Molecular Weight and Volume at Position 46 in Transmembrane Domain 1 (TM1) are Important Determinants for Ethanol Sensitivity in P2X4 Receptors

Purinergic P2X receptors (P2XRs) are a family of fast acting, cation-permeable ligand-gated ion channels, which are gated by synaptically released adenosine 5′-triphosphate (ATP). Building evidence supports the notion that P2XRs play a role in mediating and/or modulating behavioral effects of ethanol. However, this work is in the early stages and the sites and mechanisms of ethanol action in P2XRs are poorly understood. Recently, we identified an amino acid located in TM1 that was conserved across P2X3 and P2X4Rs that appeared to represent an important site for the action of ethanol (i.e., Trp41 and Trp46, respectively). The current study tests the hypothesis that physical-chemical properties of the residue at position 46 play a significant role in determining ethanol sensitivity in P2X4Rs. We expressed wildtype and mutated receptors in Xenopus oocytes and determined changes in ethanol sensitivity (200mM) using an 8-channel two-electrode voltage clamp system (OpusXpress 6000; -70mV). Exchanging Trp46 residue with other aromatic residues did not significantly alter ethanol sensitivity whereas replacing Trp46 with aliphatic residues significantly reduced the action of ethanol. Correlation analysis determined that molecular weight and volume of the residue at position 46 were significantly correlated with ethanol sensitivity whereas polarity or hydrophobicity was not. The findings suggest that the size and aromaticity of the residue at position 46 in TM1 play an important role in determining ethanol sensitivity of P2X4Rs. Identifying physical-chemical properties of residues that are important for ethanol sensitivity will increase our knowledge regarding structural requirements that are necessary for ethanol to cause changes in modulation and/or transduction of agonist action in P2X4Rs. Support: NIAAA/NIH F31 AA017029, KO1 AA017243-01A1, AA013922, AA03972 and USC School of Pharmacy.