Phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ are both required to open the Cl^- channel TMEM16A

Transmembrane member 16A (TMEM16A) is a widely expressed Ca²⁺-activated Cl^- channel with various physiological functions ranging from mucosal secretion to regulating smooth muscle contraction. Understanding how TMEM16A controls these physiological processes and how its dysregulation may cause disease requires a detailed understanding of how cellular processes and second messengers alter TMEM16A channel gating. Here we assessed the regulation of TMEM16A gating by recording Ca²⁺-evoked Cl^- currents conducted by endogenous TMEM16A channels expressed in Xenopus laevis oocytes, using the inside-out configuration of the patch clamp technique. During continuous application of Ca²⁺, we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP₂). Thus, we sought to investigate a possible role of PIP₂ in TMEM16A gating. Consistently, synthetic PIP₂ rescued the current after rundown, and the application of PIP₂ modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP₂ sequestering agents, neomycin and anti-PIP₂, applied to the intracellular surface of excised patches sped up TMEM16A current rundown to nearly twice as fast. Conversely, rephosphorylation of phosphatidylinositol (PI) derivatives into PIP₂ using Mg-ATP or inhibiting dephosphorylation of PIP₂ using β-glycerophosphate slowed rundown by nearly 3-fold. Our results reveal that TMEM16A regulation is more complicated than it initially appeared; not only is Ca²⁺ necessary to signal TMEM16a opening, but PIP₂ is also required. These findings improve our understanding of how the dysregulation of these pathways may lead to disease and suggest that targeting these pathways could have utility for potential therapies.