Determining the target of membrane sterols on voltage-gated potassium channels

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel K_v1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing K_v1.3 and K_v10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.