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

PIP2 and Ca2+ regulation of TMEM16A currents in excised inside-out patches

“…Briefly, currents were recorded during 150 ms steps to -60 and +60 mV before and during application of 2 mM Ca 2+ . As we previously reported, application of 2 mM Ca 2+ evoked robust TMEM16A-conducted currents immediately following patch excision (Tembo et al, 2022; Tembo, Wozniak, Bainbridge & Carlson, 2019). Figure 1A depicts currents recorded during steps to -60 and +60 mV at indicated time points (10-180 s) following 2 mM Ca 2+ addition.…”

“…Here we demonstrate that in addition to activating TMEM16A channels, intracellular Ca 2+ speeds current rundown by turning on membrane tethered PLCs in excised inside out patches. TMEM16A channels require both intracellular Ca 2+ and PIP2 to open and conduct Cl − currents (De Jesus-Perez et al, 2017; Le, Jia, Chen & Yang, 2019; Ta, Acheson, Rorsman, Jongkind & Tammaro, 2017; Tembo et al, 2022; Tembo, Wozniak, Bainbridge & Carlson, 2019; Yu, Jiang, Cui, Tajkhorshid & Hartzell, 2019). Like other channels regulated by PIP 2 , TMEM16A-conducted currents decay when recorded in the inside-out configuration of the patch clamp technique (Figure 1), even in the continued presence of Ca 2+ (De Jesus-Perez et al, 2017; Tembo, Wozniak, Bainbridge & Carlson, 2019).…”

“…The full length TMEM16A isoform requires the phosphate at the 4' position of PI(4,5)P2 in order to activate these channels (Ko & Suh, 2021;Tembo et al, 2022). Whereas the fulllength channel is widely expressed in mammals, a TMEM16A splice variant expressed in the brain and skeletal muscle lacks four residues (EAVK) that reside in a linker connecting transmembrane domains 2 and 3; for this variant, the phosphate position does not matter for the phospholipid to potentiate channel activity (Ko & Suh, 2021;Le, Jia, Chen & Yang, 2019).…”

“…In addition to divalent cations, TMEM16A channels also require the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to open (De Jesus-Perez et al, 2017; Le, Jia, Chen & Yang, 2019; Ta, Acheson, Rorsman, Jongkind & Tammaro, 2017; Tembo et al, 2022; Tembo, Wozniak, Bainbridge & Carlson, 2019; Yu, Jiang, Cui, Tajkhorshid & Hartzell, 2019). A hallmark of channels potentiated by PI(4,5)P2 is that their currents rundown when recorded in excised patches (Suh & Hille, 2008).…”

“…A hallmark of channels potentiated by PI(4,5)P2 is that their currents rundown when recorded in excised patches (Suh & Hille, 2008). Indeed, TMEM16A conducted currents decay in excised patches, even in the presence of continuously applied intracellular Ca 2+ (Tembo et al, 2022; Tembo, Wozniak, Bainbridge & Carlson, 2019). How PI(4,5)P2 potentiates TMEM16A conductance is unknown.…”