TMEM16A is implicated in regulating endothelial functions, including membrane potential control, Ca²⁺ signaling regulation, and cell proliferation. TMEM16A is known to be stimulated by PIP2 in the inner leaflet during the activation process. Recent reports have shown that increased cholesterol concentration can lead to endothelial dysfunction, indicating that the activity of TMEM16A is mediated by multiple membrane molecules. Relying on multiscale and high-throughput dynamic simulation methods, we found PIP2 binds significantly at α6, the key region of channel permeability, which is consistent with the existing data. Addition of cholesterol reduces the concentration of PIP2 around α4 and α6, as well as the intensity of PIP2 on α6. Moreover, cholesterol causes the lower half of α6 to turn up, reflecting the closed processing tendency of TMEM16A affected by cholesterol, in agreement to the experiment results. The binding free energy calculation further proves that the presence of cholesterol is more conducive to the upward turning of α6, which is a more energy-saving state. This study reveals the molecular regulation mechanism of different membrane molecules in the expression of TMEM16A activity from the perspective of atomic-level action, which helps to build the membrane environment basis for the activity of related ion channels.
 

cholesterol,PIP2,lipid-protein interplay,molecular dynamics