Interplay of Ca ²⁺ and PIP ₂ in TMEM16A channel function

The transmembrane member 16A (TMEM16A) forms a Ca 2+ ‐activated Cl − channel with widespread expression and diverse physiological roles. Activating these channels requires both Ca 2+ and the acidic phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP 2 ). Like other PIP 2 ‐dependent channels, TMEM16A‐mediated currents in excised patches display a characteristic time‐dependent decline, known as rundown. We have reported that TMEM16A currents exhibit rapid rundown at high intracellular Ca 2+ concentrations, providing a tractable system to study PIP 2 regulation. Here, we investigated how intracellular Ca 2+ affects rundown kinetics of TMEM16A currents by performing inside–out patch‐clamp recordings from Xenopus laevis oocytes, where these channels are naturally abundant. We found that the rate of rundown is modulated by intracellular Ca 2+ concentration, with elevated Ca 2+ accelerating rundown in part by activating membrane‐associated phospholipase C (PLC) and phosphatases. Refuelling kinases with Mg‐ATP slowed rundown, and combining ATP with a PLC inhibitor further amplified this effect, highlighting the interplay between PIP 2 resynthesis and enzymatic degradation. This study delineates the complex role of Ca 2+ in TMEM16A regulation, proposing it as a dual modulator that gates the channel directly and promotes indirect inhibition through PIP 2 dephosphorylation and hydrolysis by patch‐associated enzymes. image Key points TMEM16A currents undergo Ca 2+ ‐accelerated rundown in excised inside–out patches due to PIP 2 depletion. Rundown rate increases with intracellular Ca 2+ concentration, with half‐maximal acceleration at ∼150 µM Ca 2+ . Inhibiting phosphatases or refuelling kinases with Mg‐ATP slows rundown but does not fully prevent it. Ca 2+ ‐activated phospholipase C (PLC) activity contributes to PIP 2 degradation; PLC inhibition attenuates rundown. Co‐application of diC8‐PIP 2 and Ca 2+ prevents rundown, highlighting the dual role of Ca 2+ in channel gating and PIP 2 depletion.