Whole-cell patch recordings of Ca2+ currents in acutely dissociated dorsal root ganglion neurons

Recordings were obtained from acutely dissociated DRG neurons as described previously [¹⁰,¹¹]. To isolate calcium currents, Na⁺ and K⁺ currents were blocked with 500 nM tetrodotoxin (TTX; Alomone Laboratories) and 30 mM tetraethylammonium chloride (TEA-Cl; Sigma). Extracellular recording solution (at ~310 mOsm) consisted of the following (in mM): 110 N-methyl-D-glucamine (NMDG), 10 BaCl₂, 30 TEA-Cl, 10 HEPES, 10 glucose, pH at 7.4, 0.001 TTX, 0.01 nifedipine. The intracellular recording solution (at ~310 mOsm) consisted of the following (in mM): contained 150 CsCl₂, 10 HEPES, 5 Mg-ATP, 5 BAPTA, pH at 7.4. Activation of I_Ca was measured by using a holding voltage of −90 mV with voltage steps 200 ms in duration applied at 5-s intervals in +10 mV increments from −70 to +60 mV. Current density was calculated as peak I_Ca/cell capacitance. Steady-state inactivation of I_Ca was determined by applying an 800-ms conditioning prepulse (−100 to −20 mV in +10 mV increments) after which the voltage was stepped to −20 mV for 200 ms; a 15-s interval separated each acquisition.

Whole-cell voltage clamp recordings were performed at room temperature (RT) using an EPC 10 Amplifier-HEKA as previously described [⁶]. The internal solution for voltage clamp sodium current recordings contained (in millimolar): 140 CsF, 1.1 CsEGTA, 10 NaCl, and 15 HEPES (pH 7.3, 290–310 mOsm/L) and external solution contained (in millimolar): 140 NaCl, 3 KCl, 30 tetraethylammonium chloride, 1 CaCl2, 0.5 CdCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES (pH 7.3, 310–315 mosM/L).

DRG neurons were subjected to current-density (I-V) and fast-inactivation voltage protocols as previously described [⁴,¹²]. In the I-V protocol, cells were held at a −80 mV holding potential before depolarization by 20-ms voltage steps from −70 to +60 mV in 5-mV increments. This allowed for collection of current density data to analyze activation of sodium channels as a function of current vs voltage and also peak current density, which was typically observed near ~0 to 10 mV and normalized to cell capacitance (pF). In the fast-inactivation protocol, cells were held at a − 80 mV holding potential prior to hyperpolarizing and repolarizing pulses for 500 ms between −120 and −10 mV in 5 mV increments. This step conditioned various percentages of channels into fast-inactivated states so that a 0-mV test pulse for 20 ms could reveal relative fast inactivation normalized to maximum current. Fire-polished recording pipettes, 2 to 5 MΩ resistance were used for all recordings. Whole-cell recordings were obtained with a HEKA EPC-10 USB (HEKA Instruments Inc., Bellmore, NY); data were acquired with a Patchmaster (HEKA) and analyzed with a Fitmaster (HEKA). Capacitive artifacts were fully compensated, and series resistance was compensated by ~70%. Recordings made from cells with greater than a 5 mV shift in series resistance compensation error were excluded from analysis. All experiments were performed at room temperature (~23°C).

The Boltzmann relation was used to determine the voltage dependence for activation of I_Ca and I_Na wherein the conductance–voltage curve was fit by the equation G/G_max = 1/[1 + exp (V_0.5 − V_m)/k], where G is the conductance G = I/(V_m−E_Ca or E_Na), G_max is the maximal conductance obtained from the Boltzmann fit under control conditions, V_0.5 is the voltage for half-maximal activation, V_m is the membrane potential, and k is a slope factor. E_Ca is the reversal potential for I_Ca; E_Na is the reversal potential for I_Na and was determined for each individual neuron. The values of I_Ca and I_Na around the reversal potential were fit with a linear regression line to establish the voltage at which the current was zero. The Boltzmann parameters were determined for each individual neuron and then used to calculate the mean ± S.E.M.