Molecular dissection of the inward rectifier potassium current (IK1) in rabbit cardiomyocytes: evidence for heteromeric co-assembly of Kir2.1 and Kir2.2

Abstract

Cardiac inward rectifier K+ currents (IK1) play an important role in maintaining resting membrane potential and contribute to late phase repolarization. Members of the Kir2.x channel family appear to encode for IK1. The purpose of this study was to determine the molecular composition of cardiac IK1 in rabbit ventricle. Western blots revealed that Kir2.1 and Kir2.2, but not Kir2.3, are expressed in rabbit ventricle. Culturing rabbit myocytes resulted in an approximately 50% reduction of IK1 density after 48 or 72 h in culture which was associated with an 80% reduction in Kir2.1, but no change in Kir2.2, protein expression. Dominant-negative (DN) constructs of Kir2.1, Kir2.2 and Kir2.3 were generated and tested in tsA201 cells. Adenovirus-mediated over-expression of Kir2.1dn, Kir2.2dn or Kir2.1dn plus Kir2.2dn in cultured rabbit ventricular myocytes reduced IK1 density equally by 70% 72 h post-infection, while AdKir2.3dn had no effect, compared to green fluorescent protein (GFP)-infected myocytes. Previous studies indicate that the [Ba2+] required for half-maximum block (IC50) differs significantly between Kir2.1, Kir2.2 and Kir2.3 channels. The dependence of IK1 on [Ba2+] revealed a single binding isotherm which did not change with time in culture. The IC50 for block of IK1 was also unaffected by expression of the different DN genes after 72 h in culture. Taken together, these results demonstrate functional expression of Kir2.1 and Kir2.2 in rabbit ventricular myocytes and suggest that macroscopic IK1 is predominantly composed of Kir2.1 and Kir2.2 heterotetramers.

Figure 1. Effect of culture on I_K1 in rabbit ventricular myocytes

A, typical Ba²⁺-subtracted membrane currents elicited by 500 ms voltage steps ranging from −130 mV to −10 mV (by 10 mV) from a holding potential of −40 mV. B, peak I_K1 densities from myocytes cultured for 2 h (□) were significantly decreased (P < 0.05) after culturing for 48 h (▵) but stayed unchanged upon further culturing (72h, •) for membrane potentials between −130 and −80 mV.

Figure 2. Western blots for native K_ir2.1, K_ir2.2 and K_ir2.3 proteins in rabbit ventricular myocytes

Western blots showing typical signals obtained by probing rabbit ventricular myocyte preparations harvested after 2 or 48 h in culture along with rabbit brain controls using specific antibodies for K_ir2.1 (A), K_ir2.2 (B) or K_ir2.3 (C). The bar graphs show summarized results of densitometric measurements for K_ir2.1 (n = 3) and K_ir2.2 (n = 7) after 48 h in culture normalized to expression levels after 2 h in culture.

Figure 3. Validation of dominant-negative (DN) genes in tsA201 cells

Bar graphs showing the membrane conductance measured in tsA201 cells transfected with either wild-type K_ir2.1 (A) or K_ir2.2 (B) genes. Equimolar co-transfection with either K_ir2.1dn or K_ir2.2dn constructs significantly diminished the slope conductance of the wild-type channels. Doubling the amount of wild-type construct (‘K_ir2.1 double’ or ‘K_ir2.2 double’) increased the slope conductance correspondingly. Membrane potentials were held at −40 mV and voltage steps from −130 to −10 mV in 10 mV increments were applied for 500 ms with an interval of 1 s. Slope conductance was calculated from the linear range of the current–voltage relationship between −130 and −80 mV). * Significantly different at P < 0.05 relative to K_ir2.1 (A) or K_ir2.2 (B).

Figure 4. Effect of infection of different AdK_ir2.xdn constructs on I_K1 in cultured rabbit ventricular myocytes

A, original traces showing typical Ba²⁺-substracted I_K1 recorded from myocytes 72 h after infection with the indicated constructs. B, Ba²⁺-subtracted peak currents as a function of voltage in cultured rabbit myocytes 72 h after infection. I_K1 density (from −130 mV to −90 mV) decreased significantly in myoctyes infected with AdK_ir2.1dn, AdK_ir2.1dn or AdK_ir2.1dn plus AdK_ir2.1dn compared to the control myocytes (AdGFP, P < 0.05). No significant difference was detected between I_K1 density recorded after infection with AdK_ir2.1dn, AdK_ir2.2dn or AdK_ir2.1dn plus AdK_ir2.2dn.

Figure 5. Effects of a dominant-negative K_ir2.3 construct

A, slope conductance measured in tsA201 cells expressing K_ir2.3 or K_ir2.3 plus K_ir2.3dn. Cells were held at a membrane potential of −40 mV and voltage steps from −130 to −10 mV in 10 mV increments were applied for 500 ms with a 1 s interval. Slope conductance was calculated from the linear range of the current–voltage relationship (−130 to −80 mV). B, Ba²⁺-subtracted peak I_K1 as a function of voltage measured in rabbit ventricular myocytes expressing either AdGFP or AdK_ir2.3dn. * Significant difference at P < 0.05 between K_ir2.3 alone and K_ir2.3 plus K_ir2.3dn.

Figure 6. Effects of infection with AdK_ir2.X genes on transient outward K⁺ currents in cultured rabbit ventricular myocytes

A, typical recordings of I_to measured at +60 mV with a prepulse at +40 mV to inactivate sodium currents in myocytes infected with AdGFP or AdK_ir2.1dn plus AdK_ir2.2dn 72 h post-infection. B, summary of the effects of infection on I_to in cultured myocytes following infection with the indicated AdK_ir2.X genes.

Figure 7. Blockade of cardiac I_K1 by Ba²⁺ in cultured rabbit ventricular myocytes

A, Ba²⁺ dose–response curves for I_K1 in non-infected myocytes cultured for 2 h or 48 h at −100 mV. The data were fitted with a single binding isotherm I_Ba/I_Con = 1/(1 + [Ba²⁺]ⁿ/IC₅₀ⁿ) where I_Ba is the I_K1 in the presence of Ba²⁺ while I_Con is the I_K1 before application of Ba²⁺. B, Ba²⁺ dose–response curve for I_K1 measured at −100 mV in myocytes cultured for 48 h following over-expression of various AdK_ir2.x genes as indicated. Despite marked differences in the level of I_K1 in the various groups, there was no measurable difference in the IC₅₀ values for Ba²⁺.