x laevis oocytes  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a diC8-PI(4,5)P 2 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)P 2 , diC8-PI(4,5)P 2 , 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)P 2 with Ca 2+ (N = 8). diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.

Article Snippet: Here, we expressed Kir2.1 in X. laevis oocytes (Addgene plasmids 32669 and 32641, ( , )) and used TEVC to record whole-cell currents while ramping the voltage from −150 to +50 mV, over 2 s. Recording in a potassium-based solution comprised of (in millimolar): 90 KCl, 5 Hepes, 0.1 CaCl 2 , 1 MgCl 2 , pH 7.6 ( ).

Techniques: Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: TMEM16A Ca 2+ -evoked Cl − currents are depleted in whole Xenopus laevis oocytes by dephosphorylation of PI(4,5)P 2 . A , schematic demonstrating pseudojanin translocation to the plasma membrane. To express pseudojanin at the membrane, the membrane tether Lyn 11 -mCherry and pseudojanin-CFP RNAs were both injected into X. laevis oocytes. Lyn 11 -mCherry expresses at the plasma membrane, and pseudojanin expresses in the cytoplasm. Upon rapamycin application, rapamycin binds Lyn 11 -mCherry and induces the membrane translocation of pseudojanin-CFP. Once at the membrane, pseudojanin-CFP dephosphorylates PI(4,5)P 2 at the 4′ and 5′ position. The effects of pseudojanin on whole-cell TMEM16A Ca 2+ -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)P 2 , phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.

Article Snippet: Here, we expressed Kir2.1 in X. laevis oocytes (Addgene plasmids 32669 and 32641, ( , )) and used TEVC to record whole-cell currents while ramping the voltage from −150 to +50 mV, over 2 s. Recording in a potassium-based solution comprised of (in millimolar): 90 KCl, 5 Hepes, 0.1 CaCl 2 , 1 MgCl 2 , pH 7.6 ( ).

Techniques: De-Phosphorylation Assay, Translocation Assay, Clinical Proteomics, Membrane, Injection, Control, Expressing, Incubation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: 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. 1 C ). D , representative plot of normalized currents versus time following VSP activation. TMEM16A, TransMEMbrane 16A; Xl-VSP, Xenopus laevis voltage-sensing phosphatase.

Article Snippet: Here, we expressed Kir2.1 in X. laevis oocytes (Addgene plasmids 32669 and 32641, ( , )) and used TEVC to record whole-cell currents while ramping the voltage from −150 to +50 mV, over 2 s. Recording in a potassium-based solution comprised of (in millimolar): 90 KCl, 5 Hepes, 0.1 CaCl 2 , 1 MgCl 2 , pH 7.6 ( ).

Techniques: Patch Clamp, Expressing, Clinical Proteomics, Membrane, Control, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: Docking suggests key PI(4,5)P 2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P 2 or IP 3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A , position of diC8-PI(4,5)P 2 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)P 2 –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 IP 3 (foreground) on the PI(4,5)P 2 –xTMEM16A (transparency) interaction. diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P 2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.

Article Snippet: Here, we expressed Kir2.1 in X. laevis oocytes (Addgene plasmids 32669 and 32641, ( , )) and used TEVC to record whole-cell currents while ramping the voltage from −150 to +50 mV, over 2 s. Recording in a potassium-based solution comprised of (in millimolar): 90 KCl, 5 Hepes, 0.1 CaCl 2 , 1 MgCl 2 , pH 7.6 ( ).

Techniques: Clinical Proteomics, Membrane