysiology. Toluene potentiated currents in cells expressing homomeric P2X2 or P2X4 subunits when ATP concentrations were near or below the EC50 concentration. This potentiation was rapid in onset, voltage independent and was readily reversed upon washout of the toluene-containing solution. The toluene-induced potentiation of P2X2 currents was not altered by lowering the pH of the recording media to 5.5 or by including the heavy-metal chelator EDTA in the recording solution. At maximal ATP concentrations, toluene did not affect ATP-gated currents in cells expressing P2X2 or P2X4 receptors. ATP-gated currents were also markedly potentiated by toluene in cells transfected with both P2X4 and P2X6 subunits. In contrast, P2X3 receptor currents were inhibited by toluene at both low and high ATP concentrations. HEK293 cells transfected with both P2X2 and P2X3 subunits showed non-desensitizing currents when stimulated with alpha, beta-methylene ATP. In these cells, toluene potentiated currents only at sub-maximal concentrations of alpha, beta-methylene ATP. The results of this study suggest that the abused solvent toluene affects the function of P2X receptors in a subunit-dependent and agonist-dependent fashion.
• Multiple Sites of Ethanol Action in Alpha1 and Alpha2 Glycine Receptors Suggested by Sensitivity to Pressure Antagonism Journal of Neurochemistry. Jun, 2004  |   Pubmed ID: 15147510 The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in glycine receptors (GlyRs). The effects of 12 times normal atmospheric pressure of helium-oxygen gas (pressure) on ethanol-induced potentiation of GlyR function in Xenopus oocytes expressing human alpha1, alpha2 or the mutant alpha1(A52S) GlyRs were measured using two-electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in alpha1 GlyR by 40-200 mm ethanol, but did not antagonize 10 and 25 mm ethanol in the same oocytes. In contrast, pressure did not significantly affect potentiation of glycine by 25-100 mm ethanol in alpha2 GlyRs, nor did pressure alter ethanol response in the A52S mutant. Pressure did not affect baseline receptor function or response to glycine in the absence of ethanol. These findings provide the first direct evidence for multiple sites of ethanol action in GlyRs. The sites can be differentiated on the basis of ethanol concentration, subunit and structural composition and sensitivities to pressure antagonism of ethanol. Parallel studies with butanol support this conclusion. The mutant alpha1(A52S) GlyR findings suggest that increased attention should be focused on the amino terminus as a potential target for ethanol action.
• Ethanol Differentially Affects ATP-gated P2X(3) and P2X(4) Receptor Subtypes Expressed in Xenopus Oocytes Neuropharmacology. Aug, 2005  |   Pubmed ID: 15993446 P2X receptors are cation-selective, ligand-gated ion channels activated by synaptically released, extracellular adenosine 5'-triphosphate (ATP). ATP-gated currents are inhibited by ethanol when tested in dorsal root ganglion and CA1 neurons. Recently, we reported differences in sensitivity to ethanol inhibition between homomeric P2X(2) and P2X(4) receptors expressed in Xenopus oocytes, which suggested that subunit composition of native P2X receptors determines their ethanol sensitivity. The present study extended the investigation to P2X(3) receptors. The effects of ethanol and zinc ions (Zn(2+)) were tested on homomeric P2X(3) and P2X(4) receptors expressed in Xenopus oocytes using two-electrode voltage clamp. Ethanol potentiated ATP-gated P2X(3) receptor currents in a concentration dependent manner. In contrast, ethanol inhibited P2X(4) receptor function. Ethanol did not directly alter receptor function, nor did it alter the Hill coefficient or maximal ATP response (E(max)) in either P2X(3) or P2X(4) receptors. Ethanol increased the maximal response to Zn(2+) ATP-gated currents in P2X3 receptors which suggests that ethanol and Zn(2+) act on different sites. The differences in ethanol response of P2X(3) and P2X(4) receptors set the stage for future investigations that will use chimeric P2X receptors or other molecular manipulations of P2X structure to investigate the molecular sites and mechanisms of action of ethanol.
• Effects of Ethanol on Adenosine 5'-triphosphate-gated Purinergic and 5-hydroxytryptamine Receptors Alcoholism, Clinical and Experimental Research. Feb, 2006  |   Pubmed ID: 16441284 This report of the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism highlights the actions of ethanol on purinergic (P2XRs) and 5-hydroxytryptamine3 (5-HT3Rs) receptors. Both P2XRs and 5-HT3Rs, are modulated by pharmacologically relevant concentrations of ethanol, with inhibition or stimulation of P2XR subtypes and stimulation of 5-HT3Rs, respectively. With regard to ethanol-modulatory actions, these 2 distinctly different receptor classes have been studied to a much lesser extent than other LGICs. The organizers and chairs were Daryl L. Davies and Tina K. Machu. John J. Woodward discusses the molecular pharmacology and physiology of P2XRs and 5-HT3Rs and sets the stage for a detailed investigation into the ethanol sensitivity of these channels by the invited speakers. Daryl L. Davies discusses the results from recent electrophysiological studies conducted in his and Dr. Woodward's laboratories, highlighting the actions of ethanol on P2XR subtypes. Jiang-Hong Ye discusses results from recent studies using loose-patch and whole-cell recordings on purinergic receptors expressed on neurons from the ventral tegmental area (VTA) in rats. Tina K. Machu discusses electrophysiological studies conducted in her and Dr. David Lovinger's laboratories on nonpore lining residues of the second transmembrane domain (TM2) of the 5-HT3A receptor. Li Zhang presents data demonstrating that F-actin cytoskeletons play a critical role in 5-HT3 receptor clustering in hippocampal neurons. Collectively, the presentations provided strong evidence that P2X and 5-HT3 receptors are important targets for ethanol action.
• Evidence That Ethanol Acts on a Target in Loop 2 of the Extracellular Domain of Alpha1 Glycine Receptors Journal of Neurochemistry. Sep, 2007  |   Pubmed ID: 17561937 Considerable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one site in order to determine its effect at other site(s). In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267. Collectively, these findings parallel the evidence that established the TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the same alcohol pocket and indicate that the net effect of ethanol on GlyR function reflects the summation of its positive and negative modulatory effects on different targets.
• Cysteine Scanning of Transmembrane Domain Three of the Human Dipeptide Transporter: Implications for Substrate Transport Journal of Drug Targeting. Apr, 2007  |   Pubmed ID: 17454359 The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly-Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly-Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.
• A Charge Pair Interaction Between Arg282 in Transmembrane Segment 7 and Asp341 in Transmembrane Segment 8 of HPepT1 Pharmaceutical Research. Jan, 2007  |   Pubmed ID: 17009102 To determine whether R282 in transmembrane segment 7 (TMS7) of hPepT1 forms a salt bridge with D341 in TMS8.
• Roles for Loop 2 Residues of Alpha1 Glycine Receptors in Agonist Activation The Journal of Biological Chemistry. Oct, 2008  |   Pubmed ID: 18658152 The present study tested the hypothesis that several residues in Loop 2 of alpha1 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 alpha1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a beta-turn structure of Loop 2, with odd-numbered residues in the beta-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 alpha1GlyRs 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.
• Roles of Ectodomain and Transmembrane Regions in Ethanol and Agonist Action in Purinergic P2X2 and P2X3 Receptors Neuropharmacology. Oct, 2008  |   Pubmed ID: 18639563 The present work investigated sites of ethanol action in ATP-gated P2X receptors (P2XRs) using chimeric strategies that exploited the differences in ethanol response between P2X2R (inhibition) and P2X3R (potentiation). We tested ethanol (10-200mM) effects on ATP- and alpha,beta-methylene-ATP (alpha,beta-meATP)-induced currents in wildtype P2X2, P2X3 and chimeric P2X2/P2X3Rs expressed in Xenopus oocytes using two-electrode voltage-clamp (-70mV). Exchanging ectodomain regions of P2X2 and P2X3Rs reversed wildtype ethanol responses. Substituting back portions of the P2X2R ectodomain at TM interfaces in chimeras that contained the P2X3R ectodomain restored wildtype P2X2R-like ethanol response. Point mutations that replaced non-conserved ectodomain residues at TM interfaces of P2X3Rs with homologous P2X2R residues identified positions that reversed the direction (304) or changed the magnitude (53, 55 and 313) of ethanol response. Homologous substitutions in P2X2Rs did not significantly alter wildtype P2X2R-like ethanol responses. These findings suggest that ectodomain segments at TM interfaces play key roles in determining qualitative and quantitative responses to ethanol of P2X2 and P2X3Rs. Studies that substituted TM regions of P2X3R with respective P2X2R TMs indicate that the TM1, but not the TM2, region plays a role in determining the magnitude of ethanol response. Studies with ATP and alpha,beta-meATP support prior indications that TM regions are important in agonist desensitization and suggest that both ectodomain and TM regions play roles in determining agonist potency and selectivity. Overall, these findings are the first to identify potential targets for ethanol in P2X2 and P2X3Rs and should provide insight into the sites of ethanol action in other P2XRs.
• Purinergic Type 2 Receptors at GABAergic Synapses on Ventral Tegmental Area Dopamine Neurons Are Targets for Ethanol Action The Journal of Pharmacology and Experimental Therapeutics. Oct, 2008  |   Pubmed ID: 18583548 The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1-200 microM) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, alpha,beta-methylene ATP, a selective P2X(1) and P2X(3) receptor agonist, increased sIPSC frequency, whereas adenosine 5'-[beta-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X(1) and/or P2X(3) receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.
• Targets for Ethanol Action and Antagonism in Loop 2 of the Extracellular Domain of Glycine Receptors Journal of Neurochemistry. Aug, 2008  |   Pubmed ID: 18485105 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 alpha1GlyRs, or the homologous alpha2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in alpha1GlyRs 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 alpha2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTalpha2GlyR, thereby making it alpha1-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 alpha1- and alpha2GlyRs 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.
• Ethanol Inhibits Functional Activity of the Human Intestinal Dipeptide Transporter HPepT1 Expressed in Xenopus Oocytes Alcoholism, Clinical and Experimental Research. May, 2008  |   Pubmed ID: 18336632 The pathological effects of high alcohol (ethanol) consumption on gastrointestinal and hepatic systems are well recognized. However, the effects of ethanol intake on gastric and intestinal absorption and transport systems remain unclear. The present study investigates the effects of ethanol on the human peptide transporter 1 (hPepT1) which mediates the transport of di-and tripeptides as well as several orally administered peptidomimetic drugs such as beta-lactam antibiotics (e.g., penicillin), angiotensin-converting enzyme inhibitors, the anti-neoplastic agent bestatin, and prodrugs of acyclovir.
• Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter Pharmaceutical Research. Oct, 2009  |   Pubmed ID: 19685173 The human dipeptide transporter (hPEPT1) facilitates transport of dipeptides and drugs from the intestine into the circulation. The role of transmembrane domain 10 (TMD10) of hPEPT1 in substrate translocation was investigated using cysteine-scanning mutagenesis with 2-Trimethylammonioethyl methanethiosulfonate (MTSET).
• Loop 2 Structure in Glycine and GABA(A) Receptors Plays a Key Role in Determining Ethanol Sensitivity The Journal of Biological Chemistry. Oct, 2009  |   Pubmed ID: 19656948 The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and gamma-aminobutyric acid type A receptors (GABA(A)Rs). To test this, we mutated Loop 2 in the alpha1 subunit of GlyRs and in the gamma subunit of alpha1beta2gamma2GABA(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 alpha1GlyR subunits with Loop 2 from the deltaGABA(A)R (deltaL2), but not the gammaGABA(A)R subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the gamma subunit of GABA(A)Rs with deltaL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA(A) gamma-deltaL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA(A)Rs. The deltaL2 mutations did not affect GlyR or GABA(A)R sensitivity, respectively, to Zn(2+) or diazepam, which suggests that these deltaL2-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 deltaL2 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.
• Functional Role of the Intracellular Loop Linking Transmembrane Domains 6 and 7 of the Human Dipeptide Transporter HPEPT1 The Journal of Membrane Biology. Dec, 2010  |   Pubmed ID: 21104182 The human intestinal di-/tripeptide transporter (hPEPT1) is a 12-transmembrane protein that facilitates transport of peptides from the intestine into the circulation. hPEPT1 is also an important target in oral delivery of drugs, but mechanistic and structural data for the protein are limited. In particular, there is little information on the function of the loops of the transporter. In this study, we show that mutation of several charged residues in the largest intracellular loop of hPEPT1 (loop 6-7, amino acids 224-278) significantly reduces hPEPT1 function. This loop has an asymmetric distribution of charged residues, with only positive charges in the N-terminal half and all five negative charges in the loop located in a small part of the C-terminal half. Point mutagenesis to alanine of three positive residues in the N-terminal half of loop 6-7 and four negative residues in the C-terminal half of the loop significantly reduced glycylsarcosine uptake. E267 was particularly sensitive to mutation, and kinetic analyses of E267A- and E267K-hPEPT1 gave V (max) values 10-fold lower than that for the wild-type protein. Secondary structure prediction suggested that loop 6-7 includes two amphipathic α-helices, with net positive and negative charges, respectively. We interpret the mutagenesis data in terms of interactions of the charged residues in loop 6-7 that may influence conformational changes of hPEPT1 during and after substrate transport.
• Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors The Journal of Pharmacology and Experimental Therapeutics. Sep, 2010  |   Pubmed ID: 20543096 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) alpha-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 concentration-dependent 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.
• Molecular Targets and Mechanisms for Ethanol Action in Glycine Receptors Pharmacology & Therapeutics. Jul, 2010  |   Pubmed ID: 20399807 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.
• A Point Mutation in the Ectodomain-transmembrane 2 Interface Eliminates the Inhibitory Effects of Ethanol in P2X4 Receptors Journal of Neurochemistry. Jan, 2010  |   Pubmed ID: 19878433 ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration-response curves and ethanol (10-200 mM)-induced changes in ATP EC(10)-gated currents were determined using two-electrode voltage clamp (-70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50-61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321-337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I(max), EC(50), or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.
• Alcohol-binding Sites in Distinct Brain Proteins: the Quest for Atomic Level Resolution Alcoholism, Clinical and Experimental Research. Sep, 2011  |   Pubmed ID: 21676006 Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmitter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a "molecular lubricant" for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol.
• Implication of the Purinergic System in Alcohol Use Disorders Alcoholism, Clinical and Experimental Research. Apr, 2011  |   Pubmed ID: 21223299 In the central nervous system, adenosine and adenosine 5'-triphosphate (ATP) play an important role in regulating neuronal activity as well as controlling other neurotransmitter systems, such as, GABA, glutamate, and dopamine. Ethanol increases extracellular adenosine levels that regulate the ataxic and hypnotic/sedative effects of ethanol. Interestingly, ethanol is known to increase adenosine levels by inhibiting an ethanol-sensitive adenosine transporter, equilibrative nucleoside transporter type 1 (ENT1). Ethanol is also known to inhibit ATP-specific P2X receptors, which might result in such similar effects as those caused by an increase in adenosine. Adenosine and ATP exert their functions through P1 (metabotropic) and P2 (P2X-ionotropic and P2Y-metabotropic) receptors, respectively. Purinergic signaling in cortex-striatum-ventral tegmental area (VTA) has been implicated in regulating cortical glutamate signaling as well as VTA dopaminergic signaling, which regulates the motivational effect of ethanol. Moreover, several nucleoside transporters and receptors have been identified in astrocytes, which regulate not only adenosine-ATP neurotransmission, but also homeostasis of major inhibitory-excitatory neurotransmission (i.e., GABA or glutamate) through neuron-glial interactions. This review will present novel findings on the implications of adenosine and ATP neurotransmission in alcohol use disorders.
• Ethanol is a Fast Channel Inhibitor of P2X4 Receptors The Journal of Pharmacology and Experimental Therapeutics. Apr, 2011  |   Pubmed ID: 21212160 P2X receptors (P2XRs) are ion channels gated by synaptically released ATP. The P2X4 is the most abundant P2XR subtype expressed in the central nervous system and to date is the most ethanol-sensitive. In addition, genomic findings suggest that P2X4Rs may play a role in alcohol intake/preference. However, little is known regarding how ethanol causes the inhibition of ATP-gated currents in P2X4Rs. We begin to address this issue by investigating the effects of ethanol in wild-type and mutant D331A and M336A P2X4Rs expressed in human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp methods. The results suggest that residues D331 and M336 play a role in P2X4R gating and ethanol inhibits channel functioning via a mechanism different from that in other P2XRs. Key findings from the study include: 1) ethanol inhibits ATP-gated currents in a rapid manner; 2) ethanol inhibition of ATP-gated currents does not depend on voltage and ATP concentration; 3) residues 331 and 336 slow P2X4 current deactivation and regulate the inhibitory effects of ethanol; and 4) ethanol effects are similar in HEK293 cells transfected with P2X4Rs and cultured rat hippocampal neurons transduced with P2X4Rs using a recombinant lentiviral system. Overall, these findings provide key information regarding the mechanism of ethanol action on ATP-gated currents in P2X4Rs and provide new insights into the biophysical properties of P2X4Rs.
• Ivermectin Reduces Alcohol Intake and Preference in Mice Neuropharmacology. Aug, 2012  |   Pubmed ID: 22465817 The high rate of therapeutic failure in the management of alcohol use disorders (AUDs) underscores the urgent need for novel and effective strategies that can deter ethanol consumption. Recent findings from our group showed that ivermectin (IVM), a broad-spectrum anthelmintic with high tolerability and optimal safety profile in humans and animals, antagonized ethanol-mediated inhibition of P2X4 receptors (P2X4Rs) expressed in Xenopus oocytes. This finding prompted us to hypothesize that IVM may reduce alcohol consumption; thus, in the present study we investigated the effects of this agent on several models of alcohol self-administration in male and female C57BL/6 mice. Overall, IVM (1.25-10 mg/kg, intraperitoneal) significantly reduced 24-h alcohol consumption and intermittent limited access (4-h) binge drinking, and operant alcohol self-administration (1-h). The effects on alcohol intake were dose-dependent with the significant reduction in intake at 9 h after administration corresponding to peak IVM concentrations (C(max)) in the brain. IVM also produced a significant reduction in 24-h saccharin consumption, but did not alter operant sucrose self-administration. Taken together, the findings indicate that IVM reduces alcohol intake across several different models of self-administration and suggest that IVM may be useful in the treatment of AUDs.
• Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors The Journal of Pharmacology and Experimental Therapeutics. May, 2012  |   Pubmed ID: 22357974 Recent studies highlighted the importance of loop 2 of α1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the β hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC₅₀ but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC₅₀ while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC₅₀ and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the β hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs.
• Sociocommunicative and Sensorimotor Impairments in Male P2X4-deficient Mice Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology. Sep, 2013  |   Pubmed ID: 23604007 Purinergic P2X receptors are a family of ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP). Of the seven P2X subtypes, P2X4 receptors (P2X4Rs) are richly expressed in the brain, yet their role in behavioral organization remains poorly understood. In this study, we examined the behavioral responses of P2X4R heterozygous (HZ) and knockout (KO) mice in a variety of testing paradigms designed to assess complementary aspects of sensory functions, emotional reactivity, and cognitive organization. P2X4R deficiency did not induce significant alterations of locomotor activity and anxiety-related indices in the novel open field and elevated plus-maze tests. Conversely, P2X4R KO mice displayed marked deficits in acoustic startle reflex amplitude, as well as significant sensorimotor gating impairments, as assessed by the prepulse inhibition of the startle. In addition, P2X4R KO mice displayed enhanced tactile sensitivity, as signified by a lower latency in the sticky-tape removal test. Moreover, both P2X4R HZ and KO mice showed significant reductions in social interaction and maternal separation-induced ultrasonic vocalizations in pups. Notably, brain regions of P2X4R KO mice exhibited significant brain-regional alterations in the subunit composition of glutamate ionotropic receptors. These results collectively document that P2X4-deficient mice exhibit a spectrum of phenotypic abnormalities partially akin to those observed in other murine models of autism-spectrum disorder. In conclusion, our findings highlight a putative role of P2X4Rs in the regulation of perceptual and sociocommunicative functions and point to these receptors as putative targets for disturbances associated with neurodevelopmental disorders.
• Pharmacological Insights into the Role of P2X4 Receptors in Behavioural Regulation: Lessons from Ivermectin The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP). Jun, 2013  |   Pubmed ID: 23174033 Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind extracellular adenosine 5'-triphosphate. In particular, convergent lines of evidence have recently highlighted P2X(4) receptors as a potentially critical target in the regulation of multiple nervous and behavioural functions, including pain, neuroendocrine regulation and hippocampal plasticity. Nevertheless, the role of the P2X(4) receptor in behavioural organization remains poorly investigated. To study the effects of P2X(4) activation, we tested the acute effects of its potent positive allosteric modulator ivermectin (IVM, 2.5-10 mg/kg i.p.) on a broad set of paradigms capturing complementary aspects of perceptual, emotional and cognitive regulation in mice. In a novel open field, IVM did not induce significant changes in locomotor activity, but increased the time spent in the peripheral zone. In contrast, IVM produced anxiolytic-like effects in the elevated plus maze and marble burying tasks, as well as depression-like behaviours in the tail-suspension and forced swim tests. The agent induced no significant behavioural changes in the conditioned place preference test and in the novel object recognition task. Finally, the drug induced a dose-dependent decrease in sensorimotor gating, as assessed by pre-pulse inhibition (PPI) of the acoustic startle reflex. In P2X(4) knockout mice, the effects of IVM in the open field and elevated plus maze were similar to those observed in wild type mice; conversely, the drug significantly increased startle amplitude and failed to reduce PPI. Taken together, these results suggest that P2X(4) receptors may play a role in the regulation of sensorimotor gating.
• Recent Advances in the Discovery and Preclinical Testing of Novel Compounds for the Prevention And/or Treatment of Alcohol Use Disorders Alcoholism, Clinical and Experimental Research. Jan, 2013  |   Pubmed ID: 22671690 Alcohol abuse and dependence have a staggering socioeconomic impact, yet current therapeutic strategies are largely inadequate to treat these disorders. Thus, the development of new strategies that can effectively prevent alcohol use disorders (AUDs) is of paramount importance. Currently approved medications attempt to deter alcohol intake by blocking ethanol metabolism or by targeting the neurochemical systems downstream of the cascades leading to craving and dependence. Unfortunately, these medications have provided only limited success as indicated by the continued high rates of alcohol abuse and alcoholism. The lack of currently available effective treatment strategies is highlighted by the urgent call by the NIAAA to find new and paradigm-changing therapeutics to either prevent or treat alcohol-related problems. This mini-review highlights recent findings from 4 laboratories with a focus on compounds that have the potential to be novel therapeutic agents that can be developed for the prevention and/or treatment of AUDs.
• Glycine and GABA(A) Ultra-sensitive Ethanol Receptors As Novel Tools for Alcohol and Brain Research Molecular Pharmacology. Dec, 2014  |   Pubmed ID: 25245406 A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, we developed a novel class of ultra-sensitive ethanol receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as four residues in the extracellular loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABA(A)Rs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode voltage clamp, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABA(A)Rs. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol.
• P2X4 Receptors (P2X4Rs) Represent a Novel Target for the Development of Drugs to Prevent And/or Treat Alcohol Use Disorders Frontiers in Neuroscience. 2014  |   Pubmed ID: 25009459 Alcohol use disorders (AUDs) have a staggering socioeconomic impact. Few therapeutic options are available, and they are largely inadequate. These shortcomings highlight the urgent need to develop effective medications to prevent and/or treat AUDs. A critical barrier is the lack of information regarding the molecular target(s) by which ethanol (EtOH) exerts its pharmacological activity. This review highlights findings implicating P2X4 receptors (P2X4Rs) as a target for the development of therapeutics to treat AUDs and discusses the use of ivermectin (IVM) as a potential clinical tool for treatment of AUDs. P2XRs are a family of ligand-gated ion channels (LGICs) activated by extracellular ATP. Of the P2XR subtypes, P2X4Rs are expressed the most abundantly in the CNS. Converging evidence suggests that P2X4Rs are involved in the development and progression of AUDs. First, in vitro studies report that pharmacologically relevant EtOH concentrations can negatively modulate ATP-activated currents. Second, P2X4Rs in the mesocorticolimbic dopamine system are thought to play a role in synaptic plasticity and are located ideally to modulate brain reward systems. Third, alcohol-preferring (P) rats have lower functional expression of the p2rx4 gene than alcohol-non-preferring (NP) rats suggesting an inverse relationship between alcohol intake and P2X4R expression. Similarly, whole brain p2rx4 expression has been shown to relate inversely to innate 24 h alcohol preference across 28 strains of rats. Fourth, mice lacking the p2rx4 gene drink more EtOH than wildtype controls. Fifth, IVM, a positive modulator of P2X4Rs, antagonizes EtOH-mediated inhibition of P2X4Rs in vitro and reduces EtOH intake and preference in vivo. These findings suggest that P2X4Rs contribute to EtOH intake. The present review summarizes recent findings focusing on the P2X4R as a molecular target of EtOH action, its role in EtOH drinking behavior and modulation of its activity by IVM as a potential therapy for AUDs.
• Multiday Administration of Ivermectin is Effective in Reducing Alcohol Intake in Mice at Doses Shown to Be Safe in Humans Neuroreport. Sep, 2014  |   Pubmed ID: 25004078 Ivermectin (IVM), an FDA approved anthelmintic agent, can significantly reduce ethanol intake in mice following acute administration. The current study evaluates the sustainability and safety of multiday IVM administration in reducing 10% v/v ethyl alcohol (10E) intake in mice at a dose shown to be safe in humans. We tested the effect of 10-day administration of IVM (3.0 mg/kg/day; intraperitoneally) on reducing 10E intake in C57BL/6J mice using a 24-h, two-bottle choice paradigm. On the 10th day of IVM administration, mice were sacrificed at 0, 0.5, 2, 8, 32, 48, and 72 h after injection. Brain tissue and plasma samples were collected and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Analysis of variance (ANOVA) was used to assess the effect of 10-day IVM administration on 10E intake, 10E preference, water intake, and total fluid intake with Dunnett's multiple comparison post-hoc test. Individual Student's t-tests were also used to further quantify changes in these dependent variables. IVM significantly decreased 10E intake over a 9-day period (P
• Contribution of P2X4 Receptors to Ethanol Intake in Male C57BL/6 Mice Neurochemical Research. Jun, 2014  |   Pubmed ID: 24671605 P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels activated by synaptically released extracellular adenosine 5'-triphosphate. The P2X4 subtype is abundantly expressed in the central nervous system and is sensitive to low intoxicating ethanol concentrations. Genetic meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake and/or preference. The current study used mice lacking the p2rx4 gene (knockout, KO) and wildtype (WT) C57BL/6 controls to test the hypothesis that P2X4Rs contribute to ethanol intake. The early acquisition and early maintenance phases of ethanol intake were measured with three different drinking procedures. Further, we tested the effects of ivermectin (IVM), a drug previously shown to reduce ethanol's effects on P2X4Rs and to reduce ethanol intake and preference, for its ability to differentially alter stable ethanol intake in KO and WT mice. Depending on the procedure and the concentration of the ethanol solution, ethanol intake was transiently increased in P2X4R KO versus WT mice during the acquisition of 24-h and limited access ethanol intake. IVM significantly reduced ethanol intake in P2X4R KO and WT mice, but the degree of reduction was 50 % less in the P2X4R KO mice. Western blot analysis identified significant changes in γ-aminobutyric acidA receptor α1 subunit expression in brain regions associated with the regulation of ethanol behaviors in P2X4R KO mice. These findings add to evidence that P2X4Rs contribute to ethanol intake and indicate that there is a complex interaction between P2X4Rs, ethanol, and other neurotransmitter receptor systems.
• Avermectins Differentially Affect Ethanol Intake and Receptor Function: Implications for Developing New Therapeutics for Alcohol Use Disorders The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP). Jun, 2014  |   Pubmed ID: 24451653 Our laboratory is investigating ivermectin (IVM) and other members of the avermectin family as new pharmaco-therapeutics to prevent and/or treat alcohol use disorders (AUDs). Earlier work found that IVM significantly reduced ethanol intake in mice and that this effect likely reflects IVM's ability to modulate ligand-gated ion channels. We hypothesized that structural modifications that enhance IVM's effects on key receptors and/or increase its brain concentration should improve its anti-alcohol efficacy. We tested this hypothesis by comparing the abilities of IVM and two other avermectins, abamectin (ABM) and selamectin (SEL), to reduce ethanol intake in mice, to alter modulation of GABAARs and P2X4Rs expressed in Xenopus oocytes and to increase their ability to penetrate the brain. IVM and ABM significantly reduced ethanol intake and antagonized the inhibitory effects of ethanol on P2X4R function. In contrast, SEL did not affect either measure, despite achieving higher brain concentrations than IVM and ABM. All three potentiated GABAAR function. These findings suggest that chemical structure and effects on receptor function play key roles in the ability of avermectins to reduce ethanol intake and that these factors are more important than brain penetration alone. The direct relationship between the effect of these avermectins on P2X4R function and ethanol intake suggest that the ability to antagonize ethanol-mediated inhibition of P2X4R function may be a good predictor of the potential of an avermectin to reduce ethanol intake and support the use of avermectins as a platform for developing novel drugs to prevent and/or treat AUDs.
• Structural Models of Ligand-gated Ion Channels: Sites of Action for Anesthetics and Ethanol Alcoholism, Clinical and Experimental Research. Mar, 2014  |   Pubmed ID: 24164436 The molecular mechanism(s) of action of anesthetic, and especially, intoxicating doses of alcohol (ethanol [EtOH]) have been of interest even before the advent of the Research Society on Alcoholism. Recent physiological, genetic, and biochemical studies have pin-pointed molecular targets for anesthetics and EtOH in the brain as ligand-gated ion channel (LGIC) membrane proteins, especially the pentameric (5 subunit) Cys-loop superfamily of neurotransmitter receptors including nicotinic acetylcholine (nAChRs), GABAA (GABAA Rs), and glycine receptors (GlyRs). The ability to demonstrate molecular and structural elements of these proteins critical for the behavioral effects of these drugs on animals and humans provides convincing evidence for their role in the drugs' actions. Amino acid residues necessary for pharmacologically relevant allosteric modulation of LGIC function by anesthetics and EtOH have been identified in these channel proteins. Site-directed mutagenesis revealed potential allosteric modulatory sites in both the trans-membrane domain (TMD) and extracellular domain (ECD). Potential sites of action and binding have been deduced from homology modeling of other LGICs with structures known from crystallography and cryo-electron microscopy studies. Direct information about ligand binding in the TMD has been obtained by photoaffinity labeling, especially in GABAA Rs. Recent structural information from crystallized procaryotic (ELIC and GLIC) and eukaryotic (GluCl) LGICs allows refinement of the structural models including evaluation of possible sites of EtOH action.
• Chronic Ethanol Exposure Combined with High Fat Diet Up-regulates P2X7 Receptors That Parallels Neuroinflammation and Neuronal Loss in C57BL/6J Mice Journal of Neuroimmunology. Aug, 2015  |   Pubmed ID: 26198936 The present investigation tested the role of ATP-activated P2X7 receptors (P2X7Rs) in alcohol-induced brain damage using a model that combines intragastric (iG) ethanol feeding and high fat diet in C57BL/6J mice (Hybrid). The Hybrid paradigm caused increased levels of pro-inflammatory markers, changes in microglia and astrocytes, reduced levels of neuronal marker NeuN and increased P2X7R expression in ethanol-sensitive brain regions. Observed changes in P2X7R and NeuN expression were more pronounced in Hybrid paradigm with inclusion of additional weekly binges. In addition, high fat diet during Hybrid exposure aggravated the increase in P2X7R expression and activation of glial cells.
• Oral Delivery of Ivermectin Using a Fast Dissolving Oral Film: Implications for Repurposing Ivermectin As a Pharmacotherapy for Alcohol Use Disorder Alcohol (Fayetteville, N.Y.). Sep, 2015  |   Pubmed ID: 26095588 Individuals suffering from an alcohol-use disorder (AUD) constitute a major health concern. Preclinical studies in our laboratory show that acute and chronic intraperitoneal (i.p.) administration of ivermectin (IVM) reduces alcohol intake and preference in mice. To enable clinical investigation to use IVM for the treatment of an AUD, development of an oral formulation that can be used in animals as well as long-term preclinical toxicology studies are required. The present work explores the use of a promising alternative dosage form of IVM, fast-dissolving oral films (Cure Pharmaceutical®), to test the efficacy and safety of oral IVM in conjunction with alcohol exposure. We tested the effect of IVM (0.21 mg) using a fast-dissolving oral film delivery method on reducing 10% v/v alcohol (10E) intake in female C57BL/6 mice using a 24-h access two-bottle choice paradigm for 6 weeks (5 days per week). Differences in ethanol intake, preference for ethanol, water intake, fluid intake, food intake, changes in mouse and organ weights, as well as histological changes to kidney, liver, and brain were analyzed. The IVM group drank significantly less ethanol over the 30-day period compared to the placebo (blank strip) and the no-treatment groups. Organ weights did not differ between the groups. Histological evaluation showed no differences in the brain and kidney between groups. In the liver, there was a slight increase in the incidence of microvesicular fatty and degenerative changes of the animals receiving the thin strips. No overt hepatocellular necrosis or perivascular inflammation was noted. Overall, these data support the use of this novel method of oral drug delivery for longer-term studies and should facilitate FDA required preclinical testing that is necessary to repurpose IVM for treatment of an AUD.
• The Importance of Animals in Advancing Research on Alcohol Use Disorders Alcoholism, Clinical and Experimental Research. Apr, 2015  |   Pubmed ID: 25833015