An Additional Ca(2+) Binding Site Allosterically Controls TMEM16A Activation

Calcium (Ca(2+)) is the primary stimulus for transmembrane protein 16 (TMEM16) Ca(2+)-activated chloride channels and phospholipid scramblases, which regulate important physiological processes ranging from smooth muscle contraction to blood coagulation and tumor progression. Binding of intracellular Ca(2+) to two highly conserved orthosteric binding sites in transmembrane helices (TMs) 6–8 efficiently opens the permeation pathway formed by TMs 3–7. Recent structures of TMEM16K and TMEM16F scramblases revealed an additional Ca(2+) binding site between TM2 and TM10, whose functional relevance remains unknown. Here, we report that Ca(2+) binds with high affinity to the equivalent third Ca(2+) site in TMEM16A to enhance channel activation. Our cadmium (Cd(2+)) metal bridging experiments reveal that the third Ca(2+) site’s conformational states can profoundly influence TMEM16A’s opening. Our study thus confirms the existence of a third Ca(2+) site in TMEM16A, defines its functional importance in channel gating, and provides insight into a long-range allosteric gating mechanism of TMEM16 channels and scramblases.