Serine 550 is Involved in the Regulation of Mouse TMEM16A-Caccs by CaMKII

Abstract

Ca2+-activated Cl- channels (CaCCs) recorded from vascular smooth muscle cells (VSMCs) are regulated by phosphorylation involving CaMKII, Calcineurin and PP1 (Ayon et al., J Biol Chem 2009;284:32507-32521). TMEM16A, a recently identified anion channel gene, is believed to encode for CaCCs in VSMCs (Davis et al. Am J Physiol Cell Physiol 2010;299:C948-C959; Manoury et al. J Physiol 2010;588:2305-2314) and to contribute to vascular tone (Davis et al. Br J Pharmacol 2013;168:773-784). This study aimed to determine whether Ca2+-activated Cl- currents (IClCa) generated by transient expression of mouse TMEM16A in HEK-293 cells are regulated by kinase and phosphatase activity. Immunocytochemical experiments confirmed the presence of PP1α, PP1β/δ, PP1γ, PP2A and CaMKII in HEK-293 cells, and mRNA transcripts for all four known mammalian CaMKII isoforms (CaMKIIα, β, γ and δ) were identified by RT-PCR in these cells. Similar to VSMCs, TMEM16A-induced IClCa displayed significant rundown in cells dialyzed with 5mM ATP; this effect was potently inhibited by removing ATP. The attenuation of TMEM16A-IClCa rundown in the absence of ATP was suppressed by intracellular application of the PP1/PP2A inhibitor okadaic acid (30 nM). Intracellular application of KN-93 (10 µM) or autocamtide-2-related inhibitory peptide (ARIP; 5 µM), two specific CaMKII inhibitors led to a significant attenuation of TMEM16A-IClCa rundown in the presence of ATP. While mutating a threonine 623 of TMEM16A, a putative CaMKII phosphorylation site, to an alanine did not affect rundown, a serine to alanine mutation at position 550 attenuated ICl(Ca) rundown to an extent similar to that produced by KN-93. These data suggest that TMEM16A-IClCa is regulated by CaMKII and PP1 in a manner similar to CaCCs in VSMCs. Our data also suggest that serine 550 is an important contributor to the regulation of IClCaby CaMKII.