Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity.

Maya Popova,Larry Rodriguez,Michael Bloomfield,Daryl L Davies,James R Trudell,Sylvia Nguyen,Liana Asatryan

doi:10.3390/ijms21072471

Abstract

Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.

Highlights

Alcohol abuse and alcoholism continues to present a serious health and economic burden worldwide [1,2], due in part to the lack of effective medications for treatment and/or prevention
Within the P2XR family, P2X4 receptors (P2X4Rs) are the most ethanol-sensitive subtype; in vitro, P2X4Rs are inhibited by behaviorally relevant concentrations of ethanol (e.g., 17 mM which is equal to 0.08% blood ethanol, or the legal limit to drive in the United States of America) [10,11,12,13]. These findings indicate that P2X4Rs and ethanol are related in the behavioral, cellular, and molecular level, and represent an important therapeutic target for pharmacological strategies aimed at the prevention and/or treatment of alcohol use disorder (AUD)
Amino acid residues within the TM1 segment of rat P2X4Rs, positions 29 through 49, were individually mutated to alanine (A), excluding alanine residues at positions 34 and 41, which were mutated to tryptophan (W)

Summary

Introduction

Alcohol abuse and alcoholism (alcohol use disorder: AUD) continues to present a serious health and economic burden worldwide [1,2], due in part to the lack of effective medications for treatment and/or prevention. Medication development is complicated by a lack of understanding of the targets, target sites, and mechanisms of ethanol action (i.e., alcohol intoxication) in the central nervous system (CNS). Growing evidence suggests that ethanol affects purinergic P2X4 receptors (P2X4Rs), a member of the P2XR superfamily (for review, see [3]). Of the seven family members, P2X4Rs are the most abundant P2XR subtype in the CNS [4,5]. Gene expression profiling studies have suggested that P2X4R expression may be involved in innate alcohol preference, and, reduced regional levels of p2rx mRNA were found in alcohol-preferring vs alcohol non-preferring rats [7] as well as high alcohol drinking vs low alcohol drinking rats [8]

Methods

Results

Discussion

Conclusion