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siRNA-mediated knockdown of endogenously expressed bestrophin in smooth muscles.

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  • Danish Biomembrane Research Centre
  • Department of Molecular Biology
 We have recently characterized in smooth muscle cells a unique cGMP-dependent Ca2+-activated Cl- current (ICl(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 ICl(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 ICl(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 ICl(cGMP-Ca) while the "classical" Ca2+-activated Cl- current was not affected.

Our studies provide evidence that bestrophin-4 is responsible for the ICl(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.

Original languageEnglish
Publication year2007
Number of pages1
Publication statusPublished - 2007
EventExperimental Biology 2007 - , United States
Duration: 27 Apr 20072 May 2007


ConferenceExperimental Biology 2007
CountryUnited States

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