Heteromultimeric P2X(1/2) receptors show a novel sensitivity to extracellular pH

Abstract

Rat P2X(1) and P2X(2) subunits were coexpressed in defolliculated Xenopus oocytes and the resultant P2X receptors studied under voltage-clamp conditions. Extracellular ATP elicited biphasic inward currents, involving an initial rapidly inactivating (P2X(1)-like) component and a later slowly inactivating (P2X(2)-like) component. The maximum amplitude of P2X(1)-like ATP responses was increased in some cells by lowering extracellular pH (from 7.5 to 6.5), whereas P2X(2)-like responses and those of homomeric rP2X(1) and rP2X(2) receptors were not changed by this treatment. Concentration-response (C/R) curves for ATP for pH-enhanced P2X(1)-like responses were biphasic, and clearly distinct from monophasic ATP C/R curves for homomeric rP2X(1) and rP2X(2) receptors. Under acidic (pH 5.5 and 6.5) and alkaline (pH 8.5) conditions, ATP C/R curves for P2X(1)-like responses showed increases in agonist potency and efficacy, compared with data at pH 7.5, but the same was not true of homomeric rP2X(1) and rP2X(2) receptors. ATP C/R curves for P2X(2)-like responses overlay C/R curves for homomeric rP2X(2) receptors, and determinations of agonist potency and efficacy were identical for P2X(2)-like and P2X(2) responses at all pH levels tested. Our results show that P2X(1)-like responses possessed the kinetics of homomeric P2X(1) receptors but an acid sensitivity different from homomeric P2X(1) and P2X(2) receptors. In contrast, the P2X(2)-like responses exactly matched the profile expected of homomeric P2X(2) receptors. Thus, coexpression of P2X(1) and P2X(2) subunits yielded a mixed population of homomeric and heteromeric P2X receptors, with a subpopulation of novel pH-sensitive P2X receptors showing identifiably unique properties that indicated the formation of heteromeric P2X(1/2) ion channels.

Fig. 1

Effect of extracellular pH on evoked biphasic responses. In A, example of a subset of rP2X₁/rP2X₂ cRNA-coinjected oocytes (73 of 87 cells sampled) that responded to extracellular ATP (100 μM) with biphasic inward currents, where the initial P2X₁-like response (open arrows) was inhibited by lowering extracellular pH from 7.5 to 6.5, and the later P2X₂-like response (closed arrows) largely unaffected. Both records in A from the same cell (V_h = −60 mV). B, example of another subset of coinjected oocytes (14 of 87 cells sampled) where the P2X₁-like response was potentiated by lowering extracellular pH from 7.5 to 6.5, and later P2X₂-like response unaffected. Both records in B from another cell, but same batch as oocyte A (V_h = −60 mV).

Fig. 2

Agonist responses in oocytes expressing pH-sensitive P2X assemblies. A, concentration-dependent inward currents to ATP (10 nM-300 μM, for 30 s, and 20 min apart) recorded from a single rP2X₁/rP2X₂ cRNA-coinjected oocyte (V_h = −60 mV). Monophasic P2X₁-like responses were evoked by low ATP concentrations (< 3 μM), and biphasic (P2X₁-like and P2X₂-like) responses by higher ATP concentrations (3–300 μM). B, inward currents evoked by a saturating concentration of ATP (100 μM) and by known homomeric rP2X₁ receptor agonists Ap₆A (30 μM) and α,β-meATP (30 μM) from a single rP2X₁/rP2X₂ cRNA-coinjected oocyte. Records in B from the same cell (V_h = −60 mV).

Fig. 3

Agonist potency at P2X subunit assemblies. A, C/R curves for rapidly inactivating P2X₁-like responses evoked by ATP, Ap₆A and α,β-meATP in oocytes coexpressing rP2X₁ and rP2X₂ subunits. Data given as mean ± S.E.M. (6–9 sets of observations). B, C/R curves for ATP, Ap₆A and α,β-meATP in oocytes expressing homomeric P2X₁ receptors (n = 4–8). Where missing, error bars are occluded by symbols. Curves fitted to the Hill equation, by using Prism version 2.0 (GraphPad Software).

Fig. 4

Effects of extracellular pH of ATP activity at P2X₁-like responses. A, C/R curves for rapidly inactivating P2X₁-like responses evoked by ATP at four levels of extracellular pH (8.7, 7.5, 6.5, and 5.5) Data given as mean ± S.E.M. (4–9 sets of observations). B to D, C/R curves are redrawn to compare the effects of test pH levels (B, pH 8.5; C, pH 6.5; D, pH 5.5) against control data (pH 7.5). Curves fitted to the Hill equation, by using Prism version 2.0 (GraphPad Software). Data compared by Student’s unpaired t test (N.S., not significantly different; ⋆, p < 0.05; ⋆⋆, p < 0.01).

Fig. 5

Effects of extracellular pH of ATP activity at P2X₂-like responses. A, C/R curves for slowly inactivating inward currents evoked by ATP in oocytes either coexpressing P2X₁ and P2X₂ subunits or expressing homomeric rP2X₂ receptors. ATP activity was assessed at pH 7.5 and again at pH 6.5. Curves fitted to the Hill equation, by using Prism version 2.0 (GraphPad Software). B, relative amplitude of maximum P2X₂-like responses to ATP (100 μM), at three test pH levels (8.5, 6.5, and 5.5), compared with control data (pH 7.5), in oocytes coexpressing P2X₁ and P2X₂ subunits. Data given as mean ± S.E.M. (4–9 observations). Data compared by Student’s unpaired t test (N.S., not significantly different).

Fig. 6

Reproducibility of P2X₁-like and P2X₁-like responses. ATP (100 μM, for 30 s, and 20 min apart) evoked biphasic inward currents of consistent amplitude, at either pH 7.5 or pH 6.5, in oocytes coexpressing rP2X₁ and rP2X₂ subunits. The reproducibility of P2X₁-like responses at each pH_e level supported the conclusion that pH potentiation was not due to a relaxation of rP2X₁ receptor desensitization; the maximum amplitude of P2X₂-like responses was not significantly affected by extracellular pH (Fig. 5B).