Silencing the cardiac potassium channel Kv4.3 by RNA interference in a CHO expression system.
RNA interference (RNAi) is a powerful technique for gene silencing, in which the downregulation of mRNA is triggered by short RNAs complementary to a target mRNA sequence, with consequent reduction of the encoded protein. The aim of this study was to test the effects of silencing the expression of the cardiac potassium channel Kv4.3 in a heterologous expression system, in order to investigate the effect of RNAi on channel properties. A Chinese hamster ovary cell line stably expressing Kv4.3 and the accessory beta-subunit KChIP2 was transfected with small-interfering RNAs (siRNAs) targeting Kv4.3. Effects of RNAi were monitored at the mRNA, protein, and functional levels. Real-time PCR and immunofluorescence staining revealed significant reduction of Kv4.3 mRNA and protein expression. These results were confirmed by functional patch-clamp measurements of the transient outward current (I(to)) which was reduced up to 80% by RNAi. We conclude that the use of siRNAs reagents for post-transcriptional gene silencing is a new effective method for the reduction of the expression and function of different ionic channels which may be adapted for studying their role also in native cells.
Cotella
Diego
D
Department of Pharmacology and Toxicology, Carl Gustav Carus Medical Faculty, University of Technology, Dresden, Germany.
Jost
Norbert
N
Darna
Mahesh
M
Radicke
Susanne
S
Ravens
Ursula
U
Wettwer
Erich
E
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D020868:Gene Silencing; D006801:Humans; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051672:Shal Potassium Channels
D017931:DNA Primers; C497021:KCND3 protein, human; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051672:Shal Potassium Channels