We have recently characterized in smooth muscle cells a unique cGMP-dependent Ca²⁺-activated Cl^- current (I_Cl(cGMP-Ca)) that co-exists with a "classical" Ca²⁺-activated Cl^- current. We hypothesized that bestrophin-4 (a product of the VMD2-like 3 gene) could be responsible for the I_Cl(cGMP-Ca) based on similarities between membrane current produced by bestrophin heterologous expression and this endogenous current. This was supported by a similar distribution pattern of the I_Cl(cGMP-Ca) and the bestrophin expression.

To test this hypothesis siRNA-mediated downregulation of gene expression was used. Cultured aortic smooth muscle cells (A7r5) were transfected with siRNA directed against bestrophin-4 and cultured for 3 days. The efficiency of transfection was demonstrated by specific perinuclear fluorescence of Cy3-labelled siRNA. The downregulation of targeted protein expression was controlled by qPCR and Western blot. The downregulation of bestrophin-4 expression (by 88% in mRNA) with siRNA was a associated with significant reduction (by 83%) of the I_Cl(cGMP-Ca) while the "classical" Ca²⁺-activated Cl^- current was not affected.

Our studies provide evidence that bestrophin-4 is responsible for the I_Cl(cGMP-Ca) in smooth muscle cells. This study presents a novel efficient technique for specific downregulation of gene expression in blood vessels, much needed in studies of vascular function.