Molecular dissection of the inward rectifier potassium current (I_K1) in rabbit cardiomyocytes: evidence for heteromeric co‐assembly of K_ir2.1 and K_ir2.2

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 a ∼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.

Cardiac ion channels

“…Kir2.1 is the key component of the current I K1 in ventricular myocytes and is a critical regulator of resting membrane potential [59][60][61] ; whereas Kir2.3 is the dominant Kir2 subunit in atrial myocytes. 62 In the vasculature, Kir2.1 channels are found in smooth muscle cells of small diameter arteries, where they control vascular tone (Fig.…”

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

“…Misexpression of this dominant-negative construct specifically induced 20% heterotaxia [(n=191), controls=4% (n=422), 2 =40.6, PӶ0.001]. By contrast, injection of mRNAs encoding the Xenopus H,K-ATPase ␣ subunit (Mathews et al, 1995), or the dominant-negative Kir2.2 subunit (Zobel et al, 2003) did not cause heterotaxia (1% heterotaxia each, n=85,93 respectively). These data are consistent with the screen results, demonstrate that embryonic asymmetry is not generally labile with respect to microinjection per se (even when performed during cytoplasmic rotation), and strongly support the hypothesis that H + -V-ATPase function is necessary for LR patterning.…”

Expression of inwardly rectifying potassium channel subunits in native human retinal pigment epithelium

“…3c) polyclonal antibodies, both from Alomone Labs, revealed ~100 kDa bands in brain, RPE, and retina. Because both Kir2.1 and Kir2.2 have a predicted molecular mass of 48 kDa (Giovannardi et al, 2002), the 100 kDa bands likely represent homomeric or heteromeric dimers of Kir2.1 and Kir2.2 subunits (Giovannardi et al, 2002;Zobel et al, 2003). Although the identity of the labeled protein band in brain was confirmed to be Kir2.1 in peptide blocking experiments, preabsorption of anti-Kir2.2 antibody with antigenic peptide failed to significantly reduce labeling in brain (not shown), raising questions about its specificity.…”

“…Kir2.1 and Kir2.2 channels, which can exist as homomeric or heteromeric complexes (Preisig-Muller et al, 2002), are constitutively active and strongly inwardly rectifying (Kubo et al, 1993;Koyama et al, 1994;Rae and Shepard, 2000;Yang et al, 2000;Yang et al, 2003b;Zobel et al, 2003). The strong rectification of these channels allows them to stabilize the membrane potential near the potassium equilibrium potential in excitable cells and glia.…”