PIP₂ and Ca²⁺ regulation of TMEM16A currents in excised inside-out patches

Abstract

The Ca²⁺ activated Cl⁻ channel formed by transmembrane member 16A (TMEM16A) is broadly expressed and regulates diverse processes. In addition to Ca²⁺, TMEM16A channels require the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to open. Like other channels regulated by PI(4,5)P₂, TMEM16A-conducted currents recorded in excised patches slowly decay overtime. Here we assessed how intracellular Ca²⁺ alters the rate of this current rundown, using the channels endogenously expressed in oocytes from the African clawed frog, Xenopus laevis. We found that in excised, inside-out patches, the concentration of applied Ca²⁺ alters the rate of rundown, with high Ca²⁺ concentrations speeding rundown by activating membrane associated phospholipase C (PLC). Together, these results clarify our understanding of how Ca²⁺ regulates both TMEM16A directly, and targets PLC to regulate the membrane PI(4,5)P₂ content.