Molecular Mechanism of TMEM16A Regulation: Role of CaMKII and PP1/PP2A.

This study explored the mechanism by which Ca2+-activated Cl-channels (CaCC) encoded by the Tmem16agene are regulated by CaMKII and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl-currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCawere evoked using a pipette solution in which free Ca2+concentration ([Ca2+]i) was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCadecayed to below 50% of the initial current magnitude within 10 min after seal rupture. IClCarundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCarecorded after 20 min of cell dialysis with 0 ATP were more than 2-fold 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 non-selective 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 TMEM16Arundown 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 ...

Source: Am J Physiol Cell Ph... - August 27, 2019 Category: Cytology Authors: Ayon RJ, Hawn MB, Aoun J, Wiwchar M, Forrest AS, Cunningham F, Singer CA, Valencik ML, Greenwood IA, Leblanc N Tags: Am J Physiol Cell Physiol Source Type: research