Tembo M , Bainbridge RE , Lara-Santos C , Komondor KM , Daskivich GJ , Durrant JD , Rosenbaum JC , Carlson AE .

R01 GM125638 NIGMS NIH HHS

             chloride channel activity
             phospholipid binding
             signal transduction

	Figure 1. TMEM16A Ca2+-evoked Cl− currents rundown in excised patches and are recovered by a diC8-PI(4,5)P2 application. A, example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B, normalized plot of current measured at −60 mV versus time, fit with a single exponential (red line). C, box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D, a soluble synthetic analog of PI(4,5)P2, diC8-PI(4,5)P2, was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E, box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P2 with Ca2+ (N = 8). diC8-PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
	Figure 2. TMEM16A Ca2+-evoked Cl− currents are depleted in whole Xenopus laevis oocytes by dephosphorylation of PI(4,5)P2. A, schematic demonstrating pseudojanin translocation to the plasma membrane. To express pseudojanin at the membrane, the membrane tether Lyn11-mCherry and pseudojanin-CFP RNAs were both injected into X. laevis oocytes. Lyn11-mCherry expresses at the plasma membrane, and pseudojanin expresses in the cytoplasm. Upon rapamycin application, rapamycin binds Lyn11-mCherry and induces the membrane translocation of pseudojanin-CFP. Once at the membrane, pseudojanin-CFP dephosphorylates PI(4,5)P2 at the 4′ and 5′ position. The effects of pseudojanin on whole-cell TMEM16A Ca2+-evoked Cl− currents were measured using the two-electrode voltage-clamp technique. B, box plot distribution of the percentage remaining current observed in uninjected control and pseudojanin-CFP–expressing X. laevis oocytes after incubation in 10 μM rapamycin for 5 min. The percent of remaining currents was significantly different (p = 0.02) as determined by a two-tailed t test. ∗ denotes p < 0.05. C and D, example of whole-cell currents recorded at −80 mV before and after rapamycin application in oocytes expressing pseudojanin-CFP. Current was recorded in control solution (black) and after incubation in rapamycin for 5 min (purple). Red bar represents 250 ms duration of UV light application. CFP, cyan fluorescent protein; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
	Figure 3. Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP3 (diC8-PIP3), PI(3,4)P2 (diC8-PI(3,4)P2), and PI(3,5)P2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A, box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P2 (N = 9), diC8-PIP3 (N = 7), diC8-PI(3,4)P2 (N = 6), or diC8-PI(3,5)P2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP3 (B), diC8-PI(3,4)P2 (C), or diC8-PI(3,5)P2 (D). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P2, dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P2, dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,5)P2, dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.
	Figure 4. Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P (A), 100 μM diC8-PI5P (B), or 100 μM diC8-PI4P (D). C, box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P2, as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.
	Figure 5. Xl-VSP does not significantly change TMEM16A current rundown. Inside–out patch-clamp recordings were conducted on macropatches excised from Xenopus laevis oocytes expressing Xl-VSP. A, schematic depicting a VSP tagged with GFP. B, confocal and bright-field images of a representative X. laevis oocyte expressing GFP-tagged Xl-VSP at the plasma membrane. Bar denotes 200 μm. C, box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials for the Xl-VSP expressing (purple) (N = 6). Background gray dashed lines denote 25 to 75% of the data spread, and the solid line represents the median rate of rundown measured from patches recorded under the control conditions (plotted in Fig. 1C). D, representative plot of normalized currents versus time following VSP activation. TMEM16A, TransMEMbrane 16A; Xl-VSP, Xenopus laevis voltage-sensing phosphatase.
	Figure 6. Docking suggests key PI(4,5)P2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P2 or IP3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A, position of diC8-PI(4,5)P2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB). B, detailed view of the hypothesized PI(4,5)P2–xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C, superposition of docked IP3 (foreground) on the PI(4,5)P2–xTMEM16A (transparency) interaction. diC8-PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.
	Supplemental Figure 1. Homology model of xTMEM16A. A homology model of X. laevis TMEM16A (xTMEM16A) is highly similar to experimental structures of mouse TMEM16A (mTMEM16A, PDB 5OYB). (A) Structural alignment of xTMEM16A (cyan) and mTMEM16A (magenta). (B) Structural and sequence alignment of the hypothesized PI(4,5)P2 binding site in xTMEM16A and mTMEM16A. Arrows highlight proposed interacting residues. The mTMEM16A (∆EAVK) splice site is indicated by a red bar. (C) Modeling a missing loop region in the TM2-3 linker (mTMEM16A, orange). Alignment of this missing region with the xTMEM16A sequence reveals an insertion in xTMEM16A. Loop residues are indicated by a yellow bar.
	Figure 1. TMEM16A Ca2+-evoked Cl−currents rundown in excised patches and are recovered by a diC8-PI(4,5)P2application.A, example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B, normalized plot of current measured at −60 mV versus time, fit with a single exponential (red line). C, box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D, a soluble synthetic analog of PI(4,5)P2, diC8-PI(4,5)P2, was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E, box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P2 with Ca2+ (N = 8). diC8-PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
	Figure 2. TMEM16A Ca2+-evoked Cl−currents are depleted in whole Xenopus laevis oocytes by dephosphorylation of PI(4,5)P2.A, schematic demonstrating pseudojanin translocation to the plasma membrane. To express pseudojanin at the membrane, the membrane tether Lyn11-mCherry and pseudojanin-CFP RNAs were both injected into X. laevis oocytes. Lyn11-mCherry expresses at the plasma membrane, and pseudojanin expresses in the cytoplasm. Upon rapamycin application, rapamycin binds Lyn11-mCherry and induces the membrane translocation of pseudojanin-CFP. Once at the membrane, pseudojanin-CFP dephosphorylates PI(4,5)P2 at the 4′ and 5′ position. The effects of pseudojanin on whole-cell TMEM16A Ca2+-evoked Cl− currents were measured using the two-electrode voltage-clamp technique. B, box plot distribution of the percentage remaining current observed in uninjected control and pseudojanin-CFP–expressing X. laevis oocytes after incubation in 10 μM rapamycin for 5 min. The percent of remaining currents was significantly different (p = 0.02) as determined by a two-tailed t test. ∗ denotes p < 0.05. C and D, example of whole-cell currents recorded at −80 mV before and after rapamycin application in oocytes expressing pseudojanin-CFP. Current was recorded in control solution (black) and after incubation in rapamycin for 5 min (purple). Red bar represents 250 ms duration of UV light application. CFP, cyan fluorescent protein; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
	Figure 3. Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP3 (diC8-PIP3), PI(3,4)P2 (diC8-PI(3,4)P2), and PI(3,5)P2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A, box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P2 (N = 9), diC8-PIP3 (N = 7), diC8-PI(3,4)P2 (N = 6), or diC8-PI(3,5)P2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP3 (B), diC8-PI(3,4)P2 (C), or diC8-PI(3,5)P2 (D). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P2, dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P2, dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,5)P2, dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.
	Figure 4. Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P (A), 100 μM diC8-PI5P (B), or 100 μM diC8-PI4P (D). C, box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P2, as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.
	Figure 5. Xl-VSP does not significantly change TMEM16A current rundown. Inside–out patch-clamp recordings were conducted on macropatches excised from Xenopus laevis oocytes expressing Xl-VSP. A, schematic depicting a VSP tagged with GFP. B, confocal and bright-field images of a representative X. laevis oocyte expressing GFP-tagged Xl-VSP at the plasma membrane. Bar denotes 200 μm. C, box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials for the Xl-VSP expressing (purple) (N = 6). Background gray dashed lines denote 25 to 75% of the data spread, and the solid line represents the median rate of rundown measured from patches recorded under the control conditions (plotted in Fig. 1C). D, representative plot of normalized currents versus time following VSP activation. TMEM16A, TransMEMbrane 16A; Xl-VSP, Xenopus laevis voltage-sensing phosphatase.
	Figure 6. Docking suggests key PI(4,5)P2phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P2 or IP3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A, position of diC8-PI(4,5)P2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB). B, detailed view of the hypothesized PI(4,5)P2–xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C, superposition of docked IP3 (foreground) on the PI(4,5)P2–xTMEM16A (transparency) interaction. diC8-PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.

Askew Page, TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension. 2019, Pubmed

Askew Page, TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension. 2019, Pubmed
Bairoch, The Universal Protein Resource (UniProt). 2005, Pubmed
Benedetto, TMEM16A is indispensable for basal mucus secretion in airways and intestine. 2019, Pubmed
Berman, The Protein Data Bank. 2000, Pubmed
Bradley, Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells. 1999, Pubmed , Xenbase
Britschgi, Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling. 2013, Pubmed
Brunner, X-ray structure of a calcium-activated TMEM16 lipid scramblase. 2014, Pubmed
Caputo, TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. 2008, Pubmed
Crottès, TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis. 2019, Pubmed
Dang, Cryo-EM structures of the TMEM16A calcium-activated chloride channel. 2017, Pubmed
De Jesús-Pérez, Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1). 2018, Pubmed
Durrant, BlendMol: advanced macromolecular visualization in Blender. 2019, Pubmed
Ferrera, Regulation of TMEM16A chloride channel properties by alternative splicing. 2009, Pubmed
Forrest, Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. 2012, Pubmed
Friesner, Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. 2004, Pubmed
Friesner, Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. 2006, Pubmed
Hahn, Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research. 2018, Pubmed
Halgren, Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. 2004, Pubmed
Hammond, PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity. 2012, Pubmed
Handklo-Jamal, Andersen-Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1. 2020, Pubmed , Xenbase
Hossain, Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases. 2008, Pubmed , Xenbase
Huang, International Union of Basic and Clinical Pharmacology. LXXXV: calcium-activated chloride channels. 2012, Pubmed , Xenbase
Huang, Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction. 2012, Pubmed
Humphrey, VMD: visual molecular dynamics. 1996, Pubmed
Hwang, Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles. 2009, Pubmed
Inoue, An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. 2005, Pubmed
Jeng, Independent activation of distinct pores in dimeric TMEM16A channels. 2016, Pubmed
Jin, Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor. 2013, Pubmed
Jumper, Highly accurate protein structure prediction with AlphaFold. 2021, Pubmed
Ko, Allosteric modulation of alternatively spliced Ca2+-activated Cl- channels TMEM16A by PI(4,5)P2 and CaMKII. 2020, Pubmed
Ko, Differential Regulation of Ca2+-Activated Cl- Channel TMEM16A Splice Variants by Membrane PI(4,5)P2. 2021, Pubmed
Lam, Gating the pore of the calcium-activated chloride channel TMEM16A. 2021, Pubmed
Le, Molecular basis of PIP2-dependent regulation of the Ca2+-activated chloride channel TMEM16A. 2019, Pubmed
Lim, Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A. 2016, Pubmed
Liu, The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl- channels. 2010, Pubmed
Lomize, OPM database and PPM web server: resources for positioning of proteins in membranes. 2012, Pubmed
O'Driscoll, Increased complexity of Tmem16a/Anoctamin 1 transcript alternative splicing. 2011, Pubmed
Paulino, Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM. 2017, Pubmed
Pedemonte, Structure and function of TMEM16 proteins (anoctamins). 2014, Pubmed
Peters, The Sixth Transmembrane Segment Is a Major Gating Component of the TMEM16A Calcium-Activated Chloride Channel. 2018, Pubmed
Ratzan, Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence. 2011, Pubmed , Xenbase
Repasky, Flexible ligand docking with Glide. 2007, Pubmed
Schroeder, Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. 2008, Pubmed , Xenbase
Suh, PIP2 is a necessary cofactor for ion channel function: how and why? 2008, Pubmed
Ta, Contrasting effects of phosphatidylinositol 4,5-bisphosphate on cloned TMEM16A and TMEM16B channels. 2017, Pubmed
Tembo, Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ are both required to open the Cl- channel TMEM16A. 2019, Pubmed , Xenbase
Tien, Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC). 2013, Pubmed
Wang, PubChem BioAssay: 2014 update. 2014, Pubmed
Wang, PubChem: a public information system for analyzing bioactivities of small molecules. 2009, Pubmed
White, A Molecular Toolbox for Rapid Generation of Viral Vectors to Up- or Down-Regulate Neuronal Gene Expression in vivo. 2011, Pubmed
Wozniak, PLC and IP3-evoked Ca2+ release initiate the fast block to polyspermy in Xenopus laevis eggs. 2018, Pubmed , Xenbase
Wozniak, The TMEM16A channel mediates the fast polyspermy block in Xenopus laevis. 2018, Pubmed , Xenbase
Yang, TMEM16A confers receptor-activated calcium-dependent chloride conductance. 2008, Pubmed , Xenbase
Yu, Spontaneous neural activity is required for the establishment and maintenance of the olfactory sensory map. 2004, Pubmed
Yu, A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl- channel ANO1 (TMEM16A). 2019, Pubmed
Zhang, Patch-Clamp and Perfusion Techniques to Study Ion Channels Expressed in Xenopus Oocytes. 2018, Pubmed , Xenbase