Roles of ectodomain and transmembrane regions in ethanol and agonist action in purinergic P2X2 and P2X3 receptors

Abstract

SUMMARYThe 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 α,β-methylene-ATP (α,β-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 α,β-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.

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

“…Previous investigations suggest that the interface between the ectodomain and the TM segments in P2X4Rs contains targets for ethanol and IVM action or modulation [17,23,24]. We have identified residues Asp331 and Met336 in the ectodomain-TM2 segment interface to be important for ethanol modulation, whereas residues Asn338, Ser341, Gly342, Leu346, Gly347, Ala349, and Ile356 in the TM2 segment were found to play a role in IVM modulation of the receptor [25,26].…”

Activation and Regulation of Purinergic P2X Receptor Channels

“…ATP is a full agonist for rP2X3R with estimated EC 50 values of 1.2 M (Chen et al, 1995), 2.6 M (Pratt et al, 2005), 4.1 M (Asatryan et al, 2008), and 7.3 M (Grote et al, 2005), but the precision of these estimates is limited by profound desensitization (Khmyz et al, 2008). Similar to the rP2X1R, ␣␤-meATP is a full agonist at rP2X3R, acting with a potency similar to (Pratt et al, 2005;Asatryan et al, 2008) or slightly lower than (Chen et al, 1995) that of ATP. 2-meSATP is also a full agonist for this receptor, whereas ATP␥S acts as a partial agonist Liu et al, 2001a).…”

“…In another study, His 241 was identified as a residue responsible for the ethanol sensitivity of P2X4Rs (Xiong et al, 2005). Experiments with chimeric P2X2R and P2X3R revealed that ectodomain segments at the TM interfaces play key roles in determining the qualitative and quantitative responses to ethanol (Asatryan et al, 2008). Further experiments by the same group identified Asp 331 and Met 336 as critical TM residues for the ethanol sensitivity of P2X4R .…”

Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors

“…Two-electrode voltage-clamp recordings of oocytes were performed with an oocyte clamp amplifier (model OC-725C; Warner Instruments, Hamden, CT) using procedures published previously (Asatryan et al, 2008;Popova et al, 2010).…”

“…Previous studies found that residues contained within the ectodomain-TM segment interfaces are important for ethanol ac-tion in P2X3Rs (Asatryan et al, 2008). Extending the investigation to P2X4Rs resulted in the identification of two key residues in the TM2 segment near the ectodomain interface (Asp331 and Met336) that, when substituted to alanine, caused a significant reduction in ethanol (10 -200 mM) inhibition of ATP-gated currents (Popova et al, 2010).…”