Regulation of cardiac CFTR Cl^– channel activity by a Mg²⁺-dependent protein phosphatase

Abstract.

Dephosphorylation of the CFTR Cl^– channel is known to be induced by both okadaic-acid- (OA-) sensitive and -insensitive protein phosphatases (PPs). In the present study, the effects of cytosolic free Mg²⁺ on the cardiac CFTR Cl^– current were examined in relation to the latter PP activity in guinea pig ventricular myocytes. Even when maintaining intracellular Mg-ATP at millimolar concentrations under whole-cell patch-clamp mode, cAMP-activated Cl^– conductance was reversibly suppressed by cytosolic free Mg²⁺, with an IC₅₀ of around 2.5 mmol/l. In contrast, changes in the cytosolic concentration of free Mg²⁺ ([Mg²⁺]_i) had no effect on genistein-activated CFTR Cl^– currents. The Mg²⁺ effect on cAMP-activated CFTR Cl^– conductance was completely reversed by application of anthracene-9-carboxylic acid (9-AC), which was previously shown to inhibit an OA-insensitive PP in cardiac myocytes. A 9-AC-sensitive fraction of endogenous PP activity in the extract of guinea pig ventricle was found to be activated by free Mg²⁺ at millimolar concentrations but to be inactive at micromolar concentrations. The intracellular application of OA failed to activate basal Cl^– conductance at millimolar [Mg²⁺]_i. In the presence of OA, however, basal Cl^– conductance became activated either by reducing [Mg²⁺]_i to micromolar concentrations or by applying 9-AC. Thus, we conclude that a Mg²⁺-dependent PP sensitive to 9-AC plays a key role in the cAMP-mediated regulation of cardiac CFTR Cl^– channel at physiological [Mg²⁺]_i under both basal and cAMP-activated conditions. Also, it appears that the genistein-activated conformation of the cardiac CFTR channel is not sensitive to the Mg²⁺-dependent PP.