An Additional Ca2+ Binding Site Allosterically Controls TMEM16A Activation.

Calcium (Ca2+) is the primary stimulus for transmembrane protein 16 (TMEM16) Ca2+-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 Ca2+ 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 Ca2+ binding site between TM2 and TM10, whose functional relevance remains unknown. Here, we report that Ca2+ binds with high affinity to the equivalent third Ca2+ site in TMEM16A to enhance channel activation. Our cadmium (Cd2+) metal bridging experiments reveal that the third Ca2+ site's conformational states can profoundly influence TMEM16A's opening. Our study thus confirms the existence of a third Ca2+ 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.