Myr (98% purity) was provided by Dr Yong Ye (College of Pharmacy, Guangxi Medical University, Nanning, China). It was prepared as a 10-mM stock solution in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored in aliquots at 4°C prior to use. For use in the subsequent experiments, it was diluted to 0.5, 3 or 10 µM with extracellular fluid, as described below.

The I_kur recording solution (extracellular solution) consisted of 130 mM NaCl and 0.33 mM Na₂HPO₄ x12H₂O (both from KeLong Chemical, Chengdu, China) as well as 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (all from Sangon Biotech Co. Ltd., Shanghai, China), 5.5 mM glucose (Bio Basic Inc, Markham, OT, Canada) and NaOH (Jinshan Chemical Reagent Co. Ltd, Chengdu, China) to adjust the pH to 7.4. The electrode solution contained 110 mM l-aspartate (Sigma-Aldrich), 110 mM KOH (Sangon Biotech Co. Ltd.), 20 mM KCl, 8 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM ethylene glycol tetraacetic acid (all from Sigma-Aldrich), 10 mM HEPES (Sangon Biotech Co. Ltd.) and KOH to adjust the pH to 7.2; 5 mM Mg-adenosine triphosphate (Sigma-Aldrich) was added immediately prior to use, and the solution was filtered through 0.22-µm microporous membranes (Merck Millipore, Carrigtwohill, Republic of Ireland). The lysis buffer consisted of 250 mM glucose, 20 mM 3-(N-morpholino)propanesulfonic acid and l mM dithiothreitol (Sigma-Aldrich); 1% (v/v) protease inhibitor (Calbiochem; EMD Millipore, Billerica, MA, USA) was added prior to use.

HEK293 cells (American Type Tissue Collection, Manassas, VA, USA) were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and penicillin/streptomycin (Sigma-Aldrich). Cells were transiently transfected with hK_v1.5-overexpression vector in six-well plates (Corning-Costar, Corning, NY, USA) using 3 µl Entranster™-H transfection reagent (Engreen Biosystem Co. Ltd, Beijing, China) and 3 µg of hK_v1.5 cDNA in pEGFP-N1 vector (Sangon Biotech Co. Ltd.). A pEGFP-N1 was used as a negative control. Subsequent assays were performed at 24 h subsequent to transfection. Cells used for electrophysiology experiments were seeded at a density of 1×10⁴ cells/35-mm dish with glass cover slips.

MTT assays were used to assess the toxicity of Myr. Logarithmically growing cells were seeded at a density of 1–5×10⁴ cells/well in 96-well plates (Corning-Costar) and were allowed to adhere for 24 h at 37°C. The medium was then replaced with fresh medium containing various concentrations of Myr. The cells were maintained for two days and the toxicity of Myr was determined using MTT (Amresco LLC, Solon, OH, USA). Ten microliters of MTT (5 mg/ml stock) was added to each well followed by incubation for 4 h. The seeding medium was then discarded and 0.15 ml DMSO was added to each well. The absorbance (A) was read using a multimode microplate reader (Infinite M200; Tecan Group, Ltd., Männedorf, Switzerland) at 570 nm and the cell viability was determined as follows: (1−A_{treated group}/A_{control group} ×100.

Cells (4×10⁵/ml) were incubated with or without Myr (0–100 µM) for 24 h in six-well plates. Cells were collected and lysed in lysis buffer. After sonication in ice water, crude lysates were cleared by centrifugation at 12,000 ×g for 5 min at 4°C. The total protein concentration of the lysates was measured using a bicinchoninic acid protein assay kit (Solarbio, Beijing, China). Lysates were diluted at a 4:1 ratio with 5X loading buffer (Beyotime Institute of Biotechnology, Haimen, China) and boiled for 5 min. Equal amounts of protein (10 µg) were loaded onto 10% SDS-polyacrylamide gels and separated by electrophoresis using an electrophoresis apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The separated proteins were then electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Immun-Blot^® PVDF membrane; Bio-Rad Laboratories, Inc.). Following electrotransfer, the membranes were blocked with 5% non-fat milk (Yili Industrial Group Co. Ltd, Inner Mongolia, China) in Tris-buffered saline with Tween 20 for 60 min on an orbital shaker (Qilinbeier Instrument and Manufacture Co. Ltd, Haimen, China) and then incubated with rabbit polyclonal primary antibodies against K_v1.5 (A03K0017; 1:200 dilution; BlueGene Biotech Co., Ltd., Shanghai, China) overnight at 4°C followed by biotin (AP132B; 1:10,000; EMD Millipore) for 1 h and goat anti-rabbit horseradish peroxidase-conjugated antibodies (sc-2004; 1:50,000; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min at room temperature. Rabbit polyclonal antibody of the endogenous protein GAPDH (Santa Cruz Biotechnology) was used as a loading control. Protein bands were detected using enhanced chemiluminescence western blotting substrate (Pierce Biotechnology Inc, Rockford, IL, USA), imaged using the Universal Hood II system (Bio-Rad Laboratories, Inc.) and quantified using Quantity one software v4.6.2 (Bio-Rad Laboratories, Inc.).

Currents were recorded using the whole-cell patch-clamp technique at room temperature (~22°C). The protocol used 200-msec voltage steps between −50 mV and +60 mV from a holding potential of −80 mV, followed by a return to −40 mV (Fig. 2A). The current amplitude was measured from 150 msec to the end of the depolarization step. Borosilicate glass electrodes (1.2 mm optical density) were pulled using a Brown-Flaming puller (model P-97; Sutter Instrument Co, Novato, CA, USA); they had a tip resistance of 5–7 MΩ when filled with the electrode solution. The membrane current was recorded in voltage-clamp mode using an EPC-10 amplifier and Pulse software (Heka Elektronik, Lambrecht, Germany). A 3 M NaCl-agar salt bridge was used as the reference electrode. The tip potential was set to zero before the patch pipette touched the cell. After a GΩ seal was obtained, the cell membrane was ruptured using gentle suction to establish the whole-cell configuration. The series resistance (Rs) and membrane capacitance were compensated prior to the onset of recordings. The membrane capacity (Cm) was recorded using 10-msec voltage steps to −65 mV from a holding potential of −60 mV, followed by a return to −60 mV (Fig. 2B). The formula used to calculate the membrane capacity was as follows: Cm=0.98× A × τ/5, with amp as the current amplitude and τ as the time constant. I_kur was recorded at various time-points in the presence of various concentrations of Myr, and Myr-untreated cells were used as a control. Prior to measurements, cells were perfused with extracellular solution containing various concentrations of Myr at a rate of 3 ml/min. The perfusion volume was ~15 ml per experiment so that the original solution was replaced completely. The Myr incubation time-dependency was analyzed using the mean ± standard error of the current suppression ratio (I_C−I_A)/I_C, where I_C represents the I_kur in the control group and I_A stands for I_kur in the presence of Myr.

Electrophysiological data were analyzed using Patchmaster (Heka Elektronik), Clampfit 10.0 (Axon Instruments, Molecular Devices, Sunnyvale, CA, USA), and Origin 9 software (OriginLab, Northampton, MA, USA). The results were analyzed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA), and values are expressed as the mean ± standard error. Statistical comparisons were made using Student's t-tests for two groups of data, or analysis of variance for multiple groups of data. P<0.05 was considered to indicate a statistically significant difference.

To determine an effective, non-toxic concentration of Myr for use in the patch-clamp assay, MTT assays were performed following treatment of HEK293 cells with Myr at a range of doses. Myr caused a dose-dependent decrease in cell growth between 2 and 6 mM with an IC₅₀ of 4.66 mM (Fig. 1B). However, no cell growth inhibition was observed at 0–100 µM Myr, which was therefore used in the subsequent experiments.

A K_v1.5 wild-type overexpression plasmid was transiently transfected into HEK293 cells using Entranster™-H transfection reagent (Engreen Biosystem Co., Ltd., Beijing, China) for 24 h (Fig. 2A), and the levels of K_v1.5 protein were assessed using western blot analysis after treatment with Myr (0–100 µM) for 24 h (Fig. 2B). Myr induced a dose-dependent decrease in the expression of K_v1.5 after 24 h.

Patch clamp experiments on cells perfused with extracellular solution containing various concentrations of Myr showed that 5 µM Myr reduced the current amplitude from 215.01±40.59 to 77.72±17.94 pA/pF, suggesting that I_kur was significantly inhibited by Myr (P=0.011 vs. control; n=5) (Fig. 3).

At a clamping voltage of 60 mV, the current density (Im/Cm, pA/pF) was 142.15±37.80, 103.75±29.32 and 77.72±17.94 in the presence of 3, 5 and 10 µM Myr, respectively (Fig. 4A–C), which was significantly different from that in the control group (215.01±40.59 pA/pF; all P<0.05; n=5). This suggested that Myr inhibited I_kur at concentrations of 3–10 µM in a concentration-dependent manner.

I_kur was recorded from 5 to 30 min after the addition of Myr to the chamber to evaluate the time-dependent effects of Myr on I_kur. The results indicated that the effects of Myr were time-dependent (Fig. 5A and B). The effects of time were analyzed using the mean ± standard error of the current suppression ratio (IC-IA)/IC, where IC represents the I_kur in the control group and IA stands for I_kur in the presence of Myr. The (IC-IA)/IC was 0.3101±0.1234 at 5 min (n=7; P=0.046 vs. control), 0.4091±0.1180 at 10 min (n=7; P=0.066 vs. 5 min; P=0.013 vs. control), 0.05352±0.0978 at 15 min (n=7, P=0.004 vs. 10 min; P=0.009 vs. 5 min; P=0.002 vs. control), and 0.5497±0.1060 at 20 min (n=7; P=0.453 vs. 15 min; P=0.023 vs. 10 min; P=0.004 vs. control). These results demonstrated that the I_kur amplitude decreased with time and that the inhibitory effects of Myr increased over time.

A train of 20 pulses, each with a 200-msec single voltage step, were used from a holding potential of −80 mV to +50 mV at frequencies of 0.5, 1, 3 and 4 Hz (Fig. 6A). The inhibitory effects of Myr on I_kur increased with the continuous depolarization voltage pulses. In addition, the inhibition of I_kur by Myr was dependent on the number of pulses (Fig. 6B and C), which suggested that Myr inhibited I_kur in a rate- or frequency-dependent manner. Specifically, I_kur was 376.23±1.30, 340.01±4.25, 290.59±4.44 and 270.193±4.28 pA/pF at 0.5, 1, 3 and 4 Hz, respectively, and changes between each set of two groups were significant (n=5, P<0.001). Furthermore, the inhibition of I_kur significantly increased when the frequency was increased from 0.5–4 Hz (P<0.05) (Fig. 6D). These results suggested that Myr inhibited I_kur in HEK293 cells in a frequency-dependent manner.