Modulation of transient outward current by extracellular protons and Cd2+ in rat and human ventricular myocytes

Abstract

1. The effects of extracellular acidosis and Cd2+ on the transient outward current (Ito) have been investigated in rat and human ventricular myocytes, using the whole-cell patch-clamp technique. 2. In rat myocytes, exposure to acidic extracellular solution (pH 6.0) shifted both steady-state activation and inactivation curves to more positive potentials, by 20.5 +/- 2.7 mV (mean +/- S.E.M.; n = 4) and 19.8 +/- 1.2 mV, respectively. Cd2+ also shifted the activation and inactivation curves in a positive direction in a concentration-dependent manner. 3. In human myocytes, the steady-state activation and inactivation curves were located at more positive potentials. The effect of Cd2+ was similar, but acidosis had less effect than in rat myocytes (e.g. pH 6.0 shifted activation by only 7.2 +/- 2.2 mV and inactivation by 13.7 +/- 0.5 mV; n = 4). 4. In both species, the effect of acidosis decreased with increasing concentrations of Cd2+ and vice versa, suggesting competition between H+ and Cd2+ for a common binding site. 5. The data indicate that acidosis and divalent cations influence Ito via a similar mechanism and act competitively in both rat and human myocytes, but that human cells are less sensitive to the effects of acidosis.

Figure 1. Effect of acidosis (pH 6.0) on I_to in rat myocytes

I_Ca,L was blocked by nisoldipine (10 μm). A, decrease of I_to by acidosis. Current activated by a step from a holding potential of -80 to 0 mV. Traces in control conditions (□) and in pH 6.0 (•) are superimposed. Inset, voltage-clamp protocol (20 ms prepulse to -40 mV to inactivate I_Na not shown). B, increase of I_to by acidosis. Current activated by a step to +60 mV after a 1 s prepulse to -30 mV in control (□) and in acidosis (•). Only currents during the test pulse are shown. Inset, voltage-clamp protocol. C, effect of acidosis on steady-state activation and inactivation curves. □, control; •, acidosis; ▵, second control. The effect of acidosis was reversible.

Figure 2. Effect of acidosis on activation and inactivation kinetics in rat myocytes

I_Ca,L was blocked by 0 mM Ca²⁺_o-5 mM Mg²⁺_o. □, control; •, acidosis; ▵, second control. A, voltage dependence of the fast time constant of inactivation (τ_fast) under control and acidic conditions. Inset, the I_to inactivation time course was not satisfactorily fitted by a single exponential (dotted line), but by a double exponential (continuous line, superimposed on the tracing). Current was activated by a 900 ms step to +40 mV from a holding potential of -80 mV. B shows the voltage dependence of the slow time constant of inactivation (τ_slow) under control and acidic conditions.

Figure 3. Effect of Cd²⁺ on I_to in rat myocytes

I_Ca,L was blocked by 0 mM Ca²⁺_o-5 mM Mg²⁺_o. A, decrease of I_to by Cd²⁺ (0.3 mM). Current activated by a step from a holding potential of -80 to 0 mV. Traces in the absence (□) and in the presence of Cd²⁺ (•) are superimposed. Inset, voltage-clamp protocol (20 ms prepulse to -40 mV to inactivate I_Na not shown). B, increase of I_to by Cd²⁺. Current activated by a step to +60 mV after a 1 s prepulse to -30 mV in the absence (□) and presence of Cd²⁺ (•). Only currents during the test pulse are shown. Inset, voltage-clamp protocol. C, effect of Cd²⁺ on steady-state activation and inactivation curves. □, control; •, Cd²⁺; ▵, second control. The effect of Cd²⁺ was reversible.

Figure 4. Effect of acidosis on I_to in the presence of Cd²⁺ in rat myocytes

I_Ca,L was blocked by Cd²⁺ (0.3 mM). A, decrease of I_to by acidosis. Current activated by a step from a holding potential of -80 to +10 mV. Traces in control conditions (□) and in pH 6.0 (•) are superimposed. Inset, voltage-clamp protocol (20 ms prepulse to -40 mV to inactivate I_Na not shown). B, increase of I_to by acidosis. Current activated by a step from a holding potential of -20 to +60 mV in control (□) and in acidosis (•). Inset, voltage-clamp protocol. C, effect of acidosis on steady-state activation and inactivation curves. □, control; •, acidosis; ▵, second control. The effect of acidosis was less marked than in the absence of Cd²⁺ (with nisoldipine or 0 mM Ca²⁺_o-5 mM Mg²⁺_o to block I_Ca,L).

Figure 5. Interaction of acidosis and Cd²⁺ in rat myocytes

Concentration dependence of the effect of Cd²⁺ on the V_½ of activation (open symbols) or inactivation (filled symbols) in control (squares), acidosis (circles) and second control (triangles). With increasing Cd²⁺ concentration, the V_½ of both activation and inactivation was shifted to more positive potentials. The effect of acidosis consisted of a further shift of V_½ to a more positive value but the magnitude of this additional shift decreased with increasing Cd²⁺ concentration. At 5 mM Cd²⁺ acidosis failed to induce a further shift. The values at 0 mM Cd²⁺ were obtained with nisoldipine as the I_Ca,L blocker. Different Cd²⁺ concentrations were applied in different groups of cells; n varied between 3 and 8. Data points were fitted by eye (continuous lines).

Figure 6. Comparison of outward current in human and rat myocytes

A, depolarization-induced outward current in human ventricular cell. Inset, voltage-clamp protocol: depolarizing pulses were preceded by a 20 ms prepulse to -40 mV to inactivate the I_Na current. B, depolarization-induced outward current in rat myocytes. Same voltage-clamp protocol as in A.

Figure 7. Effect of acidosis on I_to in human myocytes

I_Ca,L was blocked by nisoldipine (10 μm). Steady-state activation and inactivation curves of I_to in control and acidosis are shown. Inset, voltage dependence of the I_to inactivation time constant in control and acidosis. □, control; •, acidosis; ▵, second control.

Figure 8. Effect of Cd²⁺ on I_to in human myocytes

I_Ca,L was blocked by nisoldipine (10 μm). Steady-state activation and inactivation curves of I_to in the absence and presence of Cd²⁺. Inset, voltage dependence of the I_to inactivation time constant in the absence and presence of Cd²⁺. □, control; •, 0.3 mM Cd²⁺; ▵, second control.

Figure 9. Effect of acidosis on I_to in the presence of Cd²⁺ in human myocytes

I_Ca,L was blocked by Cd²⁺ (0.3 mM). Steady-state activation and inactivation curves of I_to in control and acidosis. Inset, voltage dependence of the I_to inactivation time constant in control and acidosis. □, control; •, acidosis; ▵, second control.