Interplay of Ca²⁺ and PIP₂ in TMEM16A channel function

The transmembrane member 16A (TMEM16A) forms a Ca²⁺-activated Cl^- channel with widespread expression and diverse physiological roles. Activating these channels requires both Ca²⁺ and the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂). Like other PIP₂-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 Ca²⁺ concentrations, providing a tractable system to study PIP₂ regulation. Here, we investigated how intracellular Ca²⁺ 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 Ca²⁺ concentration, with elevated Ca²⁺ 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 PIP₂ resynthesis and enzymatic degradation. This study delineates the complex role of Ca²⁺ in TMEM16A regulation, proposing it as a dual modulator that gates the channel directly and promotes indirect inhibition through PIP₂ dephosphorylation and hydrolysis by patch-associated enzymes. KEY POINTS: TMEM16A currents undergo Ca²⁺-accelerated rundown in excised inside-out patches due to PIP₂ depletion. Rundown rate increases with intracellular Ca²⁺ concentration, with half-maximal acceleration at ∼150 µM Ca²⁺. Inhibiting phosphatases or refuelling kinases with Mg-ATP slows rundown but does not fully prevent it. Ca²⁺-activated phospholipase C (PLC) activity contributes to PIP₂ degradation; PLC inhibition attenuates rundown. Co-application of diC8-PIP₂ and Ca²⁺ prevents rundown, highlighting the dual role of Ca²⁺ in channel gating and PIP₂ depletion.