Abstract
Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.