Nickel inhibits ß-1 adrenoceptor mediated activation of cardiac CFTR chloride channels.

Cardiac ventricular myocytes exhibit a protein kinase A-dependent Cl(-) current (ICl.PKA) mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). There is conflicting evidence regarding the ability of the divalent cation nickel (Ni(2+)), which has been used widely in vitro in the study of other cardiac ionic conductances, to inhibit ICl.PKA. Here the action of Ni(2+) on ICl.PKA activated by ß-adrenergic stimulation has been elucidated. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. Externally applied Ni(2+) blocked ICl.PKA activated by 1 µM isoprenaline with a log IC50 (M) of -4.107 ± 0.075 (IC50=78.1 µM) at +100 mV and -4.322 ± 0.107 (IC50=47.6 µM) at -100 mV. Thus, the block of ICl.PKA by Ni(2+) was not strongly voltage dependent. Ni(2+) applied internally via the patch-pipette was ineffective at inhibiting isoprenaline-activated ICl,PKA, but in the same experiments the current was suppressed by external Ni(2+) application, indicative of an external site of Ni(2+) action. In the presence of 1 µM atenolol isoprenaline was ineffective at activating ICl.PKA, but in the presence of the ß2-adrenoceptor inhibitor ICI 118,551 isoprenaline still activated Ni(2+)-sensitive ICl.PKA. Collectively, these data demonstrate that Ni(2+) ions produce marked inhibition of ß1-adrenoceptor activated ventricular ICl.PKA at submillimolar [Ni(2+)]: an action that is likely to involve an interaction between Ni(2+) and ß1-adrenoceptors. The concentration-dependence for ICl.PKA inhibition seen here indicates the potential for confounding effects on ICl,PKA to occur even at comparatively low Ni(2+) concentrations, when Ni(2+) is used to study other cardiac ionic currents under conditions of ß-adrenergic agonism.

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101.

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl(-) current (I(Cl.PKA)) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I(Cl.PKA) and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I(Cl.PKA) in a concentration- and voltage-dependent fashion (IC(50) at +100 mV=0.3 ± 1.5 µM and at -100 mV=5.1 ± 1.3 µM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I(Cl.PKA) (30 µM) was tested on other cardiac ion currents. Inward current at -120 mV, comprised predominantly of the inward-rectifier background K(+) current, I(K1), was reduced by ∼43% (n=5). Under selective recording conditions, the Na(+) current (I(Na)) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at -40 mV of ∼82%; n=8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca(2+) channel current (I(Ca,L)); fractional block at +10 mV of ∼49% and of ∼28% at -10 mV; n=11, with a ∼-3 mV shift in the voltage-dependence of I(Ca,L) activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I(Cl.PKA) channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.