Cell line culture and gene transfection
HEK293 cells were all purchased from the Institute of Life Sciences of the Chinese Academy of Sciences (China). After harvesting using 0.25 % trypsin, the cell lines were grown at 37 °C in the presence of 5 % CO2 and 95 % air and cultured in Dulbecco’s modified Eagle’s medium (DMEM) mixed with 10 % fetal bovine serum (FBS), 75 µg/mL streptomycin, and 75 U/mL penicillin. In addition, the aseptic principle was strictly observed during the experimental operation. Before transfection, the cells were added to a plate with a density of about 2 × 105 cells/cm2. Transfection was performed when 85 % confluence was reached. The plasmids (pCDNA3/rSK Ca2) used in this study were obtained from OriGene (USA). All the transfections were performed with Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s protocols. As described previously, stable expression of the SK2 gene was established in HEK293 cells (the cells are herein referred to as SK2 cells). Before the patch-clamp experiment, SK2 cells were seeded for about 24 h in the glass cover.
Drugs and solutions
Trypsin, FBS, penicillin, streptomycin, and DMEM were all obtained from Gibco Invitrogen Corp. (USA). Bupivacaine, ropivacaine, and lidocaine were purchased from Sigma–Aldrich (USA). The Tyrode’s solution comprised the following: NaCl, 137mM; KCl, 5.4mM, MgCl2, 1.8mM; HEPES, 10mM; and glucose, 10mM; pH was maintained at 7.4 with NaOH. The pipette solution comprised the following: MgCl2, 1.15mM; potassium gluconate, 144mM; and CaCl2, 0.25mM/0.5mM/1.0mM); pH was maintained at 7.2 with KOH.
All experiments were conducted using the whole-cell patch-clamp technique. The coverslip containing SK2 cells was placed under an inverted Olympus microscope (IX70, Japan) on the cell chamber. The solutions were added to the reservoirs from the superfusion system (DADVC-8PP, ALA SCIENTIFIC, USA). The DAD-VC systems have a Micromanifold comprising eight tubes of polyamide-coated quartz glass of 100 μm ID. The Micromanifold enables up to eight solutions from the reservoirs to flow into a small common space of less than 1 µL. The Micromanifold with a micromanipulator can be easily moved around the cell preparation and pointed at the target cell.
An EPC-10 amplifier (HEKA, Germany) was used for the whole-cell patch-clamp technique. A glass electrode with 1.2-mm outer diameter was pulled out with a microelectrode puller (P-97, SUTTER, USA) to achieve a resistance of 1.5–3.0 MΩ after adding the pipette solution. Under the microscope, SK2 cells with smooth cell membranes were picked up to record the currents. After gigaseal formation, negative pressure was introduced to break the SK2 cell membrane. Voltage stimulation and data recording were performed using the Pulse 8.0 software (HEKA, Germany). All experiments were performed at 36 °C. SK2 cells could produce stable currents at 0 mV. Therefore, currents at 0 mV were used for comparisons in the following experiments. SK2 cells were recorded for currents in three different phases: baseline, inhibition, and washout. The baseline phase involved the perfusion of SK2 cells with Tyrode’s solution. The inhibition phase involved the perfusion of SK2 cells with Tyrode’s solution containing LAs. The washout phase involved the replacement of LA-containing Tyrode’s solution with normal Tyrode’s solution. The currents recorded in the three phases were defined as Currentbaseline, Currentinhibition, and Currentwashout. The normalization current was represented by Currentinhibition/Currentbaseline. The normalization inhibition was calculated as (Currentbaseline − Currentinhibition )/Currentbaseline.
The SPSS software (version 19.0, IL, USA) was used for data analysis. The normality of data was tested using the Shapiro–Wilk test, and the normally distributed data were expressed as the mean ± standard deviation. Differences between the two groups were assessed using the Student t test, and ANOVA was used for comparisons of multiple groups. A P value < 0.05 indicated a statistically significant difference.
The relationship between local anesthetic concentration and its inhibitory effect on SK2 currents was fitted in a nonlinear fashion using GraphPad Prism 5.0 software (GraphPad, CA, USA). The equation was Y = Bottom + (Top − Bottom)/(1 + 10((LogIC50−X)*HillSlope)), where HillSlope represents the steepness of the family of curves, Top and Bottom represent plateaus in the units of the y-axis, X represents the logarithm of concentrations of LAs (0, 0.5, 1, 2, 2.5, and 3), and Y represents the normalization current. Normalization current was calculated as Currentinhibition/Currentbaseline.