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.
","abstract_html":"<p>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. </p>","abstract_has_math":false,"creators":["Le, Son Cong"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Yang, Huanghe"],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021","date_published":"2021","updated_at":"2026-07-24T02:07:10Z","subjects":["Biochemistry","Biophysics","Allosteric","Ca2+ Binding Site","Channel Gating","PI(4","5)P2","Regulation","TMEM16"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/10161/23016","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Yang, Huanghe"]},{"key":"dc:creator","label":"Author","values":["Le, Son Cong"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2021-05-19T18:08:06Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2021-05-19T18:08:06Z"]},{"key":"dc:date.issued","label":"Date","values":["2021"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biochemistry","Biophysics","Allosteric","Ca2+ Binding Site","Channel Gating","PI(4","5)P2","Regulation","TMEM16"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10161/23016"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Regulatory and Gating Mechanisms of TMEM16A Chloride Channel"]}]}],"canonical_facts":{"dc:contributor.advisor":["Yang, Huanghe"],"dc:creator":["Le, Son Cong"],"dc:date.accessioned":["2021-05-19T18:08:06Z"],"dc:date.available":["2021-05-19T18:08:06Z"],"dc:date.issued":["2021"],"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":["https://hdl.handle.net/10161/23016"],"dc:subject":["Biochemistry","Biophysics","Allosteric","Ca2+ Binding Site","Channel Gating","PI(4","5)P2","Regulation","TMEM16"],"dc:title":["Regulatory and Gating Mechanisms of TMEM16A Chloride Channel"],"dc:type":["Dissertation"]},"updated_at":"2026-07-24T02:07:10Z"}