Molecular diversity of the repolarizing voltage-gated K+ currents in mouse atrial cells

Abstract

Voltage-clamp studies on atrial myocytes isolated from adult and postnatal day 15 (P15) C57BL6 mice demonstrate the presence of three kinetically distinct Ca2+-independent, depolarization-activated outward K+ currents: a fast, transient outward current (Ito,f), a rapidly activating, slowly inactivating current (IK,s) and a non-inactivating, steady-state current (Iss). The time- and voltage-dependent properties of to,f, IK,s and Iss in adult and P15 atrial cells are indistinguishable. Pharmacological experiments reveal the presence of two components of IK,s: one that is blocked selectively by 50 microM 4-aminopyridine (4-AP), and a 4-AP-insensitive component that is blocked by 25 mM TEA; Iss is also partially attenuated by 25 mM TEA. There are also two components of IK,s recovery from steady-state inactivation. To explore the molecular correlates of mouse atrial IK,s and Iss, whole-cell voltage-clamp recordings were obtained from P15 and adult atrial cells isolated from transgenic mice expressing a mutant Kv2.1 alpha subunit (Kv2.1N216Flag) that functions as a dominant negative, and from P15 atrial myocytes exposed to (1 microM) antisense oligodeoxynucleotides (AsODNs) targeted against Kv1.5 or Kv2.1. Peak outward K+ current densities are attenuated significantly in atrial myocytes isolated from P15 and adult Kv2.1N216Flag-expressing animals and in P15 cells exposed to AsODNs targeted against either Kv1.5 or Kv2.1. Analysis of the decay phases of the outward currents evoked during long (5 s) depolarizing voltage steps revealed that IK, s is selectively attenuated in cells exposed to the Kv1.5 AsODN, whereas both IK,s and Iss are attenuated in the presence of the Kv2. 1 AsODN. In P15 and adult Kv2.1N216Flag-expressing atrial cells, mean +/- s.e.m. IK,s and Iss densities are also significantly lower than in non-transgenic atrial cells. In addition, pharmacological experiments reveal that the TEA-sensitive component IK,s is selectively eliminated in P15 and adult Kv2.1N216Flag-expressing atrial cells. Taken together, the results presented here reveal that both Kv1.5 and Kv2.1 contribute to mouse atrial IK,s, consistent with the presence of two molecularly distinct components of IK,s. In addition, Kv2.1 contributes to mouse atrial Iss.

Figure 3. Expression of Kv α subunits in mouse atria and ventricles

A, RT-PCR analysis reveals that Kv1.5 and Kv2.1 are readily detected in wild-type (wt) mouse atria and ventricles, whereas Kv2.1N216Flag is only detected in the hearts of transgenic (Kv2.1N216Flag-expressing) mice. For comparisons among samples, probes against muscle-specific α actin were employed. B, forward and reverse primers used in the PCR analysis.

Figure 1. Two components of adult mouse atrial I_K,s recovery from steady-state inactivation

After inactivating the currents during 5 s prepulses to +30 mV, cells were hyperpolarized to −70 mV for times ranging from 10 ms to 30 s before a second test depolarization to +30 mV (to assess the extent of recovery) was presented; the protocol is illustrated in the lower left panel. A, representative (total outward K⁺) current waveforms recorded during the conditioning step and after varying recovery times are presented; currents recorded after brief recovery times are displayed in the inset. The amplitudes of I_to,f, I_K,s and I_ss at each recovery time (in each cell) were determined from double exponential fits to the decay phases of the total outward K⁺ currents. These values were then normalized to the amplitudes of the currents (I_to,f, I_K,s and I_ss) evoked after the 40 s recovery period (in the same cell). B, mean ±s.e.m. (n = 7) normalized I_K,s amplitudes are plotted as a function of the interpulse interval (IPI); the recovery data at early times are shown on an expanded time scale in the inset. As is evident, the mean normalized recovery data for I_K,s are well fitted (continuous line) by the sum of two exponentials with time constants of ≈0.5 and ≈10 s (see text).

Figure 2. 4-AP-sensitive and insensitive components of I_K,s in adult mouse atrial myocytes

Outward K⁺ currents were recorded during 5 s depolarizations to test potentials between 0 and +50 mV from a V_h of −60 mV; all of the records displayed were obtained from the same cell. Control currents (A) and currents in the presence of 50 μM 4-AP (B) or 5 mM 4-AP (D) were recorded. The waveforms of the 50 μM 4-AP-sensitive (C) and the 5 mM 4-AP-sensitive (E) currents were obtained by off-line digital subtraction of the records in the presence of 50 μM (B) or 5 mM (D) 4-AP from the controls (A). To determine the effect of TEA on the currents remaining in the presence of 5 mM 4-AP (D), cells were exposed to 25 mM TEA (F); as is evident, most of residual current was blocked in the presence of TEA (F). The component of the 4-AP-insensitive currents (D) that is blocked by TEA (G) was obtained by off-line digital subtraction of the records in the presence of 25 mM TEA (F) from those in the presence of 5 mM 4-AP (D). Similar results were obtained in five cells.

Figure 4. Comparison of outward K⁺ currents in adult and postnatal day 15 (P15) mouse atrial myocytes

Depolarization-activated outward currents, evoked during 100 ms (A and B) and 5 s (C and D) voltage steps to varying test potentials (−50 to +50 mV) from a holding potential of −60 mV, were measured and subsequently normalized to the whole-cell membrane capacitance (C_m) in the same cell to correct for differences in cell size and determine current densities. The decay phases of the outward K⁺ currents evoked during 5 s voltage steps in both P15 (C) and adult (D) cells were well fitted by the sum of two exponentials; the fits (continuous lines) are superimposed on the experimental data. E, normalized mean ±s.e.m. peak outward K⁺ current densities in P15 and atrial cells are plotted as a function of test potential.

Figure 5. Peak outward K⁺ currents are attenuated in atrial myocytes isolated from P15 Kv2.1N216Flag-expressing transgenics and in P15 atrial cells exposed to AsODNs targeted against either Kv1.5 or Kv2.1

Whole-cell outward K⁺ currents were recorded as described in the legend to Fig. 4 from P15 cells isolated from wild-type (WT) (A) or Kv2.1N216Flag-expressing (B) mice, and from P15 cells exposed to either the Kv2.1 AsODN (C) or the Kv1.5 AsODN (D). Representative outward K⁺ current waveforms (and densities) are presented in A-D. Compared with wild-type P15 cells, mean ±s.e.m. peak outward K⁺ current densities (E) are significantly (P < 0.01) lower in P15 cells exposed to either the Kv1.5 (n = 15) or the Kv2.1 (n = 15) AsODN. Mean ±s.e.m. peak outward currents are also significantly (P < 0.001) lower in Kv2.1N216Flag-expressing (n = 18), than in WT (n = 30) cells (E).

Figure 6. Kv1.5 contributes to I_K,s, whereas Kv2.1 contributes to both I_K,s and I_ss

Outward K⁺ currents were recorded in response to 5 s depolarizing voltage steps to 0 mV from −60 mV in P15 wild-type (WT) atrial myocytes (A), cells exposed to either the Kv1.5 AsODN (D) or the Kv2.1 AsODN (C), and in Kv2.1N216Flag-expressing cells (B). The decay phases of the currents were fitted to the sum of two exponentials to provide the amplitudes of I_to,f, I_K,s and I_ss. E, in cells exposed to the Kv1.5 AsODN (n = 8) or the Kv2.1 AsODN (n = 7) and in cells isolated from Kv2.1N216Flag-expressing transgenics (n = 11), I_K,s (density) is significantly (**P < 0.01) lower than I_K,s density in WT (n = 12) cells. F, the time constants of I_K,s inactivation, however, were not affected by the (Kv1.5 or Kv2.1) AsODNs or by the expression of the (Kv2.1N216Flag) transgene. Mean ±s.e.m.I_ss densities are also attenuated significantly (*P < 0.05, **P < 0.01) in P15 atrial myocytes exposed to the Kv2.1 AsODN and in Kv2.1N216Flag-expressing cells (E).

Figure 7. The 4-AP-sensitive component of I_K,s is evident in Kv2.1N216Flag-expressing mouse atrial myocytes

Outward K⁺ currents were recorded as described in the legend to Fig. 2; all of the records displayed were obtained from the same cell. Control currents (A) and currents in the presence of 50 μM 4-AP (B) or 5 mM 4-AP (D) were recorded. The waveforms of the 50 μM 4-AP-sensitive (C) and the 5 mM 4-AP-sensitive (E) currents were obtained by off-line digital subtraction of the records in the presence of 50 μM (B) or 5 mM (D) 4-AP from the controls (A). In contrast to the findings in wild-type cells (Fig. 2), there does not appear to be a 4-AP-insensitive component of I_K,s in Kv2.1N216Flag-expressing atrial cells. Similar results were obtained in five cells.

Figure 8. The TEA-sensitive component of I_K,s, evident in wild-type mouse atrial myocytes is not detected in cells isolated from the Kv2.1N216Flag-expressing transgenic animals

Outward K⁺ currents were recorded as described in the legend to Fig. 2; the records displayed in A, B and C were obtained from the same cell, as were the records in D, E and F. Control currents (A and D) and currents in the presence of 25 mM TEA (B and E) were recorded in wild-type (WT) (A and B) and Kv2.1N216Flag-expressing cells (D and E). The waveforms of the 25 mM TEA-sensitive (C and F) were obtained by off-line digital subtraction of the records in the presence of 25 mM TEA (B and E) from the controls (A and D). Similar results were obtained in five WT and five Kv2.1N216Flag-expressing cells.