Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes

Abstract

1. Two kinetically and pharmacologically distinct transient outward K+ currents, referred to as Ito,f and Ito,s, have been distinguished in mouse left ventricular myocytes. Ito,f is present in all left ventricular apex cells and in most left ventricular septum cells, whereas Ito,s is identified exclusively in left ventricular septum cells. 2. Electrophysiological recordings from ventricular myocytes isolated from animals with a targeted deletion of the Kv1.4 gene (Kv1.4-/- mice) reveal that Ito,s is undetectable in cells isolated from the left ventricular septum (n = 26). Ito,f density in both apex and septum cells, in contrast, is not affected by deletion of Kv1.4. 3. Neither the 4-AP-sensitive, slowly inactivating K+ current, IK,slow, nor the steady-state non-inactivating K+ current, ISS, is affected in Kv1.4-/- mouse left ventricular cells. 4. In myocytes isolated from transgenic mice expressing a dominant negative Kv4.2 alpha subunit, Kv4.2W362F, Ito,f is eliminated in both left ventricular apex and septum cells. In addition, a slowly inactivating transient outward K+ current similar to Ito,s in wild-type septum cells is evident in myocytes isolated from left ventricular apex of Kv4.2W362F-expressing transgenics. The density of Ito,s in septum cells, however, is unaffected by Kv4.2W362F expression. 5. Western blots of fractionated mouse ventricular membrane proteins reveal a significant increase in Kv1.4 protein level in Kv4.2W362F-expressing transgenic mice. The protein levels of other Kv alpha subunits, Kv1.2 and Kv2.1, in contrast, are not affected by the expression of the Kv4.2W362F transgene. 6. The results presented here demonstrate that the molecular correlates of Ito,f and Ito,s in adult mouse ventricle are distinct. Kv1.4 underlies mouse ventricular septum Ito,s, whereas Kv alpha subunits of the Kv4 subfamily underlie mouse ventricular apex and septum Ito, f. The appearance of the slow transient outward K+ current in Kv4. 2W362F-expressing left ventricular apex cells with properties indistinguishable from Ito,s in wild-type cells is accompanied by an increase in Kv1.4 protein expression, suggesting that the upregulation of Kv1.4 underlies the observed electrical remodeling in Kv4.2W362F-expressing transgenics.

Figure 1. Action potential waveforms are distinct in apex and septum cells isolated from adult mouse left ventricle

Under current-clamp, action potentials evoked by 3 ms suprathreshold current pulses were recorded from adult mouse left ventricular apex (A) and septum (B) cells. Stimuli were applied at 1.0 Hz and action potentials were recorded after reaching a steady state. Action potentials are significantly broader in the septum cells; similar results were obtained in recordings from 7 apex and 8 septum cells.

Figure 2. The waveforms of the Ca²⁺-independent, depolarization-activated K⁺ currents in myocytes isolated from the apex of wild-type (A) and Kv1.4^−/− (B) adult mouse left ventricles are indistinguishable

Whole-cell outward K⁺ currents were evoked during 400 ms (left) and 4 s (right) depolarizing voltage steps to potentials between −40 and +40 mV from a holding potential of −70 mV in 10 mV increments. Each trial was preceded by a short (25 ms) depolarization to −20 mV to eliminate contamination of inward Na⁺ currents not completely blocked by TTX. The records shown in the left and right panels in A and B were obtained from the same cell.

Figure 3. I_to,s is eliminated in left ventricular septum cells isolated from Kv1.4^−/− mice

Whole-cell outward K⁺ currents were recorded as described in the legend to Fig. 2 from left ventricular septum cells isolated from wild-type (A) and Kv1.4^−/− (B) mice. In cells isolated from the left ventricular septum of Kv1.4^−/− mice, I_to,s is undetectable (B); Kv1.4^−/− septum cells express either only I_K,slow and I_SS (Bb) or I_to,f, I_K,slow and I_SS (Ba). In addition to the elimination of I_to,s, I_to,f inactivation is slowed in septum cells isolated from Kv1.4^−/− mice (see text).

Figure 4. Effects of 50 μm 4-AP and 300 nm HpTx-3 on the outward K⁺ currents in wild-type and Kv1.4^−/− mouse left ventricular septum cells

Representative whole-cell outward K⁺ currents recorded from adult mouse left ventricular septum cells lacking and expressing I_to,f are displayed in A and B, respectively. To determine the effects of 50 μm 4-AP (A) and 300 nm HpTx-3 (B), control currents were recorded before exposure to the drug, and when the effect reached a steady state, the currents were recorded again. The waveforms of the 50 μm 4-AP- and 300 nm HpTx-3-sensitive currents were obtained by offline digital subtraction of the currents in the presence of 50 μm 4-AP or 300 nm HpTx-3 from the controls. Note that I_to,s is clearly evident in wild-type septum cells, but undetectable in Kv1.4^−/− septum cells, when I_K,slow or I_to,f is selectively blocked by micromolar concentrations of 4-AP (A) or nanomolar concentrations of HpTx-3 (B). Similar results were obtained in experiments on 3 cells of each group.

Figure 5. I_to,f is eliminated in ventricular myocytes isolated from both the apex and the septum of adult Kv4.2W362F transgenic mice

Whole-cell outward K⁺ currents were recorded from adult mouse left ventricular apex (A) and septum (B) cells as described in the legend to Fig. 2. The records shown in the left and right panels in A and B were obtained from the same cell, and only the durations of the voltage steps are different. As is evident, I_to,f is undetectable in both apex and septum cells. Note that a slowly inactivating transient outward K⁺ current (I_to,s), which was not detected in wild-type apex cells (see Fig. 2A), is evident in Kv4.2W362F-expressing apex cells.

Figure 6. Effects of 300 nm HpTx-3 and 30 μm 4-AP on the outward K⁺ currents in wild-type and Kv4.2W362F-expressing mouse left ventricular apex cells

Whole-cell outward K⁺ currents were recorded as described in the legend to Fig. 2 from left ventricular apex cells isolated from wild-type and Kv4.2W362F transgenic mice. After recording control currents, cells were exposed to either 300 nm HpTx-3 (A) or to 30 μm 4-AP (B), and when the effects reached a steady state, the currents were recorded again. The waveforms of the 300 nm HpTx-3- and 30 μm 4-AP-sensitive currents were obtained by offline digital subtraction of the currents in the presence of HpTx-3 or 4-AP from the controls. Note that the slowly inactivating transient outward K⁺ current in Kv4.2W362F-expressing apex cells is insensitive to nanomolar concentrations of HpTx-3 and micromolar concentrations of 4-AP. Similar results were obtained in experiments on 4 cells of each group.

Figure 7. The slowly inactivating transient outward K⁺ current in Kv4.2W362F-expressing mouse left ventricular apex cells recovers slowly from steady-state inactivation

After inactivating the currents by 10 s prepulses to +50 mV, adult mouse left ventricular apex cells were hyperpolarized to −70 mV for varying times (ranging from 10 ms to 8 s) before 10 s test depolarizations to +50 mV to assess the extent of recovery. Typical current waveforms recorded from wild-type and Kv4.2W362F transgenic animals are illustrated in A and B, respectively. The amplitudes of I_to,f in wild-type apex cells and the slow transient outward K⁺ current (I_to,s) in transgenic apex cells evoked at +50 mV following each recovery period were determined from double exponential fits to the decay phases of the outward currents as described in Methods, and then normalized to the current amplitudes recorded during the 10 s prepulses. Mean ±s.e.m. normalized currents (n= 5) for I_to,f (○) and I_to,s (▵) are plotted as a function of recovery time in C. All recovery data are well described by single exponentials (continuous and dotted lines) although the recovery rates for I_to,f and I_to,s are distinct (see text).

Figure 8. Western blots reveal that Kv1.4 protein expression is increased in Kv4.2W362F-expressing transgenic mice

Ventricular membrane proteins (A: 180 μg loaded in each lane; B and C: 50 μg loaded in each lane) were fractionated on SDS-PAGE gels, transferred to PVDF membranes and immunoblotted with the polyclonal anti-Kv1.4 (A), anti-Kv1.2 (B) or anti-Kv2.1 (C) antibodies (see Methods). WT, TG and KO indicate ventricular membrane proteins prepared from adult wild-type, Kv4.2W362F-expressing and Kv1.4^−/− mice, respectively. The arrows show the specific bands recognized by the antibodies.