Voltage-Clamp and Current-Clamp Recordings

Animal studies followed a protocol (SYDW-2013018) approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. For patch clamp recording, DRG neurons were isolated, as previously reported (Yang et al., 2013). Briefly, rat DRG neurons were harvested and incubated at 37°C for acute dissociation in enzymatic solution [DMEM medium (Corning, NY, United States) with 0.3% collagenase (Sigma, St. Louis, MO, United States) and 0.7% trypsin (Sigma, St. Louis, MO, United States)]. After 20 min, the enzymatic solution was replaced by DMEM complete growth medium with 0.3% trypsin inhibitor (Sigma, St. Louis, MO, United States) and maintained in short-term primary culture. All cells were used within 12 h of isolation.

Ca²⁺, K⁺, and Na⁺ currents were recorded from cells using the whole-cell patch clamp technique performed as previously described (Intlekofer et al., 2013; Leipold et al., 2015; Lolignier et al., 2015; Xu et al., 2015). The P/4 protocol was used to subtract linear capacitive and leakage currents. For sodium channel current recordings on DRG neurons, the bath solution contained the following (in mM): 30 NaCl, 25 D-glucose, 1 MgCl₂, 1.8 CaCl₂, 90 TEA-Cl, 5 CsCl, and 5 HEPES at pH 7.4; the pipettes internal solution contained (in mM): 135 CsF, 10 NaCl, and 5 HEPES at pH 7.4. Adding 300 nM TTX (tetrodotoxin) and neurons diameter (<25 μm) were used for discriminating TTX-R from TTX-S Na_V channels. Cells were activated by a 100-ms step depolarization to -10 mV from a holding potential of -80 mV for Na_V currents. For potassium channel, the external solution was (in mM): 130 NaCl, 5 KOH, 12 D-glucose, 2 CaCl₂, 2 MgCl₂, and 10 HEPES, pH 7.2; pipettes were filled with a solution containing (in mM): 120 KF, 20 NMDG (N-methyl-D-glucamine), 11 EGTA, 2 Na-ATP, 0.5 GTP, and 10 HEPES, pH adjusted to 7.2 with KOH. Cells were evoked by a 500-ms depolarizing potential of +10 mV from a holding potential of -80 mV to record K_V currents. For calcium current recording, the pipette internal solution contained the following (in mM): 110 CsCl, 5 MgCl₂, 10 EGTA, 25 HEPES, and 2 Mg-ATP, pH adjusted to 7.2 with CsOH; the bath solution contained the following (in mM): 125 TEA-Cl, 10 BaCl₂, 5 HEPES, 5 D-glucose, pH adjusted to 7.3 with CsOH. Cells were activated by a 150-ms step depolarization to +10 mV from a holding potential of -90 mV for Ca_V currents. ∼1 μM TTX was added in potassium and calcium bath solution to block Na_V current. For Na_V1.8 channel voltage-clamp recording, DRG neurons (diameter < 25 μm with 300 nM TTX) were held at -70 mV to inactivate Na_V1.9 channels. Furthermore, neurons with potential contamination of Na_V1.9 current were excluded. For Na_V1.9 channels voltage-clamp recording, Na_V1.8-null mouse DRG neurons (diameter < 20 μm with 300 nM TTX) were activated by a 100-ms step depolarization to -40 mV from a holding potential of -110 mV for Na_V1.9 currents. The bath and pipettes solution for Na_V1.8 and Na_V1.9 contained the following (in mM): 150 NaCl, 2 KCl, 5 D-glucose, 1 MgCl₂, 1.5 CaCl₂, and 10 HEPES at pH 7.3; the pipettes internal solution contained (in mM): 105 CsF, 35 NaCl, 10 HEPES, 10 MgCl₂, and 10 EGTA at pH 7.3.

Concentration-response curves were fitted using the following Variable slope model: Y = Bottom + (Top-Bottom)/(1+10^∧((LogIC₅₀-X)^∗HillSlope)) where HillSlope is the steepness of the family of curves, Top and Bottom are the fraction of current resistant to inhibition at low toxin concentration and high toxin concentration, respectively. For current–voltage (I–V) relationships, cells were held at -70 mV (-110 mV for Na_V1.9 channels) and depolarized to potentials from -80 mV (-100 mV for Na_V1.9 channels) to +40 mV in 5 mV increments for 100 ms, and peak currents were recorded. Conductance–voltage (G–V) relationships were determined from peak current (I) versus voltage relationships as G = I/(V-V_rev), where V was the test potential, and V_rev was the extrapolated reversal potential. Steady-state fast inactivation was achieved with a series of 500-ms prepulses -100 mV (-120 mV for Na_V1.9) to +10 mV in 10 mV increments, and the remaining non-inactivated channels were activated by a 40-ms step depolarization to 0 mV (-40 mV for Na_V1.9). Steady-state inactivation curves were fitted using the Boltzmann equation. The patch pipettes with DC resistances of 2–3 M were fabricated from borosilicate glass tubing (VWR micropipettes, 100 mL, VWR, Radnor, PA, United States) using a two-stage vertical microelectrode puller (PC-10, Narishige, Tokyo, Japan) and fire-polished by a heater (Narishige, Tokyo, Japan). Recordings were sampled at a rate of 50 kHz, and filtered at 3 kHz.

Before proceeding to current-clamp recordings, the same amplifier with the same configurations as voltage-clamp recordings was used. Pipette solution and bath solution was the same as previously described. The voltage threshold was determined by the first action potential elicited by a series of 1000 ms depolarizing current injections that increased in 50-pA increments. Cells with unstable (>10% variation) resting membrane and action potential overshoot of <40 mV were excluded for data collection.

All data recordings were performed at room temperature using an Axon Multiclamp 700B amplifier (Molecular Devices, Silicon Valley, CA, United States).