centrifugation to sediment cellular debris. 700 mg of each extract were immunoprecipitated with protein A-Sepharose-linked anti- FLAG-M2 antibodies (Sigma). A set of identical control experi- ments was performed using beads not linked to antibody (normal A- Sepharose, Sigma). Immunoprecipitates were washed three times with lysis buffer followed by resuspension in SDS sample buffer (0.23 M Tris, pH 6.4, 10% glycerol, 0.33% SDS, 3.3 mM DTT), sonication, and electrophoresis on a 7.5% SDS-PAGE. The gel was transferred to Hybond ECL membrane (Amersham) for Western blotting with an anti-HCN2 antibody (Alomone Labs, Israel). Single-cell RT-PCR analysis cDNA synthesis and PCR reaction from two to three isolated single cells were performed using the OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) as directed by the manufacturer with sense- and antisense primer specific for KCR1 (sense: 59- GAC GAG ATC TTC CAC CTG C-39; antisense: 59-TAG AAG TTG CCA ACA CTG AAG-39; amplifying 218 base pairs and spanning one intron) and GAPDH (sense: 59-GGT CGG TGT GAA CGG ATT TG-39; antisense: 59-GTG AGC CCC AGC CTT CTC CAT-39; amplifying 318 base pairs). Amplification was performed in a Thermocycler gradient (Eppendorf, Hamburg, Germany), starting with 95uC for 15 minutes to deactivate the Reverse Transcriptase and activate the Taq polymerase. The program consisted of 50 cycles (Denaturation: 95uC for 30 sec- onds; Annealing: 5963uC gradient for 45 seconds; Elongation: 72uC for 1 minute) with a final elongation step at 72uC for 10 minutes. Samples were analyzed on 1.5% agarose gels, stained with ethidium bromide and documented on a BioDocAnalyze 2.0 (Biometra, Go¨ ttingen, Germany). Preparation of sinus and atrioventricular node Fresh hearts from adult rats (Sprague-Dawley, 200 to 300 g) and adult pigs (freshly received from slaughterhouse) were dissected. To locate the sinoatrial node we used three intersection lines: the sulcus terminalis, the lateral border of the superior vena cava, and the superior border of the superior vena cava or the right auricle. The atrioventricular node was prepared according to the anatomic landmarks of the triangle of Koch in the right atrium: the coronary sinus ostium, the membranous septum, and the septal/posterior comissure of the tricuspid valve. Finally the left and right atria, left and right ventricular free wall and ventricular septum were dissected. RNA was directly isolated from these fresh samples. RNA isolation and Quantitative Real-time PCR Total RNA was extracted from neonatal rat cardiomyocytes, and from cardiac tissue of adult rat and pig using the RNeasy kit (Qiagen), and first strand cDNA was reverse transcribed with Omniscript Reverse Transcription Kit (Qiagen) as described previously [41]. Quantitative real-time PCR was performed on a LightCyclerTM 2.0 instrument using FastStart DNA MasterPlus SYBR Green I (Roche Diagnostics). Gene-specific primers were the following: KCR 1 for rat samples: forward-primer: 59-TTC AGG AAG ATA CAG CCC AGA-39, backward-primer: 59-GGG TTG GAA ATA CTG CTA GGG-39; KCR1 for pig samples: forward-primer: 59-GAC GAG ATC TTC CAC CTG C-39, backward-primer: 59-TAG AAG TTG CCA ACA CTG AAG-39. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and SDHA (succinate dehydrogenase complex, subunit A, flavoprotein) expression of each sample were used as endogenous controls (GAPDH - primers: 59-GGT CGG TGT GAA CGG ATT TG-39 and 59-GTG AGC CCC AGC CTT CTC CAT-39, GenBank accession No. NM_017008; SDHA-primers: 59-TGG GAA CAA KCR1 Modulates If Current GAG GGC ATC TG-39 and 59-CCA CCA CTG CAT CAA ATT CAT G-39, GenBank accession No. NM_130428) [42]. Quantitative real-time PCR was carried out under the following cycling conditions: stage 1, 95uC for 10 minutes (rep 1); stage 2, 95uC for 10 s, 61uC for 5 s, and 72uC for 10s (rep 38). Analysis of the PCR curves was performed with the second derivate maximum method of the LightCycler software [41,43,44]. All sample measurements were repeated at least three times and results are given as mean6SEM. Electrophysiology and Data Analysis Experiments were carried out using standard microelectrode whole-cell and cell-attached patch-clamp techniques with an Axopatch 200B amplifier and Digidata 1200 interface (Axon instruments, Foster City, CA, USA) at room temperature (21 to 23uC) [35,45,46]. Whole-cell and single-channel recordings were done as previously described, if not otherwise indicated [4,16,41,47]. For inside-out recordings both the pipette and bath solution were composed of (mM): KCl 160 mM, MgCl2 1 mM, HEPES 10 mM, EGTA 1 mM; pH 7.4 with KOH. In some experiments 8-bromo-adenosine 39,59-cyclic monophosphate (8Br- cAMP, 1 mM) (Sigma) or ivabradine (50 mM; kindly provided by the Institut de Recherches Servier, Suresnes, France) were added to the bath solution, as indicated. In KCR1-infected myocytes action potentials were initiated by short depolarizing current pulses (2 ms, 500–800 pA). Single-channel analysis were done using custom software as previously reported [16,45,47]. Linear leak and capacity currents were digitally subtracted using the average currents of non-active sweeps. For detailed gating analysis idealized currents were analyzed in 150 ms steps recorded at continuous pulse mode (total recording 206150 ms). Closed-time and first-latency ana- lyzes were carried out only in one-channel patches. The open probability (defined as the relative occupancy of the open state during active sweeps), the availability (fraction of sweeps containing at least one channel opening), and Ipeak (the peak ensemble average current, obtained visually) were calculated from single-channel and multi-channel patches with a maximum of three active channels in one patch. Single-channel amplitudes were determined by direct measurements of fully resolved openings (conductance-calculation) or as the maximum of Gaussian fits on amplitude histograms. n, the number of the channels in the patch, was defined as the maximum current amplitude observed, divided by the unitary current. Peak current was corrected by division through n. The availability was corrected by the square root method: (1-availibiltiycorrected) is the nth root of (1-availibiltiyuncorrected). The corrected open probability was calculated on the basis of the corrected number of active sweeps, i.e. total open time divided by (n6availabilitycorrected6number of test pulses6pulse length). Time constants of open time (topen) and closed time histograms (tclosed) were obtained by multi-exponential maximum likelihood estimates (MLE) on open and closed time distributions (shown here as logarithmic binned histograms from pooled data) is given by the ‘‘best fit’’ method [21]. Comparison of gating kinetics was performed as previously described [16]. The availability or fractions of active sweeps were used for calculation of voltage-dependent activation. The voltage-depen- dence of activation was analyzed using the Boltzmann function: Y = Ymax/{1+e (V 0.5-V/k)}, where V0.5 is the voltage of half- maximal activation and k is the slope factor [45]. Pooled data are presented as mean6SEM. An unpaired two-tailed t test was used for statistical examinations. Probability values of P,0.05 were deemed significant. One-way ANOVA was calculated for multiple comparisons and considered significant for P,0.05. PLoS ONE | www.plosone.org 11 January 2008 | Issue 1 | e1511