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FIGURE 1. Sequence line-up of the extracellular domain of human P2X1â7 receptors and the Dictostelium discoideum (Dict) P2X receptor (37). Residues conserved in at least 5 of the human P2X receptors are shown in red. Alanine or cysteine mutants of the P2X1 receptor that have no or less than a 10-fold change in ATP potency are shown in lowercase gray (12, 16). P2X1 receptor mutants with a greater than 10-fold decrease in ATP potency are shown in blue. Residues that have been suggested to be important in ATP action are boxed (12, 16). Cysteine mutants of P2X2 and P2X4 receptors characterized in this paper are shown in green.
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FIGURE 2. Effect of point cysteine P2X2 mutations on ATP potency. ATP potency was tested on WT and P2X2 receptor mutants expressed in oocytes by two-electrode voltage clamp (holding potential =â60 mV). A, trace data representative of currents observed for wild type and mutant receptors in response to ATP applications (indicated by the bar, concentrations in micromolar) at 5-min intervals. B, concentration-response curves to ATP for WT and mutants K69C, K71C, F183C, T184C, N288C, F289C, R290C, and K307C. K69C, K71C, N288C, and K307C with reduced peak currents in response to a maximal concentration of ATP, are expressed as a percentage of the amplitude of the WT maximum response, whereas all others are expressed as a percentage of their own maximum response (n = 3â10). C, Western blots of total and surface expression levels of WT and mutant P2X2 receptors with reduced peak current amplitudes. Lower panel shows densitometric analysis of the blots expressed as % of WT levels.
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FIGURE 3. Effect of MTS compounds on ATP responses of P2X2 cysteine mutants. A, trace data representative of currents observed before and during the addition of MTSEA (1 mm). ATP application (EC50 concentration) is indicated by the bar. MTSEA reagent was bath applied 5 min prior to U-tube ATP coapplication with MTSEA. B, graph representing the effect of MTSEA on the P2X2 WT and mutant receptors (n = 3â6). C, trace data representative of currents observed with the addition of MTSES (1 mm). ATP application (EC50 concentration) is indicated by the bar. MTSES reagent applied in a similar manner to MTSEA traces are shown for a given oocyte before and during MTSES application. D, graph representing the effect of MTSES on the P2X2 WT and mutant receptors (n = 3â6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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FIGURE 4. Effect of MTS compounds on ATP potency at P2X2 cysteine mutants. A, ATP concentration-response curve for R290C under control conditions or following washout of MTSEA after a 10-min incubation with MTSEA (1 mm)(n = 3â4). B, ATP concentration-response curve for K307C under control conditions or following washout of MTSEA after a 10-min incubation with MTSEA (1 mm)(n = 3â6). The data are expressed as a percentage of the amplitude of the response to 3 mm ATP under control conditions.
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FIGURE 5. Effect of point cysteine P2X4 mutations on ATP potency. ATP potency was tested on WT and P2X4 receptor mutants expressed in oocytes by two-electrode voltage clamp (holding potential = â60 mV). A, trace data representative of currents observed for wild type and mutant receptors in response to ATP applications (indicated by bar, concentration in micromolar) at 5-min intervals. B, concentration-response curves to ATP for WT and mutants K67C, K69C, F185C, T186C, N293C, F294C, R295C, and K313C. K67C, K69C, N293C, R295C, and K313C, with reduced peak currents in response to a maximal concentration of ATP, are all expressed as a percentage of peak WT maximum response whereas, all others are expressed as a percentage of their own maximum response (n = 3â4). C, Western blots of total and surface expression levels of WT and mutant P2X4 receptors with reduced peak current amplitudes. Lower panel shows densitometric analysis of the blots expressed as % of WT levels.
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FIGURE 6. Effect of MTS compounds on ATP responses of P2X4 cysteine mutants. A, trace data representative of currents observed on the addition of MTSEA (1 mm). ATP application (EC50 concentration) is indicated by the bar. MTSEA reagent was bath applied 5 min prior to U-tube ATP coapplication with MTSEA. Traces are shown in response to ATP for a given oocyte before and during the application of MTSEA. B, graph representing the effect of MTSEA on the P2X4 WT and mutant receptors (n = 3â8). C, trace data representative of currents observed on the addition of MTSES (1 mm). ATP application (EC50 concentration) is indicated by the bar. MTSES reagent was applied in a similar manner to MTSEA. Traces are shown in response to ATP for a given oocyte before and during the application of MTSES. D, graph representing the effect of MTSES on the P2X4 WT and mutant receptors (n = 3â6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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FIGURE 7. Effect of MTS compounds on ATP potency at P2X4 cysteine mutants. A, MTSEA significantly increased the potency of ATP and amplitude of responses at K67C mutants. B, MTSEA significantly increased the potency of ATP and amplitude of responses at K69C mutants. C, MTSEA significantly increased ATP potency and peak response at K313C mutants (n = 3â4). The data are expressed as a percentage of the amplitude of the response to 3 mm ATP. For MTSEA treatment (1 mm, 1 h incubation, followed by washout and construction of the concentration response in the absence of MTSEA) these have been corrected with a scaling factor corresponding to the mean -fold increase, at 3 mm ATP, in currents compared with untreated oocytes.
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FIGURE 8. Model of the ATP binding site at P2X receptors. Portions of the extracellular loop adjacent to either the first (in gray) or second (in black) transmembrane domain are shown for two adjacent P2X receptor subunits (numbering for the P2X4 receptor). The gray arrow represents the disulfide bond that can be formed between cysteine mutations of these residues at the P2X1 receptor (18). Residues that showed a decrease in ATP potency when mutated to cysteine are shown, those in black correspond to those that were modified by MTS reagents, whereas those in gray were unaffected by either MTSEA or MTSES. Positively charged MTSEA increased the amplitude and potency of responses at cysteine mutants of the P2X4 receptor at positions Lys69, Lys71 and Lys313, this suggests a role of these residues in coordinating the binding of the negatively charged phosphate of ATP, however, it is possible that they also contribute to the gating of the channel as has been suggested for the residue equivalent to Lys313 at the P2X2 receptor (32). The conserved FT region is shown as a dotted line as it has been shown to be involved in agonist potency and is unclear whether the effects result from changes in agonist binding and/or gating. One possibility is that threonine could face the agonist binding pocket.
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