KCR1 Modulates If Current Figure 8. KCR1 suppresses spontaneous action potential activity. (A) Representative original recordings of spontaneous action potentials of control neonatal rat ventricular cardiomyocytes. (B) KCR1 infection suppressed spontaneous beating activity in neonatal cells. Action potential artificially induced by a depolarizing pulse in a quiescent neonatal cardiocyte. (C) KCR1siRNA infection accelerated spontaneous beating activity in neonatal cells. (D) Overexpression and knock-down of endogenous KCR1 resulted in a significant (*, p,0.001) decrease and increase of the beating rate, respectively. For data see text. doi:10.1371/journal.pone.0001511.g008 into pAdCGI-HCN2 in place of the EGFP sequence, to generate pAdCRI-HCN2. For immunoprecipitation studies, the rat KCR1 sequence was ligated in frame at the N-terminus to the three FLAG epitopes of vector p3xFLAG-CMV7.1 (Sigma GmbH, Munich), resulting in the plasmid pCFLAG3-KCR1. The small interfering RNA (siRNA) nucleotide sequences for rat KCR1 (Accession no. GI 3513450) and control LacZ were as follows: 59-GGAAACGTAGAGTTCAGTTCTC-39 and 59-CTACACAA- ATCAGCGATTT-39 (Invitrogen), respectively. For transient trans- fection experiments the DNA oligonucleotides were cloned into pENTR/U6 (Invitrogen) to give pENTR/U6-KCR1siRNA and pENTR/U6-LacZsiRNA, respectively, and for adenovirus generation into pAdHCRed to give pAdHCRed-KCR1siRNA. Adenovirus vectors were generated by Cre-lox recombination of purified y5 viral DNA and shuttle vector DNA as previously described [4,35,36]. Transient Transfections CHO-K1 cells (ATCC, American Type Culture Collection, Manassas, VA) were transfected with 1 mg/well plasmid DNA (as indicated) using Lipofectamine Plus (Life Technologies, Gaithersburg, MD) as directed by the manufacturer [37,38]. Three days posttransfection, electrophysiological recordings, RNA isolation and immunoprecipitations from whole cell protein extracts were performed. Rat Cardiomyocyte Isolation and Primary Culture Animal experiments were performed in accordance with institutional guidelines for animal use in research. Ventricular myocytes of adult rats (Sprague-Dawley, 200 to 300 g) were isolated using Langendorff apparatus as previously described [39,40]. Neonatal cardiomyocytes of Sprague-Dawley (1–2 days old) rats were enzymatically dissoci- ated as previously described [4]. Action potential studies were conducted on 4- to 6-day-old monolayer cultures. For whole-cell patch-clamp experiments, 3- to 5-day-old monolayer cultures were dispersed by trypsin, and re-plated at a low density to study electrically isolated cells within 2 to 8 hours [4]. Adenovirus Infection Infection of adult and neonatal myocytes was performed 2 h and 1–3 days after plating, respectively, at a multiplicity of infection (MOI) of 15 to 100 p.f.u. per cell. Immunoprecipitation Whole protein extracts were prepared by lysing the cells in washing buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.25% Triton X-100, and 5 mM NaF, supplemented with the mammalian protease inhibitors Aprotinin (Sigma). Extracts were then passed through a needle 15 times, followed by high-speed PLoS ONE | www.plosone.org 10 January 2008 | Issue 1 | e1511