Methods
Chemicals. Except for BW-031, all chemicals were purchased from Sigma Aldrich or Tocris Bioscience. BW-031 (1-(1-(2, 6-dimethylphenylamino)-1-oxobutan-2-yl)-1-ethylpiperidinium) was synthesized by Acesys Pharmatech according to the pathway described in Supporting Information.
Stable cell line electrophysiology. Human embryonic kidney (HEK 293) cells stably expressing the human Nav1.7 channel (Liu et al., 2012) were grown in Minimum Essential Medium (MEM, ATCC) containing 10% fetal bovine serum (FBS, Sigma), penicillin/streptomycin (Sigma), and 800 µg/ml G418 (Sigma) under 5% CO2 at 37°C. Human embryonic kidney (HEK 293) cells stably expressing the human Nav1.1 channel (gift of Dr. Alfred L. George, Jr.) were grown in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific) containing 10% FBS (Sigma), penicillin/streptomycin (Sigma), and 3 µg/ml Puromycin (Sigma) under 5% CO2 at 37°C. Chinese Hamster Ovary (CHO-K1) cells stably expressing the human Nav1.8 channel and beta 3 subunit (B’SYS GmbH) were grown in Ham’s F-12 medium (Corning) containing 10% FBS, penicillin/streptomycin (Sigma), and 3.5 µg/ml Puromycin (Sigma) and 350 µg/ml Hygromycin (Sigma) under 5% CO2 at 37°C. For electrophysiological recordings, cells were re-plated on coverslips for 1 to 6 h before recording. Whole-cell recordings were obtained using patch pipettes with resistances of 2-2.5 MΩ when filled with the internal solution consisting of (in mM): 61 CsF, 61 CsCl, 9 NaCl, 1.8 MgCl2, 9 EGTA, 14 creatine phosphate (tris salt), 4 MgATP, and 0.3 GTP (tris salt), 9 HEPES, pH adjusted to 7.2 with CsOH. The shank of the electrode was wrapped with Parafilm in order to reduce capacitance and allow optimal series resistance compensation without oscillation. Seals were obtained and the whole-cell configuration established with cells in Tyrode’s solution consisting of (in mM): 155 NaCl, 3.5 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.4 with NaOH. After establishing whole-cell recording, cells were lifted off the bottom of the recording chamber and placed in front of an array of quartz flow pipes (250 µm internal diameter, 350 µm external diameter). Recordings were made using a base external solution of Tyrode’s solution with added 10 mM TEA-Cl to inhibit small endogenous potassium currents. Solution changes were made (in < 1 second) by moving the cell between adjacent pipes. Currents were recorded at room temperature (21-23°C) with an Axopatch 200B amplifier and filtered at 5 kHz with a low-pass Bessel filter The amplifier was tuned for partial compensation of series resistance (typically 70-80% of a total series resistance of 4-10 MΩ), and tuning was periodically re-adjusted during the experiment. Currents were digitized using a Digidata 1322A data acquisition interface controlled by pCLAMP 9.2 software (Axon Instruments).
Human iPSC-derived nociceptor neuron electrophysiology. Sensory neurons were differentiated from human induced pluripotent stem cells (IPSCs) as previously described (Chambers et al., 2012). Cells were cultured in 35 mm dishes (Falcon), coated with 0.1 mg/ml poly-d-lysine (Sigma) and 10 µg/ml laminin, and grown in DMEM/F12(1:1) media (Life Technologies) containing 10% HI FBS (Life Technologies) and 35 µg/ml Ascorbic acid (Sigma), 10 ng/ml BDNF, 10 ng/ml GDNF, 10 ng/ml NGF, 10 ng/ml NT-3 (Life technologies) for 8 weeks. Whole-cell recordings were made using patch pipettes with resistances of 1.5-2.5 MΩ when filled with the internal solution consisting of (in mM): 140 CsF, 10 NaCl, 1.1 EGTA, 10 HEPES, 20 D-glucose, pH adjusted to 7.2 with CsOH. The external solution consisted of (in mM): 130 NaCl, 20 TEA-Cl, 5 KCl, 0.1 CdCl2, 2 CaCl2, 1 MgCl2, 10 D-glucose, 10 HEPES, pH adjusted to 7.4 with NaOH, which was perfused during the recording using the ValveBank perfusion system (Automate Scientific). The size of neurons was measured with inverted microscope Eclipse Ti (Nikon), and neurons with a diameter of less than 25 µm were used for the experiments. Currents were recorded using a Multiclamp 700B amplifier (Molecular Devices). Data were collected and digitized at 50 kHz using a Digidata 1440 16-bit A/D converter controlled by pCLAMP 10.5 software (Molecular Devices).
Electrophysiology data analysis. Data were analyzed using programs written in IGOR Pro 4.0 (Wavemetrics, Lake Oswego, OR), using DataAccess (Bruxton Software) to read pCLAMP data files into Igor Pro. Currents were corrected for linear capacitive and leak currents, which were determined using 5 mV hyperpolarizations delivered from the resting potential and then appropriately scaled and subtracted. Statistical analyses were performed using IGOR Pro. Data are given as mean±SEM, and statistical significance was assessed with the Mann-Whitney Test.
Mouse dorsal root ganglion (DRG) neuron culture and electrophysiology. DRG neurons were cultured as previously described (Costigan et al., 1998). DRGs from adult male C57Bl/6 mice (8-12 weeks old, Jackson Laboratories stock #000664) were dissected from into Hank’s balanced salt solution (HBSS) (Life Technologies). DRGs were dissociated in 1 μg/ml collagenase A plus 2.4 U/ml dispase II (enzymes, Roche Applied Sciences) in HEPES-buffered saline (HBSS, Sigma) for 90 min at 37 °C and then triturated down to single-cell level using glass Pasteur pipettes of decreasing size. DRGs were then centrifuged over a 10% BSA gradient and plated on laminin-coated cell culture dishes (Sigma). DRGs were cultured overnight in B27-supplemented neurobasal-A medium plus penicillin/streptomycin (Life Technologies). On the day following plating, DRG culture dishes were treated with either HBSS, 100 µM BW-031, 1 µM capsaicin or 100 µM BW-031+1 µM capsaicin in HBSS for 30 min, followed by a 5-minute perfusion of external solution to remove extracellular compounds.
Whole-cell current-clamp and voltage-clamp recordings were performed <24 hours after DRG culture using an Axopatch 200A amplifier (Molecular Devices) at 25°C. Data were sampled at 20 kHz and digitized with a Digidata 1440A A/D interface and recorded using pCLAMP 10 software (Molecular Devices). Data were low-pass filtered at 2 kHz. Patch pipettes were pulled from borosilicate glass capillaries on a Sutter Instruments P-97 puller and had resistances of 1.5–3 MΩ. Series resistance was 3–10 MΩ and compensated by at least 80% and leak currents were subtracted. Cells were classified as TRPV1+ or TRPV1- by the presence or absence of a response to perfused 1 μM capsaicin measured in voltage clamp mode at a holding potential of −80 mV. Cells were then held at -100mV and depolarized to -10mV with a 100 ms step to activate Nav channels. The external solution for DRG electrophysiological recordings consisted of (in mM): 30 NaCl, 90 Choline-Cl, 20 TEA-Cl, 3 KCl, 1 CaCl2, 1 MgCl2, 0.1 CdCl2, 10 HEPES, 10 Dextrose; pH 7.4, 320 mOsm. The internal pipette solution consisted of (in mM): 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, 20 Dextrose; pH 7.3.
Animals for pain studies. Male CD rats (7-8 weeks old) were purchased from Charles River and male C57BL/6J mice (8-12 weeks old) were purchased from Jackson Laboratories (stock #000664) and housed for 1 week prior to experiments. Rats were housed 3 animals per cage and mice were housed 5 animals per cage in separate rooms with constant temperature (23°C) and humidity (45-55%) with food and water availablead libitum . All procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Boston Children’s Hospital.
Plantar incision surgery. Rats were placed in a chamber with 5% isoflurane and monitored until they were visibly unconscious. Once unconscious, rats were removed from the chamber and anesthesia was maintained using 2% isoflurane delivered via nose cone. A toe pinch was used to confirm that animals were fully anesthetized. The animals were then secured with surgical tape at their toes and upper leg for paw stability during surgery. The plantar surface of one hind paw was sterilized with 3 alternating wipes of betadine and ethanol. A 1.5 cm longitudinal incision was made using a scalpel along the center of the plantar surface, beginning 1 cm from the heel and extending towards the foot pad and toes. Incision was made to the minimal depth necessary to cut through skin and fascia to expose the underlying plantaris muscle, approximately 1-2 mm. Once exposed the plantaris muscle was elevated for 10 seconds with surgical forceps and gently lifted for 10 seconds. After irritation of the plantaris muscle, the wound was closed with three sutures. After surgery animals were returned to their cage and monitored until they fully recovered from anesthesia. Treatments were administered subcutaneously 24 hours after injury.
Intraplantar injection of Complete Freund’s Adjuvant. Complete Freund’s Adjuvant (CFA) was purchased from Sigma Aldrich (Cat. No. F5881). Rats were restrained and subcutaneously injected in the plantar region of the left paw with 50 µL of CFA (1 mg/ml). Animals receiving test compounds (2% QX-314 or 2% BW-031) were injected with these compounds dissolved in 50 µL of CFA.
Plantar ultraviolet (UV) burn. Mice were anesthetized with 3% isoflurane. UV irradiation was performed on the plantar surface of the left hind paw under maintenance anesthesia with 2% isoflurane at an intensity of 0.5 J/cm2 for 1 minute at a wavelength of 305–315 nm using a fluorescent UV-B light source (XR UV LEDs 308nm, RayVio, Hayward, California, USA).
Peri-sciatic injection. Mice were anesthetized with 2.5% isoflurane. Upon achieving sufficient depth of anesthesia, mice were placed in the prone position, the fur on their left hindleg was shaved and the area was cleaned with betadine and 70% isopropanol. A 1-centimeter incision was made in the skin on the upper thigh. The sciatic nerve was identified in the intermuscular interval between the biceps femoris and gluteal muscle without the dissection of the superficial fascia layers. Then, a mixture of physiological saline with 0.5% lidocaine, 0.5% QX-314 or 0.5% BW-031 (70 μL) was injected into the perineural space below the fascia using an insulin syringe with a 30-gauge needle. The surgical wounds were closed with stainless steel wound clips (EZ Clips, Stoelting Co.).