Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes

Abstract

Key points: [Ca²⁺ ]_i enhanced rabbit ventricular slowly activating delayed rectifier K⁺ current (I_Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K⁺ current (I_Kr ) amplitude and voltage dependence were unaffected by high [Ca²⁺ ]_i . When measuring or simulating I_Ks during an action potential, I_Ks was not different during a physiological Ca²⁺ transient or when [Ca²⁺ ]_i was buffered to 500 nm.

Abstract: The slowly activating delayed rectifier K⁺ current (I_Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca²⁺ ([Ca²⁺ ]_i ) and β-adrenergic receptor (β-AR) stimulation modulate I_Ks amplitude and kinetics, but details of these important I_Ks regulators and their interaction are limited. We assessed the [Ca²⁺ ]_i dependence of I_Ks in steady-state conditions and with dynamically changing membrane potential and [Ca²⁺ ]_i during an AP. I_Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca²⁺ ]_i , we found that raising [Ca²⁺ ]_i from 100 to 600 nm produced similar increases in I_Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca²⁺ ]_i increased maximally activated tail I_Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I_Ks recorded during a normal physiological Ca²⁺ transient was similar to I_Ks measured with [Ca²⁺ ]_i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca²⁺ ]_i regulates I_Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca²⁺ ]_i , in the submembrane or junctional cleft space, is not required to maximize [Ca²⁺ ]_i -dependent I_Ks activation during normal Ca²⁺ transients.

Keywords: action potential; cardiac electrophysiology; delayed rectifier current; intracellular calcium; potassium channel; rabbit; voltage-gated channels.

Figure 1. Distinction between I _Ks and I _Kr

A–C, representative traces of whole‐cell total I _K (A), I _Kr (B) and I _Ks (C) measured from isolated rabbit ventricular myocytes. Currents were recorded by applying step‐like pulses (inset) from −40 to 50 mV in 10 mV increments for 3 s, followed by a ‘tail’ pulse to −50 mV for 3 s. The inter‐pulse interval was 15 s. D, representative traces of whole‐cell currents recorded with high pipette [Ca²⁺] from the same cell as in C after perfusion of 1 μm HMR to show selective I _Ks block. E, an exemplar time course of tail I _Ks monitored before (black) and after (red) perfusion of ISO when pipette [Ca²⁺] was high.

Figure 2. Assessment of steady‐state Ca²⁺ dependence of I _Ks

A, representative traces of whole‐cell I _Ks measured from isolated rabbit ventricular myocytes. I _Ks was recorded by applying the same V _m protocol as Fig. 1 (inset). B and C, the mean peak ‘step’ (B) and ‘tail’ (C) I _Ks are plotted as a function of the step voltage for cells recorded using a pipette solution containing free [Ca²⁺]_i of 0, 100, 300, 500, and 600 nm. D–F, the tail I–V relations were described using a Boltzmann equation (grey line, C) to determine I _MAX (D), V _1/2 (E) and k (F). For all figures, number of cells (n) and significance tests are indicated in boxed insets where appropriate.

Figure 3. Representative traces of Ca²⁺‐fluorescence signal recordings during simultaneous whole‐cell patch clamp measurements

A, isolated rabbit ventricular myocytes were preloaded with Fluo‐4 AM and the patch pipette solution was loaded with Fluo‐4 K⁺ salt. The Ca²⁺ signal was recorded for 0, 100, 300, 500 and 600 nm [Ca²⁺]_i using the same V _m protocol as in Fig. 1. B, the fluorescence signal was background subtracted and fitted to a single site binding equation. C, representative traces of I _Ks recorded with pipette [Ca²⁺] 500 nm before (black) and after (red) perfusion of 50 μm W7. I _Ks was recorded by applying a step pulse from −50 to 40 mV for 3 s, followed by a tail pulse to −50 mV for 3 s (inset). D, the bar graph represents the mean peak tail I _Ks recorded before or after W7 perfusion (n = 5, P < 0.05).

Figure 4. Assessment of steady‐state Ca²⁺ dependence of I _Kr

A, representative traces of whole‐cell I _Kr measured from isolated rabbit ventricular myocytes. The same V _m protocol was used as in Fig. 1. B, the mean peak tail I _Kr are plotted as a function of the step voltage for cells recorded using a pipette solution containing free [Ca²⁺]_i of 0, 100, 300 and 600 nm. C–E, the tail I–V relations were described using a Boltzmann equation (grey line, Fig. 4 B) to determine I _MAX (C), V _1/2 (D) and k (E).

Figure 5. ISO and [Ca²⁺]_i increase I _MAX of I _Ks

A, representative traces of I _Ks measured from isolated rabbit ventricular myocytes at room temperature before (black) and after (red) 50 nm [ISO] perfusion for pipette solutions containing [Ca²⁺]_i of 100 and 500 nm. The same V _m protocol was used as in Fig. 1. B and C, the mean peak tail I _Ks before (B) and after (C) ISO are plotted as a function of step voltage for cells recorded with a pipette solution containing free [Ca²⁺]_i of 0, 100, 300, 500, or 600 nm. D–F, the tail I–V relations were described using a Boltzmann equation (grey line, Fig. 5 B and C) to determine I _MAX (D), V _1/2 (E) and k (F).

Figure 6. [Ca²⁺]_i slows deactivation of I _Ks

A, representative traces of I _Ks recorded using pipette solutions with free [Ca²⁺]_i of 100 and 500 nm before (100 nm, red; 500 nm, blue) and after (100 nm, light red; 500 nm, light blue) ISO are overlaid. I _Ks was recorded using the same V _m protocol as in Fig. 3 C by applying a step pulse from −50 to 40 mV for 3 s, followed by a tail pulse to −50 mV for 3 s. B, τ_act of I _Ks was fitted to a single exponential during the step pulse to 40 mV and is plotted as a function of free [Ca²⁺]_i in the pipette solution before (black) and after (red) ISO perfusion for 0, 100, 300, 500, and 600 nm [Ca²⁺]_i. C, the decay in tail I _Ks following the 40 mV step‐pulse was fitted to a single exponential to determine  τ_deact and is plotted as a function of free [Ca²⁺]_i in the pipette solution before and after ISO perfusion for 0, 100, 300, 500, and 600 nm [Ca²⁺]_i. The n values for each situation are the same as in Fig. 5. The [Ca²⁺]_i dependence and ISO effects (50 nm) on I _Ks (D), V _1/2, (E) and τ_deact (F) were incorporated into a model of I _Ks based on the experimental data and was scaled to 37 °C. Simulated I _Ks (dashed lines) are shown overlaid with experimental results (solid lines).

Figure 7. Ca²⁺ dependence of HMR‐sensitive I _Ks activated during AP waveform

A, rabbit ventricular AP waveform used for AP simulations and to record HMR‐1556‐sensitive I _Ks at 35–37°C. B and C, time courses of I _Ks (B) and CaT (C) during 1 Hz computational simulations using the AP waveform as in Fig. 7 A are shown with [Ca²⁺]_i clamped at 100 (red) and 500 nm (blue), or unbuffered (Free CaT, green). D, representative whole‐cell current subtracted HMR‐sensitive (1 μm) I _Ks traces are overlaid for cells recorded with free [Ca²⁺]_i of 0 (black), 100 (red) and 500 nm (blue). E, representative whole‐cell current subtracted HMR‐sensitive (1 μm) I _Ks traces are overlaid for cells recorded (using the AP waveform in Fig. 7 A as the V _m command) with [Ca²⁺]_i heavily buffered by 10 mm EGTA (grey) or unbuffered (green). F, the corresponding Ca²⁺ signal is shown. Note that free [Ca²⁺]_i is reduced at baseline and during the AP when buffered, and a normal CaT is present when unbuffered. G, the peak I _Ks during ventricular AP waveform recordings are plotted for cells recorded using a pipette solution containing free [Ca²⁺]_i of 0, 100, 300, 500, or 600 nm, and buffered with EGTA or unbuffered.

Figure 8. Ca²⁺ and ISO dependence of I _Ks contribute to rabbit ventricular AP shortening during β‐AR stimulation

Simulated time courses of membrane potential, V _m (A), I _Ks (B) and CaT (C) during steady‐state 3 Hz pacing are shown before (black) and after (red) ISO (20 nm, left; and 50 nm, right).

Figure 9. Effects of Ca²⁺ and ISO dependence of rabbit I _Ks

Dashed line represents I _Ks amplitude under control conditions when [Ca²⁺]_i is highly buffered essentially to 0 in the pipette solution (based on data in Fig. 5 D).