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

• Published in: American Journal of Physiology: Cell Physiology Vol. 317; no. 6; p. C1093
• Main Authors: Ayon, Ramon J, Hawn, Matthew B, Aoun, Joydeep, Wiwchar, Michael, Forrest, Abigail S, Cunningham, Fiona, Singer, Cherie A, Valencik, Maria L, Greenwood, Iain A, Leblanc, Normand
• Format: Journal Article
• Language: English
• Published: United States 01-12-2019
• Subjects: Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Amino Acid Sequence; Animals; Anoctamin-1 - genetics; Anoctamin-1 - metabolism; Benzylamines - pharmacology; Calcium - metabolism; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - antagonists & inhibitors; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - genetics; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - metabolism; Cantharidin - pharmacology; Chlorides - metabolism; Evoked Potentials - drug effects; Evoked Potentials - physiology; Gene Expression Regulation; HEK293 Cells; Humans; Ion Transport - drug effects; Mice; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Okadaic Acid - pharmacology; Patch-Clamp Techniques; Peptides - pharmacology; Phosphorylation - drug effects; Protein Phosphatase 1 - antagonists & inhibitors; Protein Phosphatase 1 - genetics; Protein Phosphatase 1 - metabolism; Protein Phosphatase 2 - antagonists & inhibitors; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Sulfonamides - pharmacology; Ano-1; CaMKII; TMEM16A; regulation; calcium-activated chloride channel
• ISSN: 1522-1563
• DOI: 10.1152/ajpcell.00059.2018

This study explored the mechanism by which Ca
-activated Cl
channels (CaCCs) encoded by the
gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca
-activated Cl
currents (
) were recorded from HEK-293 cells expressing mouse TMEM16A.
were evoked using a pipette solution in which free Ca
concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP,
decayed to <50% of the initial current magnitude within 10 min after seal rupture.
rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP.
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
by CaMKII.