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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.
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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.
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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.
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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.
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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.
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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.
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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.
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