The role of histidine residues in modulation of the rat P2X(2) purinoceptor by zinc and pH

Abstract

P2X(2) receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X(2) receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC(50) of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 microM ATP of wild-type P2X(2) and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the pK(a) (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X(2) and revealed a substantial pH-dependent decrease in the maximum response with a pK(a) near 6.0. Substituting a lysine for H319 reduced the EC(50) for ATP 40-fold. Zinc (20 microM) potentiated the responses to 10 microM ATP of wild-type P2X(2) and seven histidine mutants by approximately 8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.

Figure 3. The ATP concentration-response relations for wild-type and H319A change with pH

A and D, representative current responses of single oocytes expressing P2X₂ (A) or H319A (D) to increasing concentrations of ATP at pH 7.5 and then at pH 6.5 after allowing a 5 min recovery between traces. The numbers and the filled and open bars above the graphs indicate the concentration and duration of ATP application. B and E, the ATP concentration-response relations for P2X₂ (B) and H319A (E) were derived from experiments like those shown in A and D. All currents were normalized to the maximum current amplitude of the initial concentration response at pH 7.5. The concentration-response relations at pH 7.5 following a 5 min incubation (pH 7.5 control) were similar to the initial concentration-response relations at pH 7.5. C and F, sample traces of the responses of oocytes expressing wild-type (C) or H319A (F) receptors to either 5 or 100 μm ATP at pH 7.5 (open bars) and pH 6.5 (filled bars). The time scales in C and F apply to both traces. The arrows in D and F indicate a transient increase in the current response of H319A after switching from a high concentration of ATP at pH 6.5 back to the recording solution without ATP at pH 7.5. For some points the error bars (s.e.m.) were smaller than the symbols.

Figure 5. Additional characterization of mutations at H319

A and B, comparison of the effect of zinc and pH on single oocytes expressing P2X₂ (A) or H319A (B). For P2X₂ the potentiation elicited by switching from 5 μm ATP at pH 7.5 to either 5 μm ATP plus 20 μm zinc at pH 7.5 or to 5 μm ATP alone at pH 6.5 was very similar, but for H319A the potentiation elicited by switching to acidic pH was much smaller than the potentiation that could be elicited by zinc. In addition, potentiation by 20 μm zinc was mostly occluded by pH 6.5 potentiation in P2X₂ but not in H319A. C, currents elicited from an oocyte expressing H319K in response to step concentration increases in ATP at pH 7.5. D, concentration-response relations of a series of oocytes from a single frog expressing P2X₂, H319A or H319K. Each graph is the average for 5–6 oocytes studied as in C. For some points the error bars (s.e.m.) were smaller than the symbols.

Figure 8. ATP concentration-response relations for P2X₂ (A), H120A (B) and H213A (C) with and without 20 μm zinc

After obtaining a concentration-response relation in the absence of zinc (○, No Zinc), 5 min was allowed for recovery and then a second concentration-response relation was obtained in either the absence (▵, No Zinc Control) or presence of 20 μm zinc (•). All currents were normalized to the maximum current amplitude of the initial concentration-response relation in the absence of zinc.

Figure 2. Potentiation of currents by protons was greatly diminished for H319A

The pH potentiation ratio is the ratio of the current amplitude in the presence of 10 μm ATP at pH 6.5 to the response at pH 7.5. Lines within the boxes indicate the median potentiation ratio (n = 7–27 oocytes for each mutant construct, n = 52 oocytes for wild-type). The upper and lower boundaries of each box plot demarcate the 75th and 25th percentiles, respectively. The horizontal lines above and below each box indicate the 90th and 10th percentiles. The dashed line indicates a ratio of 1 (no potentiation).

Figure 7. Zinc potentiation was greatly reduced in two histidine mutants, H120A and H213A

The zinc potentiation ratio is the ratio of the current amplitude in the presence of 10 μm ATP and 20 μm zinc to the current amplitude in response to 10 μm ATP alone. Lines within the boxes indicate the median potentiation ratio (n = 15–25 oocytes per mutant construct, 55 oocytes for wild-type). The upper and lower boundaries of each box demarcate the 75th and 25th percentiles, respectively. The horizontal lines above and below each box indicate the 90th and 10th percentiles. The dashed line indicates a ratio of 1 (no potentiation).

Figure 1. Potentiation of wild-type and mutant receptor currents by pH

Each trace is the response of a single oocyte expressing wild-type or mutant receptors to 10 μm ATP at pH 7.5 and pH 6.5. The bars above each trace indicate the duration of the applications. The time scale applies to all traces.

Figure 4. The relation between pH and current responses of P2X₂ and H319A receptors at 5 μm ATP (A) and 100 μm ATP (B)

The symbol legend in A also applies to B. All currents were normalized so that the maximum ratio of the fit was 100 %. The curves shown for responses to 5 μm ATP and for H319A at 100 μm ATP were fitted to the data using the Hill equation. To fit the data for potentiation of wild-type P2X₂ at 100 μm ATP, we scaled the data using the inhibitory pH-response relation of H319A at 100 μm. The fit of this adjusted pH response of P2X₂ receptor currents is shown as a dashed line in B.

Figure 6. Potentiation of wild-type and mutant receptors by zinc

Each trace is the response of a single oocyte expressing wild-type or mutant receptor to 10 μm ATP followed by 10 μm ATP with 20 μm zinc. The bars above each trace indicate the duration of the applications. The time scale applies to all traces.

Figure 9. Zinc concentration-response relations for H120A, H213A and wild-type P2X₂ in the presence of 10 μm ATP

All currents were normalized to the currents induced by ATP in the absence of zinc, and each point represents the mean ± s.e.m. of 5–13 oocytes. The continuous line represents a fit to all data from H120A and H213A (IC₅₀ = 112 μm, Hill coefficient = 1.38). The dashed line represents the predicted relation between zinc concentration and potentiation of wild-type P2X₂ (EC₅₀ = 16.5 μm, Hill coefficient = 0.92) based on the assumption that an inhibitory process identical to that seen in H120A and H213A also occurs in wild-type, but is partially masked by the potentiating effect of ATP.

Figure 10. The inhibition of H120A and H213A mutant receptor currents by high concentrations of zinc (200 μm) is voltage independent

A and B, the response of H120A (A) and H213A (B) to 10 μm ATP followed by 10 μm ATP with 200 μm zinc at two different potentials. The lengths of the bars above the graphs indicate the duration of application. C, percentage inhibition of mutant receptor currents by 200 μm zinc at different potentials.