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Appendix  S1   Experimental procedures.

Fig.  S1   Schematicrepresentation of a P2X receptor subunit structure. A P2X subunit consists oftwo transmembrane (TM) domains, a large extracellular domain (ectodomain) andintracellular amino- (N) and carboxy (C)-terminals. The ectodomain areas, near TM domains, where the alanine scanning mutagenesis was performed are in gray and pointed to by arrows.

Fig.  S2   Concentration–response for ATP-activated currents in Xenopus oocytes expressing WT and (M336A and M336Q) P2X4Rs in the absence and presence of 200 mM ethanol. a) Co-application of ethanol significantly right-shifted the ATP –response curve of WT P2X4Rs in a parallel manner (ATP EC50:.3 ± 1.1 μM in the absence of ethanol; ATP EC50:.7 ± 1.2 μM in the presence of ethanol. Co-application of minimally right shifted the ATP concentration–response curve of the: (b) (M336A) P2X4Rs (ATP EC50: 7.6 ± 1.1 μM in absence of ethanol; ATP EC50: 8.7 ± 1.0 μM in the of ethanol) and (c) (M336Q) P2X4Rs (ATP EC50: 5.6 ± 1.2mu;M in the absence of ethanol; ATP EC50: 6.1 ± 1.2 μM the presence of ethanol). As expected, the maximal amplitude (Imax) and Hill’s slope values not differ from the ones reported in Table  S3. Data the mean ± SEM of at least four different oocytes. Error not visible are smaller than the size of the symbols.

Table  S1   Alignment of the -TM interfaces of P2X3 and P2X4 receptors. The non-conserved residues were mutated to alanine are highlighted in gray (A). Alignment of the of the ectodomain-TM1 interface. (B) Alignment of residues of the -TM2 interface.

Table  S2   Imax, 50, and Hill’s slope values for ATP concentration–response curves WT and mutant P2X4 receptors at the ectodomain-TM domain interfaces. Imax peak currents generated by 100 μM ATP. EC50 and Hill’s values were determined from ATP concentration–response curves. Data shown mean ± SEM of at least five experiments per data point.

Table  S3   Imax, 50, and Hill’s slope values for ATP concentration–response curves WT and D331 and M336 mutant P2X4 receptors. Imax represent currents generated by 100 μM ATP. EC50 and Hill’s slope were determined from ATP concentration–response curves. Data shown are  ± SEM of at least five experiments per data point; *p < 0.05 to the EC50 values of WT P2X4 receptors.

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