Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of CFTR Channel Activity

Abstract

CFTR has recently been shown to exist in two distinct populations in the plasma membrane of primary human bronchial epithelial (HBE) cells: one population is diffusely distributed and the other is in cholesterol-dependent lipid rafts. Cholesterol homeostasis has been found to be altered in Cystic Fibrosis patients, although some controversy remains. For example, epithelial cells expressing F508del-CFTR exhibit increased cholesterol; however, neutrophils derived from CF patients show reduced cholesterol levels in the plasma membrane. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms, including direct cholesterol-channel protein interactions, indirect changes in physical properties of the membrane, and more. Thus far, whether cholesterol directly modulates CFTR channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized Fischer Rat Thyroid (FRT) cells and HBE cells. We found that cholesterol depletion with 5 mM methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol in FRT cells. This cholesterol reduction by MβCD significantly decreased sensitivity of WT- and P67L-CFTR forskolin (FSK)-mediated activation. This is also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of P67L-CFTR by FSK. In addition, alteration of cholesterol increased efficacy of VX-770, a clinically-used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations, but not at high FSK concentrations. This change in efficacy of VX-770-mediated potentiation likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the cholesterol level of the plasma membrane significantly modulate CFTR channel function and consequently affect potentiator efficiency in CF patients.