Silencing of Kir2 channels by caveolin-1: cross-talk with cholesterol

Key points Inwardly rectifying potassium channels (Kir) play key roles in regulating membrane excitability and K+ homeostasis in multiple cell types. Our earlier studies showed that Kir2 channels, one of the major subfamilies of Kir, are suppressed by membrane cholesterol and that cholesterol stabilizes these channels in a closed silent' state. This paper addresses a fundamental question of how Kir2 channels are regulated by caveolins, the major structural proteins of caveolae, and the relationship between the sensitivity of the channels to caveolin and to cholesterol. In this study, we present direct evidence that caveolin-1 is a negative regulator of Kir2 function and that cholesterol and caveolin-1 regulate the channels by a common mechanism. This study also challenges a general notion that cholesterol depletion alters ion channel function by disrupting caveolae, demonstrating that neither caveolin-1 nor intact caveolae are required for cholesterol sensitivity of Kir2 channels. Furthermore, we present first insights into the structural determinants of the cross-talk between the sensitivity of Kir2 channels to caveolin and to cholesterol. AbstractA growing number of studies show that different types of ion channels localize in caveolae and are regulated by the level of membrane cholesterol. Furthermore, it has been proposed that cholesterol-induced regulation of ion channels might be attributed to partitioning into caveolae and association with caveolin-1 (Cav-1). We tested, therefore, whether Cav-1 regulates the function of inwardly rectifying potassium channels Kir2.1 that play major roles in the regulation of membrane potentials of numerous mammalian cells. Our earlier studies demonstrated that Kir2.1 channels are cholesterol sensitive. In this study, we show that Kir2.1 channels co-immunoprecipitate with Cav-1 and that co-expression of Kir2.1 channels with Cav-1 in HEK293 cells results in suppression of Kir2 current indicating that Cav-1 is a negative regulator of Kir2 function. These observations are confirmed by comparing Kir currents in bone marrow-derived macrophages isolated from Cav-1(-/-) and wild-type animals. We also show, however, that Kir2 channels maintain their sensitivity to cholesterol in HEK293 cells that have very low levels of endogenous Cav-1 and in bone marrow-derived macrophages isolated from Cav-1(-/-) knockout mice. Thus, these studies indicate that Cav-1 and/or intact caveolae are not required for cholesterol sensitivity of Kir channels. Moreover, a single point mutation of Kir2.1, L222I that abrogates the sensitivity of the channels to cholesterol also abolishes their sensitivity to Cav-1 suggesting that the two modulators regulate Kir2 channels via a common mechanism.