The Maguk Scaffolding Protein CASK Regulates TMEM16A Channel Function by Phosphorylation

Abstract

Ca-activated Cl channels (CaCCs) encoded by TMEM16A (Ano1) play essential roles in many physiological processes including epithelial fluid secretion, gut motility, and smooth muscle tone. Using a quantitative SILAC proteomics approach, we discovered that CASK, a unique MAGUK protein containing an N-terminal CaM-kinase homology domain, is very highly enriched in the TMEM16A interactome. CASK contains multiple protein-protein interaction domains linking receptors and signaling molecules at membrane-cytoskeletal interfaces and has unique protein kinase activity that is inhibited by Mg2+. Here we show that CASK co-immunoprecipitates with TMEM16A and regulates the voltage-dependence of TMEM16A in Mg2+-sensitive manner. In the presence of 1 μM free intracellular Ca2+, decreasing intracellular Mg2+ from 2 mM to 0 mM shifts the conductance-voltage (G-V) relationship 98.4 mV in a hyperpolarizing direction without changing GMAX or Ca2+ sensitivity. The Mg2+-dependent shift in the G-V curve is dependent on CASK protein kinase activity because shRNA-knockdown of CASK or replacement of ATP with AMP-PNP eliminates the shift. Mutation of S639 in TMEM16A identifies this amino acid is the site phosphorylated by CASK. The G-V curve of the S639A mutant is unaffected by Mg2+ or ATP and has a V0.5 value similar to WT in the presence of 2 mM Mg2+ [WT(+Mg2+) 98.6 mV; S639A(0Mg2+) 78.8 mV]. The G-V curve of the phosphomimetic S639E mutant is unaffected by Mg2+ or ATP, but the V0.5 value is similar to WT in the absence of Mg2+ [WT(0 Mg2+) −3.5 mV; S639A(2 Mg2+) 7.7 mV]. Inhibitory autocamtide-2 (AIP-2), a CaMKII inhibitory substrate that also interferes with the catalytic activity of CASK in the internal solution efficiently abolishes the effect of Mg2+ on TMEM16A currents. These data suggest that CASK may regulate TMEM16A channels, although the physiological significance in cells remains to be determined.