Inactivation of Anoctamin-1 by CaMKII phosphorylation is hampered by the presence of alternatively spliced variant d.

Abstract

Ca2+-activated Cl- currents (ICl(Ca)) of vascular smooth muscle cells (VSMC) display rundown after seal rupture in patch clamp experiments (Hawn et al. Channels. 15:569-603, 2021). A portion of this rundown is attributed to involve at least one phosphorylation step mediated by CaMKII. Alternative splicing is known to regulate the properties of Ca2+-activated Cl- channels encoded by the Ano1 gene. The a variant of mouse Anoctamin-1 (ANO1) expressed in HEK-293 cells displayed a similar pattern to ICl(Ca) in VSMC with prominent rundown that was attenuated by blocking CaMKII with KN-93 or ARIP, or by mutating Serine 528 to Alanine (S528A), suggesting that the rundown of ICl(Ca) is in part due to direct CaMKII-induced phosphorylation of ANO1 protein. In this study, we found that the rundown of ICl(Ca) was attenuated when splice variant d (ANO1-ad, located in the first intracellular loop) of ANO1 was concomitantly expressed with splice variant a in HEK-293 cells (ANO1-a: 60 ± 5%, n=10; ANO1-ad: 16 ± 10%, n=7; P < 0.05). Since splice variant d is near S528, and the rundown of ANO1-ad was similar to that of ANO1-a in the presence of CaMKII inhibitors, or the ANO1-a S528A mutant, we hypothesized that the presence of variant d may limit access of CaMKII to phosphorylate Serine 528. Consistent with this hypothesis was the observation that blocking CaMKII with ARIP (5 μM) or KN-93 (10 μM) had no effect on the rundown of ANO1-ad. These results indicate that splice variant d has a considerable influence on the regulation of ANO1 by CaMKII, which may partially explain why ANO1-induced ICl(Ca) recorded in the same cell type in different species exhibit variable stability in patch clamp recordings.