Cholesterol Regulates the Basal Functions and Ethanol Sensitivity of Large Conductance, Ca 2+-Sensitive K+ channel through Specific Cholesterol-Protein Interaction

Abstract

Membrane cholesterol plays an important role in regulation of a variety of ion channels and in maintaining the normal functions of cell membranes. The mechanism underlying this influence on ion channels, however, remains poorly understood. Cholesterol can act on ion channel proteins through either: 1) direct interaction with the protein, or 2) indirect effects on the biophysical properties of lipid bilayer. To differentiate between these alternatives, we used a synthesized enantiomer of cholesterol (ent-CHS). Enantiomeric forms of cholesterol have been used in the study of cholesterol-protein interaction, based upon the assumption that the effect on the lipid bilayer will be identical to that of natural cholesterol (nat-CHS), steric interaction directly with the protein will be abolished. We employed planar bilayer recording techniques to study the interaction of nat-CHS and ent-CHS with the large conductance, calcium-sensitive potassium channel (BK) in lipid bilayers of POPE/POPS (3/1, weight) and DOPE/SPM (3/2, weight), and examined how the presence of nat-CHS and ent-CHS affected the basal function and ethanol sensitivity of the BK channel. We found that ent-CHS increased BK channel conductance in both lipid bilayers similarly to that of nat-CHS. However, they are strikingly different in their effects on BK channel gating and ethanol sensitivity. In the POPE/POPS bilayer, nat-CHS dramatically reduced the open probability (Po) while ent-CHS did not. Both ent-CHS and nat-CHS reduced the ethanol sensitivity of the BK channel, but ent-CHS did so to a lesser extent. In the DOPE/SPM bilayer, nat-CHS dramatically changed the ethanol response of BK, depending on cholesterol concentration in the membrane. However, ent-CHS had little effect on ethanol sensitivity. We conclude that membrane cholesterol has a specific interaction with the BK channel that can directly influence ethanol's actions.