328 HA Lee, et al Fig. 1. Structure of sertraline. METHODS Reagents Solutions Sertraline and all compounds used to prepare external and internal solutions were purchased from Sigma-Aldrich (MO, USA). The stock solutions of sertraline were prepared using eth- yl alcohol (EtOH), aliquoted and stored frozen. Test concen- trations were prepared fresh daily by diluting stock sol- utions into normal Tyrode (NT) solution. The concentration of EtOH in Tyrode’s solution was always kept at 0.1%. The external solution for recording the IKr, IKs and INa channel currents was NT solution as follows (in mM)：143 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 5 HEPES (4-(2-hydrox- yethyl)-1-piperazineethanesulfonic acid ), 0.33 NaH2PO4 and 16.6 glucose (pH adjusted to 7.4 with NaOH). The in- ternal solution for IKr contained the following (in mM)：130 KCl, 5 ethylene glycol tetraacetic (EGTA), 10 HEPES, 1 MgCl2, 5 Mg-ATP (pH adjusted 7.25 with KOH), and for IKs in the KCNQ1/KCNE1-cotransfected Human Embryonic Kidney 293 (HEK293) cells, 150 KCl, 5 EGTA, 10 HEPES, 2 MgCl2, 1 CaCl2 and 5 Na2-ATP (pH adjusted 7.25 with KOH). The internal solution for IK1 in KCNJ2-transfected HEK293 cells contained (in mM): 130 K-Asp, 15 KCl, 10 HEPES, 1 MgCl2, 5 Na2-ATP, 5 EGTA (pH adjusted 7.25 with KOH), and for the INa in SCN5A-transfected HEK293 cells, 105 CsF, 35 NaCl, 10 EGTA, 10 HEPES (pH adjusted to 7.25 with NaOH). The ICa was measured in native rat ventricular myocytes, cells were superperfused with an ex- ternal solution that consisted of (in mM)：137 cholin-Cl, 5 CsCl, 0.5 MgCl2, 2, 4-Aminopyridine (4-AP), 10 HEPES, 10 glucose and 1.8 CaCl2 (pH adjusted to 7.4 with NaOH), whereas the solution used to fill the pipette had the follow- ing ionic solution (in mM)：20 CsCl, 100 Cs-Asp, 10 EGTA, 10 HEPES, 20 TEA-Cl, 5 Mg-ATP (pH adjusted to 7.25 with KOH). Kraft-Bruhe (KB) solution for storage of the freshly isolated rat ventricular myocytes contained (in mM)：70 K-glutamate, 55 KCl, 10 HEPES, 3 MgCl2, 20 taurine, 20 KH2PO4, 0.5 EGTA (adjusted to pH 7.2 with KOH). Cell preparation For various aspects of cardiac ion channel study, HEK293 (ATCC, Manassas, VA, USA) cells were transiently trans- fected through the lipofectamine method [15], using lipofectAmin2000 (Gibco BRL, USA) as transfection reagent according to the manufacturer’s instructions. The hERG (the gene corresponding to IKr), KCNQ1/KCNE1 (the gene corresponding to IKs), KCNJ2 (the gene corresponding to IK1) or SCN5A (the gene corresponding to INa) cDNA was co-transfected with Green Fluorescence Protein (GFP), the surface marker protein, to allow assessment of the trans- fection efficiency. L-type calcium currents were recorded form acutely isolated, enzymatically dispersed rat ventri- cular myocytes. Recording of ionic currents The cells were placed in a recording chamber on the stage of a Nikon inverted microscope (Nikon Instruments Inc., Tokyo, Japan), and continuously perfused (5±1 ml/min) with 37±1oC bath solution. Ionic currents were recorded in a whole-cell configuration with a standard patch clamp technique [16] using a HEKA EPC8 amplifier (Electronik, Lambrecht, Germany). Data were recorded during the ap- proximately 5 minutes following initial application of the bath solution to verify currents stability. Test drug sol- utions were subsequently perfused for approximately 5 mi- nutes to achieve steady-state blocks. To investigate the ef- fect of sertraline on the ion channel currents, various con- centrations (0.01∼30 μM) were tested. Voltage-clamp pro- tocol generation and data acquisition were controlled by computers equipped with an A/D converter, Digidata (Axon Inc., USA) and Rclamp software developed in Seoul Natio- nal University (Seoul, Korea). The patch pipettes were made from borosilicate glass capillaries (Clark Electromedi- cal Instruments, UK) using a pipette puller (PP-830, Narishige, Japan). Their resistances were 3∼4 MΩ when filled with pipette solution. The current signals were fil- tered at a sampling rate of 5 kHz, and they were low-pass filtered at 1 kHz and stored on computer. All experimental parameters, such as pulse generation and data acquisition, were controlled using the Rclamp software. Data analysis and statistical test Data analysis and curve fitting were carried out using GraphPad InStat (GraphPad Software, San Diego, CA) and SigmaPlot 2000 (Systat Software, Inc. San Jose, CA). All data are expressed as mean±SEM and an n indicated num- ber of replicates. Student’s t tests or ANOVA were used for statistical comparisons when appropriate, and differ- ences were considered significant when p＜0.05, or p＜0.01. Current amplitudes were measured before and after appli- cation of sertraline. The relative remaining currents were calculated according to the following equation: Initial cur- rent amplitude/Current amplitude in the presence of com- pound=Relative remaining current. Effects were calculated from the results of 4 experiments per concentration of sertraline. Concentration response relations were calcu- lated by a non-linear least squares fit of equation [Hill equation; f=xH/(IC50H＋xH); H=Hill coefficient, IC50=IC50, x=concentration, f=inhibition ratio] using the SigmaPlot 2000 program. The half-maximum inhibiting concentration (IC50) was calculated with this function. RESULTS Effect of sertraline on hERG, IKs, and IK1 currents Fig. 2 shows the effects of sertraline on hERG K＋ channel