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.).