Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A

Abstract

This study explored the mechanism by which Ca2+-activated Cl− channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl− currents ( IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

Calcium-Activated Chloride Channel ANO1/TMEM16A: Regulation of Expression and Signaling

“…Phospho-serine binding protein 14-3-3 directly interacts with ANO1 and also stabilizes ANO1 on cell surface by unknown mechanism, both in heterologous models and in glioblastoma U251 cells (Lee et al, 2016b). Cell surface expression of ANO1 is also promoted by Ca 2+/ Calmodulin-dependent protein kinase II (CaMKII) in heterologous models and in glioblastoma U251 cells (Sim et al, 2020), potentially (but not proven) through phosphorylation of ANO1 by CAMKII (Ayon et al, 2019). In contrast, β-COP, a subunit of Coat Protein Complex I (COPI), directly interacts with ANO1 and reduces its surface expression by promoting the retrograde transport, which was also shown at the endogenous levels of ANO1 and β-COP in U251 cells (Lee et al, 2016a).…”

Molecular mechanisms of activation and regulation of ANO1-Encoded Ca2+-Activated Cl- channels

“…Ayon et al 177 to support channel activity. 161 The rundown of ANO1 was partially attenuated by inhibiting CaMKII with KN-93 or the peptide inhibitor ARIP.…”

“…4B). 54-56, 59, 84 Additionally, Ayon et al 177 carried out sitedirected mutagenesis to identify one or several potential sites for CaMKII phosphorylation. Of four sites bearing the consensus CaMKII sequence RxxS/T, only the S528A (identical to S525 in Lin et al 176 and corresponding to S471 in mouse ANO1ac; see Fig.…”

“…4B). 177 The reasons for such differences are unclear. However, they are likely due to the combination of many factors, including the exact composition and level of expression of CaMKII, Calcineurin and PP1/PP2A, the molecular architecture of the microdomain comprising ANO1, the splice variants of ANO1 that are expressed, whether KCNE1 or 5 is co-expressed with ANO1, and many other possibilities.…”

Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells

“…While direct a CAM-regulation of TMEM16A is discussed controversially, several studies demonstrate that CAM-dependent kinase II (CAMKII) regulates CaCC/TMEM16A. However, CAMKII was found to activate [ 8 , 9 , 10 , 11 , 12 ] and to inhibit [ 13 , 14 , 15 , 16 , 17 ] CaCC and TMEM16A, respectively. Greenwood and Leblanc found a cell-type-dependent activation/inhibition of CaCC by CAMKII [ 18 ], while Ko et al found differential regulation of TMEM16A by CAMKII, depending on the splice variant [ 19 , 20 ].…”