Molecular dissection of the inward rectifier potassium current(/K1)in rabbit cardiomyocytes:: evidence for heteromeric co-assembly of Kir2.1 and Kir2.2

Cardiac inward rectifier K+ currents (I-K1) play an important role in maintaining resting membrane potential and contribute to late phase repolarization. Members of the K-ir2.x channel family appear to encode for I-K1. The purpose of this study was to determine the molecular composition of cardiac I-K1 in rabbit ventricle. Western blots revealed that K-ir2.1 and K-ir2.2, but not K-ir2.3, are expressed in rabbit ventricle. Culturing rabbit myocytes resulted in a similar to50 % reduction of I-K1 density after 48 or 72 h in culture which was associated with an 80% reduction in K-ir2.1, but no change in K-ir2.2, protein expression. Dominant-negative (DN) constructs of K-ir2.1, K-ir2.2 and K-ir2.3 were generated and tested in tsA201 cells. Adenovirus-mediated over-expression of K-ir2.1dn, K-ir2.2dn or K-ir2.1dn plus K-ir2.2dn in cultured rabbit ventricular myocytes reduced I-K1 density equally by 70% 72 h post-infection, while AdK(ir2.3dn) 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, K-ir2.2 and K-ir2.3 channels. The dependence of I-K1 on [Ba2+] revealed a single binding isotherm which did not change with time in culture. The IC50 for block of I-K1 was also unaffected by expression of the different DN genes after 72 h in culture. Taken together, these results demonstrate functional expression of K-ir2.1 and K-ir2.2 in rabbit ventricular myocytes and suggest that macroscopic I-K1 is predominantly composed of K-ir2.1 and K-ir2.2 heterotetramers.