PH-dependent promotion of phospholipid flip-flop by the KcsA potassium channel

KcsA is a pH-dependent potassium channel that is activated at acidic pH. The channel undergoes global conformational changes upon activation. We hypothesized that the open-close conformational changes of the transmembrane region could promote the flip-flop of phospholipids. Based on this hypothesis, we measured the flip-flop of NBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of C₆NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH4.0 than at pH7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were non-selectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA channel, tetrabutylammonium locks the channel in the open conformation at acidic pH; however, it did not alter the flip rate of C₆NBD-PC. Thus, the open-close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked an N-terminal amphipathic helix (M0-helix) was found to show reduced ability to flip C₆NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of M0-helix, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation. These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.