Tmem16a‐induced ClCa Currents Are Regulated Via Phosphotransferase Activity

Ca2+‐activated Cl− currents (ICl(Ca)) represent a major excitatory mechanism in vascular smooth muscle cells (SMCs). In arterial SMCs, ICl(Ca) are down‐regulated by calmodulin‐dependent protein kinase II (CaMKII) and up‐regulated by calcineurin (CaN) and PP1. Recently, Tmem16a has emerged as a gene candidate for the native ICl(Ca) in pulmonary artery SMCs. HEK‐293 cells over‐expressing Tmem16a displayed time‐ and voltage‐dependent Cl− currents when dialyzed with 500 nM free Ca2+ that resembled the properties of ICl(Ca) from vascular SMCs. Similar to ICl(Ca) from VSMCs, Tmem16a‐induced ICl(Ca) also displayed enhanced rundown when cells were dialyzed with 5 mM ATP and this process was attenuated in the absence of ATP. Similar to ICl(Ca) in rabbit VSMCs (Ayon et al., J Biol Chem 284:32507–32521, 2009), dialysis of 30 nM okadaic acid, a nonselective PP1/PP2A inhibitor, impeded the recovery of Tmem16a‐dependent Cl− currents in the absence of ATP. Analysis of mouse Tmem16a protein sequence revealed 5 consensus sites for phosphorylation by CaMKII, which are believed to lie on protein segments facing the cytoplasm. A single point mutation of one of these putative CaMKII phosphorylation sites (Threonine 610 to Alanine) did not alter rundown of the channels. Taken together, these data suggest that much like native ICl(Ca), Tmem16a‐dependent Cl− currents also appear to be regulated by at least one phosphorylation step.