Popova M , Rodriguez L , Trudell JR , Nguyen S , Bloomfield M , Davies DL , Asatryan L .

	Figure A1. ATP concentration-response curves for WT and select P2X4R mutants. (A) Hill slopes were significantly reduced for F48A, W46A and R33A mutants (See Table 1). There was a left-shift for F48A and W46A mutants in the ATP concentration-response curves and a tendency for right-shift for R33A compared to that of the WT receptor. (B) A right-shift in the ATP concentration-response curves shown for V49G and V49L mutants. (C) Position 33 mutants demonstrated WT-like ATP concentration responses. (D) WT-like ATP concentration responses for position 33 and 354 reciprocal mutant:WT receptor mixtures.
	Figure 1. Mutations at positions 49 and 33 do not significantly affect total and surface expression of mutant P2X4Rs. Representative Western Blots showing the P2X4R bands at ~60 kDA for total and biotinylated fractions for WT and mutant receptors.
	Figure 2. Effects of 10 mM and 100 mM ethanol in WT and TM1 segment mutant P2X4Rs. (A) A bar graph of ethanol responses. The response to 10 mM ethanol was significantly increased in the R33A and A34W mutants’ receptors, and the inhibitory effect to 100 mM ethanol was significant decreased in the W46A mutant receptor. The data are presented as mean ± SEM, n = 5–18. * p < 0.05 and ** p < 0.001 compared to ethanol effects in the WT P2X4R. (B) Representative ATP-induced current tracings for the WT and R33A mutant P2X4Rs; the effects of 10 and 100 mM ethanol are shown.
	Figure 3. Effects of ethanol (10–100 mM) in mutant receptors at position 33. (A) A bar graph of ethanol responses. Alanine or serine substitution at position 33 increased the inhibitory effect of ethanol at low concentrations (10 mM and 25 mM). The increases were significant for 10 mM but not 25 mM ethanol. The data are expressed as mean ± SEM, n = 4–17. * p < 0.05, ** p < 0.005 compared to ethanol effects in the WT P2X4R. (B) Representative ATP-induced current tracings for the WT, R33S, and R33K mutant P2X4Rs; the effects of 10 mM ethanol are shown.
	Figure 4. Homology models of rat P2X4Rs based on the closed (4DW0) and open (4DW1) zebrafish structures [22]. (A) A model illustrating the transmembrane (TM) segments in the closed state (red solid ribbon of backbone with residues at positions 33, 46, and 49 in the TM1 segment of one subunit as well as 354 in the TM2 segment of the neighboring subunit depicted as a ball and stick. Residue 33 of subunit 1 (S1) and 354 of subunit 2 (S2) face each other on the closed structure. (B) A model illustrating the TM segments in the open state (blue line ribbon of backbone) with the same residues shown in A. The residues at positions 33 (S1) and 354 (S2) face away from each other.
	Figure 5. Effects of reciprocal mutations at position 33 and/or 354 as well as the mixtures of reciprocal and WT on ethanol and ivermectin (IVM) responses. (A) A representative Western Blot showing the total and surface expression (~60 kDA) for R33D, D354R, and R33D-D354R mutant receptors. The expression of the mutants was not much different compared to the WT receptor. (B) A bar graph of the responses to 10–200 mM ethanol for mutant:WT receptor mixtures (at a ratio of 1:1). The data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01 compared to ethanol effects in the WT P2X4R. (C) A bar graph of the responses to 3 µM IVM for mutant:WT receptor mixtures. The data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01 compared to IVM effects in the WT P2X4R.

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