The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes

Abstract

1. Ventricular myocytes demonstrate a steeply inwardly rectifying K(+) current termed I(K1). We investigated the molecular basis for murine I(K1) by removing the genes encoding Kir2.1 and Kir2.2. The physiological consequences of the loss of these genes were studied in newborn animals because mice lacking Kir2.1 have a cleft palate and die shortly after birth. 2. Kir2.1 (-/-) ventricular myocytes lack detectable I(K1) in whole-cell recordings in 4 mM external K(+). In 60 mM external K(+) a small, slower, residual current is observed. Thus Kir2.1 is the major determinant of I(K1). Sustained outward K(+) currents and Ba(2+) currents through L- and T-type channels were not significantly altered by the mutation. A 50 % reduction in I(K1) was observed in Kir2.2 (-/-) mice, raising the possibility that Kir2.2 can also contribute to the native I(K1). 3. Kir2.1 (-/-) myocytes showed significantly broader action potentials and more frequent spontaneous action potentials than wild-type myocytes. 4. In electrocardiograms of Kir2.1 (-/-) neonates, neither ectopic beats nor re-entry arrhythmias were observed. Thus the increased automaticity and prolonged action potential of the mutant ventricular myocytes were not sufficiently severe to disrupt the sinus pacing of the heart. The Kir2.1 (-/-) mice, however, had consistently slower heart rates and this phenotype is likely to arise indirectly from the influence of Kir2.1 outside the heart. 5. Thus Kir2.1 is the major component of murine I(K1) and the Kir2.1 (-/-) mouse provides a model in which the functional consequences of removing I(K1) can be studied at both cellular and organismal levels.

Figure 1. Transcript analysis of cardiac tissue

Each lane was loaded with 25 μg of total neonatal ventricular RNA from wild-type (WT), Kir2.1^−/− or Kir2.2^−/− mice and probed with a portion of the open reading frame of Kir2.1, Kir2.2 or Kir2.3. Kir2.1 and 2.3 are each encoded by a single transcript while Kir2.2 is encoded by two. Equal loading of RNA was confirmed by re-probing the blots with a fragment of the GAPDH open reading frame.

Figure 2. Inwardly rectifying K⁺ currents in 4 mm external K⁺

A-D, inwardly rectifying currents were recorded in 4 mm external K⁺ from voltage-clamped neonatal myocytes isolated from wild-type (WT), Kir2.1^−/−, Kir2.2^−/− and Kir2.1^−/−-Kir.2.2^−/− mice. For each family of current traces, the voltage was stepped from a holding potential of −40 mV to potentials of −70 to −130 mV (in 15 mV increments). Leak currents were subtracted using currents evoked by small hyperpolarizing pulses (P/4 method). Cell capacitances were 14, 18, 19 and 19 pF, respectively. E, steady state currents (means ±s.e.m.) plotted as a function of voltage in wild-type (n = 11), Kir2.1^−/− (n = 8), Kir2.2^−/− (n = 11) and Kir2.1^−/−-Kir.2.2^−/− (n = 4) myocytes. Current amplitudes were measured at the end of the 500 ms voltage-clamp step and were normalized to cell capacitance to control for cell surface area.

Figure 3. Inwardly rectifying K⁺ currents in 60 mm external K⁺

A–D, inwardly rectifying currents were recorded in 60 mm external K⁺ from voltage-clamped neonatal myocytes isolated from wild-type, Kir2.1^−/−, Kir2.2^−/− and Kir2.1^−/−-Kir.2.2^−/− mice. For each family of current traces, the voltage was stepped from a holding potential of −10 mV to potentials of −10 to −70 mV (in 15 mV increments). Leak currents were subtracted using currents evoked by small depolarizing pulses (P/4 method). Cell capacitances were 15, 14, 11 and 15 pF, respectively. E, steady state currents (means ±s.e.m.) were plotted as a function of voltage in wild-type (n = 4), Kir2.1^−/− (n = 8), Kir2.2^−/− (n = 11) and Kir2.1^−/−-Kir.2.2^−/− (n = 9) myocytes. Current amplitudes were measured at the end of the 500 ms voltage-clamp step and were normalized to cell capacitance to control for cell surface area.

Figure 4. Inward Ba²⁺ currents in wild-type and Kir2.1^−/− neonatal ventricular myocytes

A and B, Ba²⁺ currents through T- and L-type Ca²⁺ channels were recorded in the presence of external TTX and internal Cs⁺ from voltage-clamped neonatal myocytes isolated from wild-type and Kir2.1^−/− mice. For each family of current traces, the voltage was stepped from a holding potential of −80 mV to potentials of −40 to +50 mV (in 15 mV increments). Cell capacitances were 17 and 16 pF, respectively. C, steady state currents (means ±s.e.m.) were plotted as a function of voltage in wild-type (n = 9) and Kir2.1^−/− (n = 9) myocytes, with and without the addition of 1 μm nifedipine to reduce L-type currents. Currents were measured 50 ms into the voltage pulse and were normalized to cell capacitance to control for cell surface area.

Figure 5. Outward K⁺ currents in wild-type and Kir2.1^−/− neonatal ventricular myocytes

A and B, outward K⁺ currents were recorded from voltage-clamped myocytes isolated from wild-type and Kir2.1^−/− mice. For each family of current traces, the voltage was stepped from a holding potential of −40 mV to potentials of −40 to +80 mV (in 20 mV increments). The Na⁺-free external solution contained 500 μm Cd²⁺ to block Ca²⁺ currents. Cell capacitances were 22 and 23 pF, respectively. C, steady state currents (means ±s.e.m.) plotted as a function of the depolarizing voltage in wild-type (n = 6) and Kir2.1^−/− (n = 6) myocytes. Currents were measured at the end of the 500 ms voltage-clamp step and were normalized to cell capacitance to control for cell surface area.

Figure 6. Triggered action potentials in wild-type and Kir2.1^−/− neonatal ventricular myocytes

A and B, representative triggered action potentials recorded from current-clamped myocytes isolated from wild-type and Kir2.1^−/− mice. The myocytes were held at approximately −80 mV by the injection of a small hyperpolarizing current and action potentials were triggered by brief injections of depolarizing current. C, superimposed average action potentials from wild-type (n = 17) and knockout (n = 11) myocytes. Though the holding potentials of cells occasionally varied by up to 5 mV, the broadening of the action potential correlated consistently with the genotype of the cell and not with the minor variations in holding potential.

Figure 7. Net current during the falling phase of the action potential as a function of voltage

A, individual triggered action potential traces like those in Fig. 6 were differentiated with respect to time and multiplied by the capacitance of each cell to calculate the net current flowing at each time point of the falling phase of the action potential. The resultant net currents were determined for each membrane potential and average values ±s.e.m. are shown for wild-type (n = 16) and knockout (n = 10) myocytes. B, the difference current was calculated by subtracting the curves in part A to reveal the net current change resulting from the loss of Kir2.1.

Figure 8. Spontaneous activity of wild-type and Kir2.1^−/− myocytes

A, cumulative probability histogram of the beat frequency of spontaneously active wild-type (n = 20) and Kir2.1^−/− (n = 19) ventricular myocytes. The frequency of the beats was calculated by determining the number of spontaneously generated action potentials seen in a 30 s period of current clamp recordings (I = 0). B, individual myocytes at 37 °C were observed for 3 s and were scored as beating or quiescent. Approximately 60 cells were examined per dish from wild-type (n = 12) and Kir2.1^−/− (n = 12) animals. The observer was blind to the phenotype of the mouse from which the cells were isolated.

Figure 9. Examples of control and Kir2.1^−/− ECGs

Representative ECGs from a wild-type (A) and a Kir2.1^−/− (B) pup. The mutants are consistently bradycardic but have a normal sinus rhythm. Time intervals, analysed quantitatively in Table 1 and described in Methods, are indicated on the wild-type trace.