Regulatory and Gating Mechanisms of TMEM16A Chloride Channel
| dc.contributor.advisor | Yang, Huanghe | |
| dc.contributor.author | Le, Son Cong | |
| dc.date.accessioned | 2021-05-19T18:08:06Z | |
| dc.date.available | 2021-05-19T18:08:06Z | |
| dc.date.issued | 2021 | |
| dc.department | Biochemistry | |
| dc.description.abstract | The TMEM16 protein family comprises two novel classes of structurally conserved but functionally distinct membrane transporters that function as Ca2+-dependent Cl- channels (CaCCs) or dual functional Ca2+-dependent ion channels and phospholipid scramblases. The TMEM16A and TMEM16B CaCCs conduct Cl- across the membrane and regulate transepithelial fluid transport, smooth muscle contraction, neuronal excitability and sensory signal transduction. Most other TMEM16 members are scramblases that mediate the flip-flop of phospholipids across the membrane to allow phosphatidylserine externalization, which is essential in a plethora of important processes such as blood coagulation, bone development and viral and cell fusion. Activation of TMEM16 proteins requires the binding of intracellular Ca2+ to two highly conserved orthosteric binding sites in transmembrane helices (TM) 6-8 opens the permeation pathway formed by TMs 3-7. However, prolonged Ca2+-dependent simulation of TMEM16 channels results in current desensitization or rundown, the mechanism of which is unclear. In addition, recent structural studies of TMEM16 scramblases revealed yet another Ca2+ binding site in TM2 and TM10 whose functional relevance remains unknown. By combining electrophysiology, systematic mutagenesis and molecular dynamics simulation, we first demonstrate that the phosphatidylinositol-(4,5)-bisphosphate (or PI(4,5)P2) plays a critical role in TMEM16A’s Ca2+-dependent activation and desensitization. We identify key basic residues at the cytosolic interface of transmembrane segments (TMs) 3–5 as the putative PI(4,5)P2-binding site, which is supported by our molecular dynamics studies. These studies reveal that TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6–8 and a PI(4,5)P2-binding regulatory module formed by TMs 3–5, which mediate channel activation and desensitization, respectively. Next, we show that Ca2+ binds with high affinity to the putative third Ca2+ site in TM2 and TM10 of TMEM16A to enhance channel activation. Our cadmium (Cd2+) metal bridging experiments further show that the third Ca2+ site’s conformational states can profoundly influence TMEM16A’s opening. Taken together, our studies not only establish the molecular bases for the PI(4,5)P2-dependent regulation of TMEM16A and the long-range allosteric gating mechanism via the third Ca2+ binding site; they also provide functional insight into the structural organization of TMEM16 ion channels and lipid scramblases. | |
| dc.identifier.uri | ||
| dc.subject | Biochemistry | |
| dc.subject | Biophysics | |
| dc.subject | Allosteric | |
| dc.subject | Ca2+ Binding Site | |
| dc.subject | Channel Gating | |
| dc.subject | PI(4 | |
| dc.subject | 5)P2 | |
| dc.subject | Regulation | |
| dc.subject | TMEM16 | |
| dc.title | Regulatory and Gating Mechanisms of TMEM16A Chloride Channel | |
| dc.type | Dissertation |