Opening of Ca²⁺-activated Cl^– channels encoded by the Tmem16a or Anoctamin-1 (ANO1) gene is thought to play a key role in the depolarization, Ca²⁺ signaling and contraction of vascular smooth muscle cells (VSMC) triggered by vasoconstricting agonists linked to G_q-protein-coupled receptors.¹ Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in VSMC display rundown after seal rupture in whole-cell patch clamp experiments, with a significant portion of this rundown involving at least one phosphorylation step mediated by CaMKII.² Interestingly, the rundown of I_Cl(Ca) recorded under similar conditions in pulmonary arterial smooth muscle cells (PASMC) from different species is variable, with I_Cl(Ca) in rabbit and rat PASMC running down by ~ 80%³ and 20%⁴ over 5-10 min, respectively. Alternative splicing is known to regulate the biophysical properties of I_Cl(Ca) resulting from ANO1 expression.²  I_Cl(Ca) resulting from the expression of the a variant of mouse ANO1 in HEK-293 cells displayed similar rundown that was partially attenuated by blocking CaMKII with KN-93 or ARIP, or by mutating Serine 528 to Alanine (S528A), suggesting that the rundown of I_Cl(Ca) is in part due to direct CaMKII-induced phosphorylation of ANO1 protein.⁵ In this study, we found that the rundown of ANO1 was greatly attenuated when splice variant d (ANO1-ad), a 26 amino acid segment located in the first intracellular loop of ANO1, was 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 very 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 the dvariant could limit access of CaMKII to phosphorylate Serine 528, or alternatively eliminate the impact of the phosphorylation on channel activity. Consistent with this hypothesis, blocking CaMKII with ARIP (5 mM) had no effect on the rundown of ANO1-ad (ANO1-ad + ARIP: 17 ± 10%, n=6; P > 0.05). We next modeled the impact of incorporating splice variant d on stable interaction energies in the vicinity of S528 using the AlphaFold Protein Structure Database. Adding splice variant d increased the ΔG of S528 from -27.98 to -55.11 kj/mol. Incorporating splice variant d to phosphoserine 528 led to a similar but more profound stabilization of local interactions with the ΔG of this residue increasing from -55.85 to -113.23 kj/mol. These simulations indicated that the stabilization of energies by splice variant d was likely the product of the residue being locked in a more rigid alpha helical structure, which may hamper the ability of CaMKII to phosphorylate this site, or prevent phosphorylation from exerting its down-regulating effect on channel activity.  These results show that splice variant d has a profound influence on the regulation of ANO1 by CaMKII, an observation that may partially explain why ANO1-induced I_Cl(Ca) recorded in different cell types or in the same cell type in different species, exhibit variable stability in patch clamp recordings.