Construction of expression vector pGEX-4T-1-Ktx-Sp2
Expression plasmid pGEX-4T-1-Ktx-Sp2 was constructed on the basis of the full-length cDNA of Ktx-Sp2 (Fig. 1a). Primers were designed to match the mature region of Ktx-Sp2. A second PCR used the products of the overlapping PCR as templates. The primers used were: Sense primer 1, 5′-CTGGGATCCGATGACGATGACAAGTCACCGCTGCATGGTGCAAAATGT-3′ with a BamHI restriction enzyme site (underlined) and corresponding to five codons encoding an enterokinase cleavage site (underlined twice); Sense primer 2, 5′-CATGGTGCAAAATGCTCATCCTCTAATCAGTGTACCCGTCCGTGCCGT-3′; Antisense primer 1, 5′-CCGCTCGAGTCAGCCATAACAGCGACAACGACCATTCATGCATTTACC-3′;
Antisense primer 2, 5′-ATTCATGCATTTACCATGGGTACCACCTCCATAACGGCACGGA CGGGT-3′ with XhoI restriction enzyme site (underlined). The PCR products were inserted into expression vector pGEX-4T-1.
Expression and purification of Ktx-Sp2 peptides
Escherichia coli Rosetta (DE3) cells containing pGEX-4T-1-Ktx-Sp2 were proliferated at 37 °C in LB with 100 mg/ml ampicillin. Fusion protein synthesis was induced by the addition of 1 mM isopropyl β-d-thiogalactoside (IPTG) at 28 °C for 4 h. Cells were harvested and resuspended in glutathione (GSH) wash buffer (pH 8.0, 50 mM Tris–HCl, 20 mM EDTA), digested by 1 mg/ml lysozyme for 30 min. After a brief sonication, the extract was clarified by a centrifugation at 10,000×g for 15 min. The fusion protein was purified by GSH affinity chromatography and enriched by centrifugal filter devices (Millipore, 10 kDa). High performance liquid chromatography (HPLC) was used to further purify peptide, under the 230 nm wavelength to monitor the absorbance of the eluate at room temperature (22–25 °C). After cleavage of the fusion protein by enterokinase (More Biotechnology, Wuhan) for 8 h at 37 °C, the mixture was filtered (Millex-HV, 0.45 mm, Millipore) and separated on a C18 column (Elite HPLC, China, 10 mm × 250 mm, 5 μm) using a linear gradient from 10 to 80% CH3CN with 0.1% TFA in 60 min with a constant flow rate of 5 ml/min. Peaks were collected manually.
Cell isolation, culture and ion channels expression
Mouse spinal columns were removed and placed in ice cold HBSS, then laminectomies were performed and bilateral DRG were dissected out. After removal of connective tissues, DRG were transferred to a 1 ml Ca2+/Mg2+-free HBSS containing 2 μl saturated NaHCO3, 0.35 mg l-cysteine and 20 U papain (Worthington, Lakewood, NJ, USA), and incubated at 37 °C for 10 min. The suspension of DRG was centrifuged, the supernatant was removed, 1 ml Ca2+/Mg2+-free HBSS containing 4 mg collagenase type II and 1.25 mg dispase type II (Worthington) was added and incubated at 37 °C for 10 min. After digestion, neurons were pelleted, suspended in neurobasal medium containing 2% B-27 supplement, 1% l-glutamine, 100 U/ml penicillin plus 100 μg/ml streptomycin, and 50 ng/ml nerve growth factor, plated on a 12 mm coverslip coated with poly-l-lysine (10 μg/ml) and cultured under a humidified atmosphere of 5% CO2/95% air at 37 °C for 18–24 h before use. Jurkat E6-1 T cells (ATCC TIB152) and HEK293T cells (ATCC ACS4500) were maintained in RPMI medium 1640 (Invitrogen, Carlsbad, CA, USA) and Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies,GrandIsland, NY, USA), supplemented with 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin, 100 μg/ml streptomycin, respectively. Cells were cultured in a humidified incubator at 37 °C with 5% CO2. The cDNAs encoding mKv1.1, mKv1.2, mKv1.3, mNav1.4, mNav1.5 and mNav1.7 were subcloned into the XhoI/BamHI sites of a bicistronic vector, pIRES2-EGFP (Clontech, USA), then transiently transfected into HEK293-T cells using Lipofectamine 2000 (Invitrogen) for electrophysiological experiments.
Whole-cell patch-clamp recordings
Whole-cell patch-clamp recordings were performed using an EPC 9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) at room temperature (22–24 °C). Pipettes pulled from borosilicate glass (BF 150-86-10; Sutter Instrument Company, Novato, CA, USA) had resistances of 2–4 MΩ when filled with the internal solution. The internal pipette solution for recording Kv currents contained: KCl 140 mM, MgCl2 1 mM, EGTA 1 mM, Na2ATP 3 mM, HEPES 10 mM (pH 7.3 with KOH); for recording Na currents contained: CsCl 100 mM, CsF 40 mM, MgCl2 2 mM, HEPES 10 mM, EGTA 10 mM, d-Glucose 10 mM, Na2ATP 3 mM (pH 7.3 with CsOH). The external solution for recording Kv currents contained: KCl 5 mM, NaCl 140 mM, HEPES 10 mM, CaCl2 2 mM, MgCl2 1 mM, d-Glucose 10 mM (pH 7.4 with NaOH); for recording Na currents contained: NaCl 132 mM, KCl 5.4 mM, CaCl2 1.8 mM, MgCl2 0.8 mM, HEPES 10 mM, d-Glucose 5 mM (pH 7.4 with NaOH). Kv currents were elicited by + 50 mV, 400 ms depolarizing pulse from the holding potential of − 60 mV every 20 s, and Na currents were elicited by + 10 mV, 100 ms depolarizing pulse from the holding potential of − 60 mV every 10 s. Using IGOR (WaveMetrics, Lake Oswego, OR) software, concentration–response relationships were fitted according to modified Hill equation: Itoxin/Icontrol = 1/1 + ([peptide]/IC50), where I is the steady-state current and [peptide] is the concentration of toxin. The parameter to be fitted was concentration of half-maximal effect (IC50).
Live cell Ca2+ imaging
Jurkat T cells were loaded with 4 μM Fura-2 AM (Life Technologies) for 60 min at 37 °C. Cells were then washed 3 times and incubated in Hank’s Balanced Salt Solution (HBSS) for 30 min at room temperature before use. Fluorescence at 340 nm and 380 nm excitation wavelengths was recorded on an inverted Nikon Ti-E microscope equipped with 340, 360 and 380 nm excitation filter wheels using NIS-Elements imaging software (Nikon Instruments Inc., Melville, NY, USA). Fura-2 ratios (F340/F380) reflect changes in [Ca2+]i upon stimulation. Data were obtained from 100 to 250 cells in time-lapse images from each coverslip.
IL-2 secretion measurements
IL-2 secretion from Jurkat T cells was measured using an ELISA kit (eBioscience, San Diego, CA, USA) following manufacturer’s instructions. Cells were centrifuged at 1500 rpm for 10 min, and the supernatants were collected to measure IL-2 concentrations. Reactions were performed in 96-well plates coated with the capture antibody and stopped with phosphoric acid (1 M). Absorbance was measured at 450 nm. Each experiment was repeated at least three times in duplicate.
Statistical analysis
All data are presented as mean ± SEM for n independent observations. Statistical analysis of differences between groups was carried out using paired t-test or ANOVA. P < 0.05 was considered significantly different.