Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and Ca 2+ are both required to open the Cl - channel TMEM16A.

Transmembrane member 16A (TMEM16A) is a widely expressed Ca ²⁺ -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 Ca ²⁺ -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 Ca ²⁺ , we found that TMEM16A-conducted currents decay shortly after patch excision. Such current rundown is common among channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP ₂ ). Thus, we sought to investigate a possible role of PIP ₂ in TMEM16A gating. Consistently, synthetic PIP ₂ rescued the current after rundown, and the application of PIP ₂ modulating agents altered the speed kinetics of TMEM16A current rundown. First, two PIP ₂ sequestering agents, neomycin and anti-PIP ₂ , 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 PIP ₂ using Mg-ATP or inhibiting dephosphorylation of PIP ₂ 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 Ca ²⁺ necessary to signal TMEM16a opening, but PIP ₂ 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.
(© 2019 Tembo et al.)