Interplay Between Lipid Modulators of Kir2 Channels: Cholesterol and PIP2

We have shown earlier that Kir2 channels are suppressed by the elevation of membrane cholesterol. Moreover, it is also well known that activation of Kir channels is critically dependent on a regulatory phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2). In this study we examined the cross-talk between cholesterol and PIP2 in the regulation of Kir2 channels. The strength of Kir2-PIP2 interactions was assessed by acute sequestering of PIP2 with neomycin dialyzed into cells through a patch pipette while simultaneously recording whole cell currents. Consistent with a reduction in PIP2 levels, dialysis of neomycin resulted in a decrease in Kir2.1 and Kir2.3 current amplitudes (current rundown), however, this effect was significantly delayed by cholesterol depletion for both types of channels suggesting that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2. Furthermore, mutation of Kir2.1 that renders the channels' cholesterol insensitive abrogated cholesterol depletion-induced delay in the current rundown whereas reverse mutation in Kir2.3 has the opposite effect. These observations provide further support for the functional cross-talk between cholesterol and PIP2 in regulating Kir2 channels. Consistent with these observations, there is a significant structural overlap between cytosolic residues that are critical for the sensitivity of Kir2 channels to the two lipid modulators but based on recent studies, there is little or no overlap between cholesterol and PIP2 binding sites. Taken together, these observations suggest that cholesterol and PIP2 regulate the channels through distinct binding sites but that the signals generated by the binding of the two modulators converge.