Abstract
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. 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.