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Biochemical and Biophysical Research Communications 330 (2005) 555–560

BBRC

Silencing the cardiac potassium channel Kv4.3 by RNA interferencein a CHO expression system

Diego Cotella a,1, Norbert Jost b,1, Mahesh Darna a, Susanne Radicke a,Ursula Ravens a, Erich Wettwer a,*

a Department of Pharmacology and Toxicology, Carl Gustav Carus Medical Faculty, University of Technology, Dresden, Germanyb Department of Pharmacology and Pharmacotherapy, Medical University, Szeged, Hungary

Received 25 February 2005Available online 18 March 2005

Abstract

RNA interference (RNAi) is a powerful technique for gene silencing, in which the downregulation of mRNA is triggered by shortRNAs complementary to a target mRNA sequence, with consequent reduction of the encoded protein. The aim of this study was totest the effects of silencing the expression of the cardiac potassium channel Kv4.3 in a heterologous expression system, in order toinvestigate the effect of RNAi on channel properties. A Chinese hamster ovary cell line stably expressing Kv4.3 and the accessoryb-subunit KChIP2 was transfected with small-interfering RNAs (siRNAs) targeting Kv4.3. Effects of RNAi were monitored at themRNA, protein, and functional levels. Real-time PCR and immunofluorescence staining revealed significant reduction of Kv4.3mRNA and protein expression. These results were confirmed by functional patch-clamp measurements of the transient outwardcurrent (Ito) which was reduced up to 80% by RNAi. We conclude that the use of siRNAs reagents for post-transcriptional genesilencing is a new effective method for the reduction of the expression and function of different ionic channels which may be adaptedfor studying their role also in native cells.� 2005 Elsevier Inc. All rights reserved.

Keywords: Ito; Kv4.3; Ion channels; RNA interference; siRNA; Gene silencing; CHO cell

The transient outward current (Ito) is a potassiumcurrent that plays an essential role in the regulation ofthe cardiac action potential (AP), particularly in initiat-ing the early repolarization phase of the AP. The ionconducting a-subunit responsible for Ito current in hu-man heart is Kv4.3 [1]. There is evidence that severalaccessory b-subunits like KChIPs, MiRPs, KChAPs,etc., can significantly alter the electrophysiological prop-erties of Ito (reviewed in [2]). Several attempts to revealthe role of these subunits have been described. One ap-proach is to express/co-express these genes in heterolo-gous expression systems like Xenopus oocytes [3] or

0006-291X/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2005.03.018

* Corresponding author. Fax: +49 351 458 6315.E-mail address: wettwer@rcs.urz.tu-dresden.de (E. Wettwer).

1 These two authors have contributed equally in writing this paper.

cell lines, e.g., HEK293, CHO, etc. [4,5]. However, thisexperimental model is limited by the number of differentgenes that can be simultaneously co-expressed, and arte-facts may occur due to the presence of endogenous pro-teins [6]. Gene silencing may serve as an alternativemethod for studying the functional role of ion channelsubunits. However, few attempts have been made in thisdirection, one successful method being antisense oligo-deoxynucleotides (AsODN) technique [7]. The recentlydeveloped RNA interference (RNAi) method is a pow-erful tool for post-transcriptional gene silencing(PTGS). With this approach downregulation of mRNAis induced by short double-stranded RNAs (small-inter-fering RNA, siRNA) complementary to the targetmRNA with the subsequent reduction of expression ofthe encoded protein [8]. Here we studied the effectiveness

556 D. Cotella et al. / Biochemical and Biophysical Research Communications 330 (2005) 555–560

of the RNAi technique in silencing Kv4.3 in a CHO cellline stably transfected with Kv4.3 and KChIP2. The aimwas to estimate whether RNAi could be a useful methodfor exploring ion channel subunit function.

Materials and methods

siRNA construction. Enzymes for gene cloning (Fermentas) wereused according to themanufacturer�s instructions or published protocols[9]. Commercially available PCR kits were obtained from Roche. TheSilencer siRNA cocktail kit (RNaseIII) was from Ambion and usedaccording to the manufacturer�s instructions. HumanKv4.3 cDNAwasa gift from Professor D. Snyders (Antwerp, Belgium). The 2.0 kb cDNAfragment was excised from its original vector withXhoI and PstI, blunt-ended with T4 DNA polymerase, and cloned into the EcoRV restrictionsite of pcDNA3.1(+) vector (Invitrogen), in order to obtain the ‘‘sense’’and the ‘‘antisense’’ constructs (with respect to the T7 promoter). Theorientation of the insert was checked by digestion with StuI. cDNAencoding human KCNE2 (used as negative control in RNAi experi-ments)was amplified fromhumanatrium cDNA(previously prepared inour laboratory) with the following primers: KCNE2_ORF_F (5 0-GGAAGCATGTCTACTTTATC-3 0) and KCNE2_ORF_R (5 0-TTATCAGGGGGACATTTT-3 0). PCR conditions were: 5 min at 94 �C; 30 s at94 �C, 30 s at 47 �C, and 40 s at 72 �C (30 cycles); and 7 min at 72 �C.The380 bp fragment was purified from agarose gel, polished with T4DNA polymerase, and cloned into the EcoRV restriction site ofpcDNA3.1(+) vector. Orientation of the cloned fragment was checkedby digestion with XbaI. Two populations of siRNA targeting hKv4.3and hKCNE2 were prepared by the in vitro transcription/RNAseIIIdigestion method, using the Silencer siRNA cocktail kit.

Cell culture and siRNA transfections. A CHO cell line stably trans-fected with Kv4.3 and KChIP2 was a gift from Dr. K. Steinmeyer(Aventis Pharma, Frankfurt, Germany). Cells were maintained at 37 �Cin Basal Iscove�s medium (Biochrom) supplemented with 10% fetal calfserum and kept under selection with 100 lg/ml zeocin and 400 lg/mlG418. Twenty-four hours before transfection, cells were seeded on 24-well plates at a density of 75,000 cells/well (when used for immunoflu-orescence experiments, cells were plated on 10 mm round coverslips).siRNAs were transfected with the TransIT-TKO transfection reagent(Mirus) according to the manufacturer�s instructions, using 2 ll oftransfection reagent per well. siRNAs were cotransfected with 200 ng ofa pEGFP-N1 vector (Clontech) in order to label the transfected cells.Twelve to 24 h after transfection the medium was replaced with freshmedium. Cells were analysed for RNAi effects 48 h after transfection.

Real-time PCR. Total RNA was extracted with the RNApure kit(Peqlab) according to the manufacturer�s protocol. Three hundrednanograms of total RNA was used in each RT-PCR. To make a cali-bration curve, theRNA from the negative control was diluted in twofoldserial dilutions, and the undiluted sample was considered 100%. Real-time PCR was performed using the QuantiTect SYBR Green RT-PCRkit (Qiagen) and a Rotor Gene thermal cycler (Corbett Research). Thefollowing primers were used: hKv4.3_F2 (5 0-TCCACCATCAAGAACCACG-30) and hKv4.3_R (5 0-AGCAGGTGGTAGTGAGGCC-3 0).RT-PCR conditions were: 30 min at 50 �C; 15 min at 95 �C; and 30 s at94 �C, 30 s at 58 �C, and 30 s at 72 �C (40 cycles).

Immunofluorescence. Cells were fixed with methanol/DAPI, washedwith PBS, and incubated for 1 h at room temperature in blockingbuffer (PBS containing 1% BSA) and then for 1 h with primary anti-bodies specific for Kv4.3 (Alomone) and for KChIP2 (a gift from Dr.D. Isbrandt, Hamburg, Germany) (both antibodies were diluted 1:200in PBS). Samples were washed three times with PBS and then incu-bated for 1 h with a Cy3-labelled secondary antibody (Amersham).Samples were washed with PBS as above, mounted using Fluoro-mount-G (SouthernBiotech), and then visualized under an Axiovert135 fluorescence microscope (Zeiss).

Whole-cell patch-clamp recordings. The whole-cell configuration ofthe patch-clamp technique was used to record membrane currents.Forty-eight hours after transfection with hKv4.3 siRNA (50 and100 nM) and with 100 nM hKCNE2 siRNA (negative control), thecells were trypsinized and harvested. Cells were allowed to settle at thebottom of a small perfusion chamber mounted on the stage of an in-verted microscope (Olympus IX50), equipped with an epifluorescenceassembly (Olympus U-RFL-T) to observe the green fluorescence oftransfected cells. Tyrode solution served as normal superfusate (inmM): NaCl 150, KCl 5.4, MgCl2 2, Hepes 10, glucose 10, and CaCl20.5 (pH, adjusted to 7.4 with NaOH). Patch-clamp micropipettes weremade from borosilicate capillaries (Hilgenberg) with a programmablepuller (DMZ) and had 1.5–2.5 MX resistance when filled with thefollowing solution: (in mM): K-aspartate 100, NaCl 8, KCl 40, Mg-ATP 5, EGTA 5, CaCl2 2, GTP–Tris 0.1, and Hepes 10 (pH 7.2, ad-justed with KOH). Current measurements were performed using aHEKA-EPC-8 patch-clamp amplifier (HEKA-Elektronik) under thecontrol of ISO2 software (MFK) at room temperature. Analyses wereperformed using the same software. Currents were normalized to cellcapacitance which was determined with fast voltage ramp steps in a±10 mV range at �50 mV. All data are expressed as means ± SEM.Statistical analysis was performed using one-way ANOVA Bonfer-roni�s multiple comparison test. Results were considered significantwhen p < 0.05 (*) or p < 0.001 (**).

Results

In vitro synthesis of siRNA

To prepare a heterogeneous siRNA population thatcould potentially target multiple sites per mRNA mole-cule for inhibition, we used the Silencer siRNA Cocktailkit (Ambion). The principle is based on the T7 polymer-ase-driven in vitro transcription of two complementaryRNA molecules, annealing into a dsRNA and successivecleavage into siRNA catalysed by a recombinant Esche-richia coli RNaseIII. The expected result is a populationof short dsRNA with a length ranging from 12 to 30 bp[10] with the majority of siRNA in the range 12–15 bp(see manufacturer�s protocol). As shown in Fig. 1A,the in vitro transcription of two templates correspond-ing to the full coding sequences of hKv4.3 and hKCNE2results into two dsRNA molecules of 2500 and 500 bp,respectively, resistant to DNaseI and RNaseA digestion.The successive cleavage by the RNaseIII of both dsR-NAs results in a common pattern of two diffuse bandsabout 12–15 bp long (Fig. 1B). These siRNA moleculesare shorter than the fragments produced by the nativeDicer enzyme of 21–30 bp [10,11] which may explainthe necessity of relatively high concentrations of siRNA(50–100 nM) to get a satisfactory RNAi efficiency, com-pared to those of other published methods for the pro-duction of siRNA [10].

Kv4.3 siRNA reduces Kv4.3 expression at both mRNA

and protein levels

Reduction in Kv4.3 mRNA content in Kv4.3 siRNA-transfected cells was analysed with real-time PCR and

Fig. 1. Preparation of siRNA from dsRNA by hydrolysis with E. coli

RNaseIII. dsRNA for hKv4.3 and hKCNE2 was prepared by the T7-driven in vitro transcription of two DNA templates (A). Afterannealing of the two complementary strands, dsRNA was digestedwith RNaseIII to obtain siRNA 12–15 bp long (B).

Fig. 2. hKv4.3 mRNA is downregulated by siRNA in stably trans-fected Kv4.3/KChIP2 CHO expression system. A calibration curvewas obtained by plotting the fluorescence signal of serial dilutions oftotal RNA from ‘‘scrambled’’ control cells against the PCR cyclenumber (filled circles) and defining the fluorescence of the undilutedsample as 100% (see Materials and methods). A single exponentialcurve with a time constant of 1.13 cycles was fitted to the data (solidcurve). From this theoretical curve the reduction of Kv4.3 mRNA wascalculated with 67% after transfection with 50 and 86% with 100 nMsiRNA (open triangles) in real-time PCR measurements.

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was expressed as percent of Kv4.3 mRNA compared tothat of the negative control cells transfected with theKCNE2 siRNA. KCNE2 was used as negative controlsince it is not expressed in CHO cells (own investiga-tions, data not shown), and the use of KCNE2 siRNAshould not affect the expression of endogenous genes.

A calibration curve was generated by plotting thefluorescence intensity of the real-time PCR signal of se-rial dilutions from the mRNA of KCNE2-transfectedcells against the PCR-cycle number and defining theundiluted sample as 100%.

With 50 and 100 nM siRNA, residual Kv4.3 mRNAwas reduced to 33% and 15% resulting in a silencing ef-fect of mRNA expression of 67% and 85%, respectively(Fig. 2). Forty-eight hours post-transfection with thenegative control KCNE2 siRNA, both Kv4.3 andKChIP2 proteins are expressed as demonstrated byimmunofluorescence staining, while in Kv4.3 siRNA-transfected cells Kv4.3 expression was strongly reducedwithout affecting KChIP2 staining (Fig. 3).

Kv4.3 siRNA reduces to a similar extent current densityand mRNA

Fig. 4A shows typical current recordings in a ‘‘scram-bled’’ negative control cell and from cells 48 h post-transfection with 50 and 100 nM siRNA, respectively.Ito was activated by 1000 ms long depolarizing voltagepulses from the holding potential of �80 mV to testpotentials ranging from �40 to +60 mV, with a pulsefrequency of 0.2 Hz. The amplitude of Ito was measuredas the difference between the peak and steady-state cur-rent. The amplitude at +50 mV in control cells was

about 15 nA, while the calculated current density was386 ± 71 pA/pF (n = 18). In cells transfected with50 nM Kv4.3 siRNA, Ito measured at +50 mV wasabout 7 nA, therefore the current was 48% smaller com-pared to that of the ‘‘scrambled’’ transfected cells. Thecorresponding current density was also significantlysmaller (186 ± 37 pA/pF (n = 17), p < 0.05). One hun-dred nanomolar Kv4.3 siRNA was even more potent,the Ito current amplitude at +50 mV was only about2.5 nA, which corresponds to 75% of reduction com-pared to controls. Current density was significantly re-duced to 111 ± 33 pA/pF (n = 20), p < 0.001. Fig. 4Brepresents the corresponding current–voltage (I–V) rela-tionships. The current reduction level was similar at allmembrane potentials between �10 and +60 mV (Fig.4C). The reduction in Ito current amplitude was notassociated with any changes in the kinetics includinginactivation, steady-state inactivation, and recoveryfrom inactivation (data not shown).

Discussion

The aim of the present study was to test the effects ofsilencing the expression of the a-subunit Kv4.3 in aCHO cell line stably expressing Kv4.3/KChIP2 subunitsby using the RNAi technique. Hereby, we designed andvalidated the method only for the pore forming a-sub-units because the silencing effects could be easily testedby direct current measurements. RNAi effects were mea-sured 48 h after transfection. No significant knock-downresulted at shorter time of transfection (data not shown),possibly due to Kv4.3 protein turnover. In addition, ifculture medium was not replaced at least 12 h before

Fig. 3. hKv4.3 protein expression is downregulated by siRNA in stably transfected Kv4.3/KChIP2 CHO expression system. Immunofluorescence ofsiRNA-transfected cells shows a significant reduction of Kv4.3 channel expression (upper right), while KChIP2 expression is not affected (bottomright). For a better overview, each fluorescence picture is accompanied by the corresponding direct light microscopy image.

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patch-clamp experiments, the cells became more difficultto patch, the cell membranes appeared more fragile be-cause cells disrupted when touched with the pipette. Ifmedium was replaced at least 12 h before patch-clampexperiments, recordings from transgenic Kv4.3/KChIP2cells were undistinguishable from those observed in cellstransfected with the negative control siRNA, suggestingthat current reduction was not an artefact due to thetransfection method, e.g., endocytosis of membrane pro-teins. Also, at longer exposure time (>48 h) RNAi wasnot efficient, suggesting siRNA degradation or dilutiondue to cell division (not shown) [12]. In patch-clampexperiments, we were able to analyse the silencing effi-ciency on the single-cell level by measuring outwardpotassium currents. Results from current measurementswere in good agreement with mRNA analysis: the silenc-ing effect was concentration-dependent, as tested withtwo concentrations of siRNA. Silencing of Kv4.3 onprotein level was shown qualitatively by immunofluores-cence staining, indicating that silencing by siRNA wasspecific in diminishing biosynthesis of the channel pro-

tein and excluding unspecific effects like translocationof protein. Compared to other silencing techniques suchas AsODN and ribozyme techniques, RNAi is a veryefficient technique: siRNA technology is more specific,effective, and more stable than AsODN [13]. siRNA isalso effective at lower concentrations than AsODN,which need higher, and therefore toxic concentrationsto knock down the gene [14]. A further advantage ofsiRNAs is that they can be synthesized in vivo by usingexpression vectors. This is not possible with AsODN.siRNA technology was already used to dissect differentpotassium currents in muscle cells of Caenorhabditis ele-gans [15]. Watanabe et al. [16] investigated the efficacy ofthe RNAi method in the silencing of the phospholam-ban gene expression and restoration of Ca2+-uptakefunction in cultured neonatal rat cardiomyocytes. Herewe have demonstrated the successful silencing of Itocurrent function in a CHO expression system. Animportant application of the RNAi technology is theknock-out of different genes encoding ion current sub-units in native cells in order to determine their function.

Fig. 4. Current measurements in stably transfected Kv4.3/KChIP2 CHO expression system. (A) Superimposed original current traces of Ito fromthree different cells after 48 h post-transfection with 100 nM of ‘‘scrambled’’ control siRNA (left), 50 nM (centre), and 100 nM Kv4.3 siRNA (right),respectively. The inset (right) shows the applied voltage protocol; holding potential �90 mV, depolarizing clamp steps to �40 to +60 mV, duration1 s, and only first 500 ms of current records are shown. (B, left) Current–voltage relation curves for Ito in CHO cells transfected with ‘‘scrambled’’control (rectangles, n = 18), 50 nM siRNA (triangles, n = 17), and 100 nM hKv4.3 siRNA (circles, n = 20), respectively. Values representmeans ± SEM. (B, right) Non-silenced Ito current in percentage (%) of ‘‘scrambled’’ controls calculated from mean current values at potentialsbetween 10 and +60 mV, for 50 and 100 nM siRNA, respectively.

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A prerequisite is, however, that native cells can be keptin culture for at least 48 h. Culturing of adult cardio-myocytes from several species was successfully per-formed for 6–7 days. However, during culturing thesecells may dedifferentiate [17], and therefore substantialchanges can be observed in the cell�s biophysical proper-ties [18]. Moreover, isolated myocytes are difficult totransfect by chemical transfection as we applied in thisstudy, although the chemical transfection of mouseadult ventricular myocytes and neonatal rat cardiomyo-cytes was reported [7,19]. An alternative method is ade-novirus infection, which was successfully applied forcardiomyocytes [20]. We conclude that applying RNAitechnology for post-transcriptional gene silencing is anovel tool to reduce the expression and function of dif-ferent ionic channel subunits. This method could beadapted for studying the role of different ion channelcomponents in native cells.

Acknowledgments

We are grateful to Dr. K. Steinmeyer (Aventis Phar-ma, Frankfurt, Germany), Dr. D. Isbrandt (Hamburg,Germany), and Professor D. Snyders (Antwerp, Bel-gium) who kindly provided us with the transgenicCHO cells, the KChIP2 antibody, and the Kv4.3 cDNA,respectively. A Marie Curie Postdoctoral Fellowshipfrom the EU Commission (HPMD-CT-2001-00119)supports D.C. and N.J.

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