Targeting of Kir2.1 and Downregulation of Inward Rectifier K+ Current by miR-212

Abstract

Downregulation of inwardly rectifying K+ channels contributes to an increased risk of cardiac arrhythmia in heart failure and to impaired cerebral arterial dilation in chronic alcohol consumption. The downregulation mechanism is unknown, although post-transcriptional regulation of gene expression by microRNAs is a strong possibility that has not been fully investigated. miR-212 is markedly augmented in heart failure and in chronic alcoholism, and is predicted by bioinformatic algorithms to target Kir2.1, the predominant inward rectifier K+ channel expressed in heart and arterial smooth muscle. We developed a fluorescence-based assay for identifying microRNA targets, using mCherry red fluorescent protein to report target sequence expression and enhanced green fluorescent protein for microRNA expression. Using this assay, we demonstrated a functional target for miR-212 in the 3′ untranslated region of Kir2.1. Red/green fluorescence intensity ratio was significantly lower in miR-212-expressing HEK293 cells compared to non-targeting control (miR-212 0.72 ± 0.024 (mean ± sem), n = 550; control 1.21 ± 0.025, n = 731; p<.001, log transformed data). The effect of miR-212 was attenuated by mutating the predicted target site (% inhibition 58.0 ± 14.51, n = 3 wild-type; 22.7 ± 1.25, n = 3 mutant). Expression of miR-212 downregulated endogenous Kir2.1 protein in HeLa cells, as shown by quantitative western blot of membrane extracts (band intensity vs Na+/K+-ATPase loading control: miR-212 0.0647 ± 0.0047; non-targeting control 0.0895 ± 0.0045; n=3, p<.05). Endogenous inward rectifier K+ current in HeLa cells was isolated by extracellular application of 100 µM Ba2+ during whole-cell patch-clamp recording. Ba2+-sensitive current density was significantly smaller in miR-212-transfected (n=13) vs control-transfected cells (n=8); p<.01. In conclusion, downregulation of inwardly rectifying K+ current and Kir2.1 expression in heart failure and alcoholic cerebrovascular dysfunction may be functionally linked to upregulation of miR-212.