Deleting the accessory subunit KChIP2 results in loss of I(to,f) and increased I(K,slow) that maintains normal action potential configuration

Abstract

Background: Four voltage-gated potassium currents, I(to,f) (K(V)4.2), I(to,s) (K(V)1.4), I(K,slow) (K(V)1.5+K(V)2.1), and I(SS) (TASK1), govern murine ventricular repolarization. Although the accessory subunit KChIP2 influences I(to,f) expression, in preliminary experiments we found that action potential duration (APD) is maintained in KChIP2 knockout mice.

Objective: We tested the role of KChIP2 in regulating APD and studied the underlying ionic currents.

Methods: We used microelectrode techniques, whole-cell patch clamp studies, and real-time polymerase chain reaction amplification to characterize ventricular repolarization and its determinants in wild-type and KChIP2(-/-) mice.

Results: Despite comparable baseline action potentials, APD was more markedly prolonged by 4-aminopyridine (4-AP) in KChIP2(-/-) preparations. Peak K(+) current densities were similar in wild-type and KChIP2(-/-) cells (mean +/- SEM I(P): 28.3 +/- 2 (n = 27) vs. 29.2 +/- 2 pA/pF (n = 24), respectively; P > .05). Heteropodatoxin-2 (HpTx-2, 1 microM) had no effect on current amplitude in KChIP2(-/-) myocytes. The current fractions sensitive to 4-AP (50 microM and 1 mM) were larger in KChIP2(-/-) than wild-type (P < .05). Real-time polymerase chain reaction showed absence of KChIP2 and increased K(V)1.5 expression in KChIP2(-/-) ventricular myocardium.

Conclusion: KChIP2 deficiency eliminated HpTx-2-sensitive I(to,f), but had little impact on total APD, secondary to upregulation of 4-AP-sensitive I(K,slow) in association with increased K(V)1.5 expression. There is increased sensitivity to 4-AP-mediated APD prolongation in KChIP2(-/-). Thus, KChIP2 seems important for murine repolarization in circumstances of reduced repolarization reserve.

Figure 1

APD prolongation by 4-AP is enhanced in KChIP2^-/- ventricles. Representative action potentials recorded from intact WT and KChIP2^-/- LV and RV in control and after addition of 1 mM 4-AP.

Figure 2

Comparison of K_V currents in WT and KChIP2^-/- myocytes. A, Representative current traces normalized to cell capacitance recorded from WT and KChIP2^-/- cells using 4.5-s depolarizing steps from V_H=-70 mV to V_step=-40 to +50 mV (Inset). Each step was preceded by a 25 ms depolarization to -20 mV to eliminate contamination by inward Na⁺ current not completely blocked by TTX. Arrowheads indicate 0 pA. B, I_P densities plotted as a function of V_test in WT (n=27) and KChIP2^-/- (n=24) myocytes. C, Time interval from cell depolarization to peak current as a function of V_test. *, P<0.05 versus WT. D, Representative current traces normalized to cell capacitance recorded from a WT and a KChIP2^-/- cell using 400-ms depolarizing steps. Although peak current amplitude is comparable, the WT cell displays faster current inactivation within the first ∼100 ms.

Figure 3

Similar inward rectifying K⁺ currents in WT and KChIP2^-/- myocytes. A, Representative current traces recorded from a WT and KChIP2^-/- myocyte. Arrowheads indicate 0 pA. Inset, voltage protocol. B, Peak inward I_K1 (left) and steady state (right) I_K1 in WT (n=7) and KChIP2^-/- (n=10) myocytes.

Figure 4

No effect of HpTx-2 on K_V currents in KChIP2^-/- myocytes. A, Representative baseline K_V currents recorded using 400-ms pulses from V_H=-70 mV to V_step between -40 and +50 mV (inset) in myocytes from WT and KChIP2^-/- mice. Arrowheads indicate 0 pA. B, Currents recorded in the same myocytes after addition of 1 μM HpTx-2 to the superfusion solution. C, The HpTx-2-sensitive currents obtained by digital offline subtraction of the current traces shown in A and B.

Figure 5

Larger fraction of K_V current is sensitive to 4-AP in KChIP2^-/- myocytes. A, Current traces normalized to cell capacitance recorded using 400-ms pulses from V_H=-70 mV to V_step=+40 mV from a WT and a KChIP2^-/- cell, before and after addition of 50 μM 4-AP to the superfusate. Arrowheads indicate 0 pA. Note the prominent inactivation of the 4-AP resistant current in WT. B, The current fraction sensitive to 50 μM 4-AP obtained by digital offline subtraction of the current traces shown in A. C, 4-AP-sensitive current relative to the full I_P in WT and KChIP2^-/- cells. *, P<0.05.

Figure 6

Compensatory remodeling potentially includes elevated K_V1.5 transcription. Expression levels of the major ion channels underlying murine repolarization in KChIP2^-/- (n=6) relative to WT (n=6) ventricles. Grey horizontal line indicates equal expression levels in WT and KChIP2^-/- ventricles. KChIP2 was not detected in KChIP2^-/- myocytes. *, P<0.05.