Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor

Abstract

P2X receptors are ATP-gated cation channels that are widely expressed in the brain. The extracellular domains of all seven P2X receptors contain 10 conserved cysteines, which could form disulfide bonds or binding sites for transition metals that modulate P2X receptors. To test whether these cysteines are critical for receptor function, we studied wild-type rat P2X(2) receptors and 10 mutant P2X(2) receptors, each containing an alanine substituted for a cysteine. Nine mutants were functional but had reduced maximum currents compared with wild-type P2X(2) expressed in either Xenopus oocytes or human embryonic kidney (HEK) 293 cells. The 10th mutant (C224A) did not respond to ATP when expressed in oocytes and gave very small currents in HEK 293 cells. Seven mutants (C113A, C124A, C130A, C147A, C158A, C164A, and C214A) showed rightward shifts (9- to 30-fold) in their ATP concentration-response relationships and very little potentiation by zinc. In contrast, C258A and C267A had EC(50) values similar to those of wild-type P2X(2) and were potentiated by zinc. Acidic pH potentiated wild-type and all mutant receptor currents. Despite the loss of zinc potentiation in seven mutants, these cysteines are unlikely to be exposed in the zinc-binding site, because [2-(trimethylammonium)ethyl] methanethiosulfonate bromide did not prevent zinc potentiation of wild-type receptor currents. On the basis of correlations in the maximum current, EC(50), zinc potentiation, and pH potentiation, we suggest that the following cysteine pairs form disulfide bonds: C113-C164, C214-C224, and C258-C267. We also suggest that C124, C130, C147, and C158 form two disulfide bonds, but we are unable to assign specific cysteine pairs to these two bonds.

Fig. 1.

Effect of DTT and MTSET on P2X₂receptor currents. A, A 5 min incubation with 10 mm DTT did not affect the current responses of oocytes expressing P2X₂ to 10 μm ATP.B, The response of P2X₂ receptors to 10 μm ATP was potentiated fourfold by 5 μmzinc, and a 5 min incubation with 1 mm MTSET did not affect the potentiation of P2X₂ receptor currents by 5 μm zinc. C, Potentiation of P2X₂ receptor currents by 5 μm zinc was reduced but not eliminated by a 5 min incubation with 10 mmDTT followed by a 2 min wash. D, Recovery of zinc potentiation after treatment with DTT. The closed circles show the potentiation of P2X₂ receptor responses to 10 μm ATP by 5 μm zinc after a 5 min incubation with 10 mm DTT and variable wash times (n = 5–8 per time point; the error bars were smaller than the size of the circles). For comparison, the average zinc potentiation of P2X₂ receptors is shown as an open circle at the first time point and extrapolated as a dashed line for other time points (n = 7).

Fig. 2.

Zinc potentiation was greatly reduced or eliminated in seven cysteine mutants. A, Zinc potentiation of ATP-induced currents of single oocytes expressing wild-type and mutant receptors. The ATP concentration used for each mutant (∼EC₁₀) is indicated in B. The zinc concentration was 20 μm. B, Mean zinc potentiation ratio of 6–19 oocytes per construct. The zinc potentiation ratio is the ratio of the current amplitude in the presence of ATP and 20 μm zinc to the current amplitude of ATP alone. The dashed line indicates no potentiation. Asterisks indicate values significantly different from wild type (p < 0.01).

Fig. 3.

Zinc-resistant mutants were significantly potentiated by acidic pH (a pH of 6.5). A, Potentiation of ATP-induced currents of single oocytes expressing wild-type and mutant receptors by acidic pH. The ATP concentration used for each mutant (∼EC₁₀) is indicated in Band for each mutant was the same concentration as used to test zinc potentiation in Figure 2. B, Mean potentiation of 6–19 oocytes per construct. The pH potentiation ratio is the ratio of the current amplitude in the presence of ATP at a pH of 6.5 to the current amplitude in the presence of ATP at a pH of 7.5. The dashed line indicates no potentiation. Asterisksindicate potentiation ratios significantly different from wild type (p < 0.01).

Fig. 4.

The concentration–response relationships for ATP of the cysteine mutants. Concentration–response relationships were determined as described in Materials and Methods. Each point represents the mean ± SEM. The EC₅₀ and Hill coefficients of these fits are summarized in Table 1.

Fig. 5.

Model of the P2X₂ receptor. All of the cysteines are shown as open circles. The solid lines indicate three proposed disulfide bonds. Thedotted box connects four cysteines that are proposed to form two additional disulfide bonds but among which we were unable to predict the pattern of pairing. The black circlesindicate two histidines (120 and 213) that are suggested by Clyne et al. (2002) to form part of a site that binds zinc (Zn). Thehatched circles indicate three lysines (69, 71, and 308) suggested to bind to the phosphates of ATP (asterisks) (Jiang et al., 2000; Ennion et al., 2002). The transmembrane domains (residues F31 to V51 and I331 to L353) are represented ascylinders, all other amino acids are indicated asgray circles, and the threeN-glycosylation sites of P2X₂ are shown asY shapes.