Coexpression of rat P2X2 and P2X6 subunits in Xenopus oocytes

Abstract

Transcripts for P2X(2) and P2X(6) subunits are present in rat CNS and frequently colocalize in the same brainstem nuclei. When rat P2X(2) (rP2X(2)) and rat P2X(6) (rP2X(6)) receptors were expressed individually in Xenopus oocytes and studied under voltage-clamp conditions, only homomeric rP2X(2) receptors were fully functional and gave rise to large inward currents (2-3 microA) to extracellular ATP. Coexpression of rP2X(2) and rP2X(6) subunits in Xenopus oocytes resulted in a heteromeric rP2X(2/6) receptor, which showed a significantly different phenotype from the wild-type rP2X(2) receptor. Differences included reduction in agonist potencies and, in some cases (e.g., Ap(4)A), significant loss of agonist activity. ATP-evoked inward currents were biphasic at the heteromeric rP2X(2/6) receptor, particularly when Zn(2+) ions were present or extracellular pH was lowered. The pH range was narrower for H(+) enhancement of ATP responses at the heteromeric rP2X(2/6) receptor. Also, H(+) ions inhibited ATP responses at low pH levels (<pH 6.3). The pH-dependent blocking activity of suramin was changed at this heteromeric receptor, although the potentiating effect of Zn(2+) on ATP responses was unchanged. Thus, the rP2X(2/6) receptor is a functionally modified P2X(2)-like receptor with a distinct pattern of pH modulation of ATP activation and suramin blockade. Although homomeric P2X(6) receptors function poorly, the P2X(6) subunit can contribute to functional heteromeric P2X channels and may influence the phenotype of native P2X receptors in those cells in which it is expressed.

Fig. 1.

Expression of homomeric and heteromeric P2X receptors. A, Whole-cell inward currents by homomeric rP2X₂ and rP2X₆ receptors activated by a near-saturating ATP concentration (100 μm, for 60 sec), at the given holding potentials (V_h).B, Whole-cell inward currents by ATP-activated heteromeric rP2X_2/6 receptors. ATP responses were often biphasic, showing a transient component (filled arrow) followed by a sustained current. The deactivation of inward current occasionally showed two phases of current decay (open arrow). C, Averaged whole-cell inward currents by homomeric rP2X₂, rP2X₆, and heteromeric rP2X_2/6 receptors activated by ATP (100 μm). The y-axis of the histogram has been truncated to help reveal the small responses by rP2X₆ receptors. Data are expressed as mean ± SEM for six to seven cells per determination.

Fig. 2.

ATP activity at homomeric and heteromeric P2X receptors. A, C–R relationship for ATP-activated inward currents at rP2X₂ and rP2X_2/6 receptors, at pH 7.5. B, The relationship between the amplitude of ATP responses (rP2X₂, 3 μm; rP2X_2/6, 10 μm; each producing 5% of the maximum response) and the extracellular pH level (range, pH 8.3–5.0) at homomeric and heteromeric P2X receptors. C, The C–R curves for ATP activation of rP2X_2/6 receptors at the pH_e levels indicated. ATP efficacy was markedly reduced at pH 5.5. D, The C–R curves for ATP activation of rP2X₂ receptors. ATP efficacy was not altered at pH 5.5. Curves were fitted by the Hill equation inA–D (solid lines) and by a single exponential function in B (dashed line). Data given as mean ± SEM for four to six cells per curve.

Fig. 3.

Nucleotide activation of rP2X_2/6receptors. In A, whole-cell inward currents at the heteromeric rP2X_2/6 receptor were evoked by ATP, ATPγS, 2-MeSATP, ATPαS, and BzATP (30 μm), each of which is a known agonist of rP2X₂ receptors (King et al., 1997). InB, the rP2X_2/6 receptor was activated weakly by Ap₄A (30 and 300 μm), and the kinetics of activation and deactivation were considerably slower than ATP responses. C, C–R relationship for agonist activation of rP2X_2/6 receptors at pH 7.5. Estimates of EC₅₀ values (micromolar concentration) were made using the “2 + 2 assay” method of Arunlakshana and Schild (1959): ATP, 29.9 ± 1.9; ATPγS, 30.8 ± 2.9; ATPαS, 40.6 ± 8.0; 2-MeSATP, 34.8 ± 5.1; BzATP, 399 ± 66; Ap₄A, >1000 (n = 4–6). The dashed lineshows the position of the full C–R curve for ATP (redrawn from Fig.2A). Open and filled arrows (in A and B) draw attention to biphasic components of receptor activation and deactivation. Data are given as mean ± SEM for four to six cells per determination.

Fig. 4.

Suramin antagonism of rP2X_2/6receptors. Shown is antagonism of ATP responses (V_h = −50 mV) at heteromeric rP2X_2/6 receptors by suramin at pH 7.5 (A) and pH 6.5 (B). Suramin was effective at micromolar concentrations at pH 7.5, but the concentration range for suramin blockade was extended at pH 6.5.C, Inhibition curves for suramin blockade of ATP responses at rP2X₂ and rP2X_2/6 receptors at the given pH levels. At pH 6.5, the inhibition curve for rP2X_2/6 was fitted best by a biphasic curve. IC₅₀ values are given in Table 2. Open arrows draw attention to biphasic current decays. Data are expressed as mean ± SEM for four to eight cells per curve. The biphasic curve for rP2X_2/6 was constructed from eight sets of data, using the results from the first two log₁₀ units of concentration (suramin, 0.001–0.01 μm) to represent the first component of the inhibition curve.

Fig. 5.

Modulation of ATP responses by Zn²⁺ and H⁺ at rP2X_2/6 receptors. A, Concentration-dependent potentiation and inhibition of agonist-evoked inward currents by extracellular Zn²⁺ (1–100 μm) given 5 min before and during ATP application at rP2X_2/6 receptors, at pH 7.5. EC₅₀ values (micromolar concentration) for Zn²⁺ potentiation of ATP responses was rP2X_2/6, 6.8 ± 1.0 versus rP2X₂, 6.9 ± 1.1 (n = 4).B, Concentration-dependent potentiation of ATP-evoked inward currents by extracellular Zn²⁺ (1–100 μm) applied simultaneously with the agonist. Under these circumstances, biphasic currents were rarely seen, and the inhibitory action of Zn²⁺ was lost. EC₅₀ values (micromolar concentration) for Zn²⁺ potentiation of ATP responses were rP2X_2/6, 8.2 ± 0.5 versus rP2X₂, 11.7 ± 2.8 (n = 6).C, Concentration-dependent potentiation and inhibition of ATP-evoked inward currents by extracellular H⁺ions (pH 7.0–5.5) at rP2X_2/6 receptors. pK_a values (−log₁₀[H⁺] causing 50% potentiation) were rP2X_2/6, 7.04 ± 0.05 versus rP2X₂, 7.05 ± 0.05 (n = 4). D, Paired biphasic inward currents evoked by ATP (100 μm, at pH 7.5) at rP2X_2/6 receptors with either Mg²⁺ or Ca²⁺ (1.8 mm) present in the bathing solution. Substitution of Ca²⁺ with Mg²⁺ resulted in a reduction of ATP potency [as shown for rP2X₂ receptors (King et al., 1997)] without significantly altering the appearance of biphasic currents. Filled arrows draw attention to transient component of ATP-evoked inward currents (A, C). Data are expressed as mean ± SEM for four to six cells per determination.