Chemicals, drugs and solutions
VGB ((±)-y-vinyl-GABA, C6H11NO2) was acquired from Sigma-Aldrich (Merck Ltd., Taipei, Taiwan), GAL-021 was from MedChemExpress (Everything Biotech Ltd., New Taipei City, Taiwan), while DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) and TRAM-34 (1-((2-chlorophenyl)-(diphenyl)methyl)-1H-pyrazole) were from Tocris (Union Biomed, Taipei, Taiwan). Unless stated otherwise, for cell preparations, all culture media, fetal bovine serum, L-glutamine, and trypsin/EDTA were acquired from HyCloneTM (Thermo Fisher; Level Biotech, Tainan, Taiwan); and, all other chemicals or reagents were of analytical grade.
The composition of the bathing solution (i.e., HEPES-buffered normal Tyrode’s solution) was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES adjusted with NaOH to pH 7.4. To measure K+ currents, we backfilled the patch pipettes with an internal solution consisting of 130 mM K-aspartate, 20 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 100 μM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES adjusted with KOH to pH 7.2 [16, 17]. To avoid the contamination of whole-cell Cl- currents, we substituted Cl- ions inside the pipette solution for aspartate.
For recording large-conductance Ca2+-activated (BKCa) channels, we kept cells in a high K+-bathing solution, and its composition was 145 mM KCl, 0.53 mM MgCl2, and 5 mM HEPES adjusted with KOH to 7.2, and the pipette solution contained 145 mM KCl, 2 mM MgCl2, and 5 mM HEPES titrated with KOH to 7.2. In this study, we obtained the reagent water from a Milli-Q water purification system (Merck, Ltd., Taipei, Taiwan). The culture medium and pipette solution were filtered on the day of use with an AcrodiscÒ syringe filter with a SuporÒ membrane (Bio-Check; New Taipei City, Taiwan).
Cell preparations
The glioblastoma multiforme cell line (13-06-MG) used in this study was kindly provided by Professor Dr. Carol A. Kruse (Department of Neurosurgery, Ronald Reagan UCLA Medical Center, LA, U.S.A). The 13-06-MG cells were cultured at a density of 106/ml in high glucose (4 g/l) Dulbecco’s modified Eagle media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 10 μg/ml streptomycin. Cells were maintained at 37˚C in a 5% CO2 incubator as monolayer cultures and thereafter sub-cultured weekly. Fresh media was added every 2-3 days in order to ensure a healthy cell population. To verify the presence of glial cells, we identified them by displaying glial fibrillary acidic protein, which is a cytoskeletal protein.
To evaluate concentration-dependent inhibition of VGB on the probability of IKCa channels that would be open, we kept 13-06-MG cells to be bathed in normal Tyrode’s solution containing 1.8 mM CaCl2, and each cell examined was voltage-clamped at -80 mV relative to the bath. The probability of channel opening was measured in the control or during cell exposure to different concentrations (0.3-100 μM) of VGB; and, these values were then compared with those taken after the addition of TRAM-34 (3 μM). TRAM-34 is a known selective blocker of IKCa channels. The concentration required to suppress 50% of channel activity was determined by means of a Hill function:
where IC50 or nH is the concentration required for a 50% inhibition or the Hill coefficient, respectively; [C] the VGB concentration; and Emax the maximal reduction in channel opening probability (i.e., TRAM-34-sensitive channel activity) caused by VGB.
Statistical analyses
Linear or nonlinear curve-fitting (e.g., sigmoidal or exponential curve) to the data sets collected was performed by using either Microsoft ExcelÒ (Redmond, WA) or OriginPro 2016 (Microcal). The experimental data are presented as the mean ± standard error of the mean (SEM) with sample sizes (n) indicating the number of 13-06-MG cells from which the results was acquired. The Student’s t-test (paired or unpaired) or one-way analysis of variance (ANOVA) followed by a post-hoc Fisher’s least-significant difference test, was performed to analyze multiple groups. The data were examined using a nonparametric Kruskal-Wallis test, subject to possible violation in the normality underlying ANOVA. Differences were considered statistically significant when the P-value was below 0.05.