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

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Abstract

ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and two-electrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.

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Acknowledgments

We would like to thank Miriam Fine, Sylvia Nguyen, and Angeline Chen for technical and laboratorial assistance. This work was supported, in part, by research grants NIAAA/NIH F31 AA017029 (M.P.), KO1 AA017243 (L.A.), AA03972 (R.L.A.), AA013922 (D.L.D.), and AA013378 (J.R.T.) and the USC School of Pharmacy. This work was conducted as partial fulfillment of the requirements for the Ph.D. degree in Pharmaceutical Sciences, University of Southern California (M.P.).

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Authors and Affiliations

  1. Alcohol and Brain Research Laboratories, Titus Family Department of Clinical Pharmacy & Pharmaceutical Economics and Policy, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA, 90089, USA

    Maya Popova, Kaixun Li, Ronald Alkana, Daryl Davies & Liana Asatryan

  2. Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA, 90089, USA

    Ronald Alkana

  3. Department of Anesthesia and Beckman Program for Molecular and Genetic Medicine, Stanford School of Medicine, Stanford, CA, 94305, USA

    James Trudell

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  1. Maya Popova
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Correspondence to Liana Asatryan.

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Popova, M., Trudell, J., Li, K. et al. Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors. Purinergic Signalling 9, 621–632 (2013). https://doi.org/10.1007/s11302-013-9373-4

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