Preprint Article Version 1 This version is not peer-reviewed

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

Version 1 : Received: 4 March 2020 / Approved: 6 March 2020 / Online: 6 March 2020 (04:45:49 CET)

A peer-reviewed article of this Preprint also exists.

Popova, M.; Rodriguez, L.; Trudell, J.R.; Nguyen, S.; Bloomfield, M.; Davies, D.L.; Asatryan, L. Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity. Int. J. Mol. Sci. 2020, 21, 2471. Popova, M.; Rodriguez, L.; Trudell, J.R.; Nguyen, S.; Bloomfield, M.; Davies, D.L.; Asatryan, L. Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity. Int. J. Mol. Sci. 2020, 21, 2471.

Journal reference: Int. J. Mol. Sci. 2020, 21, 2471
DOI: 10.3390/ijms21072471

Abstract

Mouse models of alcohol use disorder (AUD) revealed a subtype of purinergic receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)-ectodomain interface and within 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 TM segments, which are important for 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 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. Further work with mixtures of wild-type (WT) and reciprocal single (R33D:WT, D354R:WT) and double (R33D-D354R:WT) mutants confirmed the importance of this interaction for ethanol sensitivity, ivermectin action and channel function. Additionally, our findings suggest that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility during channel opening. Collectively, these findings identified new activity sites, and suggest the importance of TM1-TM2 interaction for channel function and ethanol sensitivity of P2X4Rs.

Subject Areas

purinergic P2X4 receptor; TM1 and TM2 segments; ethanol and agonist action; mutagenesis; molecular model

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