Comparative Analysis of Cholesterol Sensitivity of Kir Channels: Role of the Cytoplasmic Domain

Abstract

Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir2 channels and several other ion channels that has emerged in recent years is that they are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. Yet, the mechanism by which cholesterol affects channel function is not clear.Here we show that in addition to Kir2 channels, members of other Kir subfamilies are also regulated by cholesterol. Interestingly, while similarly to Kir2 channels, several Kir channels are suppressed by an increase in membrane cholesterol, the function of others is enhanced following cholesterol enrichment. Furthermore, similarly to Kir2.1, and independent of the impact of cholesterol on channel function, we find that mutation of residues in the CD loop affect cholesterol sensitivity of Kir channels.Among Kir2.1 CD loop residues, we have recently shown that the L222I mutation has the strongest effect on cholesterol sensitivity. This result is surprising since Kir2.2, which is as cholesterol sensitive as Kir2.1, already has an isoleucine at the corresponding position. Here we obtain further insight regarding the role of the cytosolic domain of Kir2 channels by examining mutations in adjacent cytosolic regions that also lead to loss of cholesterol sensitivity. In addition, we trace the source of the difference between Kir2.1 and Kir2.2 to a residue in the EF loop, N251, whose mutation to an aspartate reverses the effect of the L222I residue, and restores cholesterol sensitivity.These findings suggest an indirect role of the cytosolic domain of Kir channels in regulating the effect of cholesterol on channel function and provide insight into the structural determinants of their gating mechanism.