A novel anionic conductance affects action potential duration in isolated rat ventricular myocytes

Abstract

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current-clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl(-) concentration and shortened by replacement of extracellular Cl(-) with I(-). Under voltage-clamp, membrane potential steps or ramps evoked an anionic background current (I(AB)) carried by either Cl(-), Br(-), I(-) or NO(3)(-). Activation of I(AB) was Ca(2+)- and cyclic AMP-independent, and was unaffected by cell shrinkage. I(AB) was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca(2+)-, cyclic AMP- and swelling-activated Cl(-) currents in ventricular cells of other mammals. These results suggest that I(AB) may be carried by a novel class of Cl(-) channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that I(AB) could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl(-) channels as potential targets for novel antiarrythmic agents.

Figure 1

Electrophysiological recordings in the presence of different extracellular anions. (a) APs recorded during superfusion with solution A containing either 155 mM Cl⁻ (dotted), 35 mM Cl⁻ plus 120 mM aspartate⁻ (•), or 10 mM Cl⁻ plus 145 mM I⁻ (□). (b) The same APs as shown in part (a) normalized relative to their maximum to eliminate variation in RMP; lines and symbols as in (a). (c) Mean current density current-voltage (I–V) plots at different [Cl⁻]_e (in solution B). ○, [Cl⁻]_e=152 mM (n=7), ⧫, [Cl⁻]_e=37 mM (n=3). Inset: currents generated at test potentials of −100 and −25 mV in the presence of 152 mM Cl⁻ (bar indicates current averaging period). (d) Mean I–V curves obtained in solution B containing 135 mM [Br⁻]_e (□, n=3), [NO₃⁻]_e (▵, n=4) and [I⁻]_e (⧫; n=3). The arrows shown in panels (c) and (d) indicate the reversal potential for the currents. In this, and the subsequent figure, error bars represent s.e.mean.

Figure 2

Voltage-clamp experiments using ramp protocols in the presence of different anions. (a) Anion-sensitive difference currents (I_m) obtained from hyperpolarizing ramps during superfusion with solution C. The current recorded in 145 mM aspartate⁻ was subtracted from the current observed in either 145 mM I⁻ or NO₃⁻. (b) Iodide-sensitive difference currents as in (a) before and after 5 min superfusion with 50 μM DIDS in the solution C. (c) difference currents as in (a) before and after 5 min superfusion with 50 μM niflumic acid in solution C. (d) histograms showing outward current amplitude at +60 mV before (control) and after increasing intracellular cyclic AMP levels (test; β-stimulation), and also before (control) and after cell shrinkage (test; hypertonicity) cell shrinkage (right). Details given in main text.