P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes

Abstract

The intracellular signalling pathways and molecular mechanisms responsible for P2-purinoceptor-mediated chloride (Cl(-)) currents (I(Cl,ATP)) were studied in mouse ventricular myocytes. In standard NaCl-containing extracellular solutions, extracellular ATP (100 microm) activated two different currents, I(Cl,ATP) with a linear I-V relationship in symmetrical Cl(-) solutions, and an inwardly rectifying cation conductance (cationic I(ATP)). Cationic I(ATP) was selectively inhibited by Gd(3+) and Zn(2+), or by replacement of extracellular NaCl by NMDG; I(Cl,ATP) was Cl(-) selective, and inhibited by replacement of extracellular Cl(-) by Asp(-); both currents were prevented by suramin or DIDS pretreatment. In GTPgammaS-loaded cells, I(Cl,ATP) was irreversibly activated by ATP, but cationic I(ATP) was still regulated reversibly. GDPbetaS prevented activation of the I(Cl,ATP,) even though pertussis toxin pretreatment did not modulate I(Cl,ATP). These results suggest that activation of I(Cl,ATP) occurs via a G-protein coupled P2Y purinergic receptor. The I(Cl,ATP) persistently activated by GTPgammaS, was inhibited by glibenclamide but not by DIDS, thus exhibiting known pharmacological properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. In ventricular cells of cftr(-/-) mice, extracellular ATP activated cationic I(ATP), but failed to activate any detectable I(Cl,ATP). These results provide compelling evidence that activation of CFTR Cl(-) channels in mouse heart are coupled to G-protein coupled P2Y purinergic receptors.

Figure 1. Cation sensitivity of extracellular ATP-induced membrane currents in mouse ventricular myocytes

A, time course of extracellular ATP-induced whole-cell currents at +80 mV (•) and −80 mV (○) in single mouse ventricular cells exposed to standard extracellular solution during application of 100 μm ATP to the bath. [Na⁺]_o and [Cs⁺]_o were replaced with equimolar [NMDG⁺]_o during the period indicated by the bar. Here and in subsequent similar figures, the pulse protocol is the same as shown in the inset of A. B, whole-cell current recordings induced by applying 400 ms voltage-clamp steps to membrane potentials between −100 mV and +100 mV in +20 mV steps from a holding potential of 0 mV every 2 s. In subsequent similar figures, the step-pulse protocol is the same as shown in the inset of B. Currents were recorded at the time points indicated in A. C, mean I–V relationships of [NMDG⁺]_o-sensitive (b–c) and -insensitive (c–a) currents in 6 different cells.

Figure 2. Anion sensitivity of extracellular ATP-induced membrane currents in mouse ventricular myocytes

A, time course of extracellular ATP-induced whole-cell currents in NMDG-Cl solutions during application of 100 μm ATP to the bath. [Cl⁻]_o was replaced to equimolar [Asp⁻]_o during the period indicated by the bar. B, whole-cell current recordings at the time points b (Cl⁻) and c (Asp⁻) in A. C, I–V relationships of currents recorded at the time points indicated in A. D, reversal potential–log [Cl⁻]_o relationships of whole-cell currents in the presence of extracellular ATP in 4 different cells in which [Cl⁻]_o was changed from 90 to 10 mm in [NMDG⁺]-rich conditions. D, dose–response relationships of anion-sensitive [NMDG⁺ solutions] extracellular ATP-induced (difference) currents in 4 different cells. Responses were normalized to the maximum current density obtained at 100 μm ATP. The EC₅₀ and n_H coefficient correspond to the fitted curve.

Figure 3. Role of G-protein coupled purinergic receptors in activation of I_Cl,ATP

A, time course of extracellular ATP-induced whole-cell currents in standard extracellular solution during application of 100 μm ATP to the bath. The tested cell was dialysed with 0.1 mm GTPγS, and was exposed to ATP as indicated by the bar. B, I–V relationships of ATP-induced currents (b–a) and the persistently activated I_Cl,ATP (c–a). Whole-cell currents were activated by voltage-clamp pulses as in Fig. 1B, at time points a, b and c in A. C, mean current densities at +100 mV of I_Cl,ATP in GTPγS-dialysed (n = 4), GDPβS-dialysed (n = 4) or PTX-pretreated (n = 4) cells. * signifies significantly smaller than control with P < 0.05.

Figure 4. Extracellular ATP-induced membrane currents in ventricular cells from cftr^−/− mice

A, time course of extracellular ATP-induced whole-cell currents in standard extracellular solution in ventricular cells from cftr^−/− mice. B, whole-cell current recorded using same protocol as in Fig. 1B at time points a (control) and b (ATP) in panel A. Difference currents obtained by subtracting currents obtained at time b from currents obtained at time a. C, mean I–V relationships of ATP-induced difference currents in 10 cells from 4 cftr^−/− mice.

Figure 5. Effects of DIDS and suramin on I_Cl,ATP in ventricular cells of wild-type mice

A, time course of extracellular ATP-induced whole-cell currents in standard extracellular solutions in intracellular GTPγS dialysed cell of wild-type mouse. ATP (100 μm), DIDS (100 μm) and glibenclamide (100 μm) were applied during the times indicated by each bar. B, mean I–V relationships of DIDS- (e–f) and glibenclamide-sensitive currents (e–g), which were obtained from whole-cell recordings induced using the same voltage clamp pulse protocol shown in Fig. 1B, at time points e, f and g in A. C, the left side shows the mean (n = 4) difference current density at +100 mV in DIDS, DIDS and ATP, and ATP following the removal of DIDS in control (absence of GTPγS) cells. The right side shows the mean (n = 4) difference current densities at +100 mV in suramin, suramin and ATP, and ATP following removal of suramin. * and ** signify significantly smaller than ATP alone with P < 0.05 and 0.01, respectively.