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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 (I_ClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. I_ClCa 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, I_ClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. I_ClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. I_ClCa 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 I_ClCa by CaMKII.

SUBMITTER: Ayon RJ

PROVIDER: S-EPMC6957382 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Ayon Ramon J RJ Hawn Matthew B MB Aoun Joydeep J Wiwchar Michael M Forrest Abigail S AS Cunningham Fiona F Singer Cherie A CA Valencik Maria L ML Greenwood Iain A IA Leblanc Normand N

American journal of physiology. Cell physiology 20190828 6

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 ...[more]

PMID: 31461344

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

Publications

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

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