4.2. Patch-Clamp Method

The patch-clamp method was applied to quantitively describe the effects of the attacking molecules on the Na_V1.8 channel activation gating system by registering the Na_V1.8 channel peak current–voltage characteristics before and after ligand–receptor binding. The method accuracy strongly depends on the series resistance, Rs [22]. Our prior data [4] indicate that if the Rs value is less than 3 MOhm, the effective charge transferred by the activation gating system of the Na_V1.8 channel during its opening (Z_eff, measured in electron charge units, e₀) can be evaluated using the Almers [10] logarithmic potential sensitivity method [9] (see below). Microelectrodes with relatively large tip diameters were used in the experiments, which allowed us to maintain Rs practically constantly and below 3 MOhm. Another significant factor that might affect the accuracy of measurements is the rundown effect, manifested in a decrease in the sodium current amplitude. This effect is caused by the slow substitution of the intracellular solution with the solution inside the electrode, which results in a positive shift of the inactivation characteristics along the voltage axis and in a slow decrease in the Na_V1.8 channel density in the neuron membrane [7,23]. It should be stressed that the Na_V1.8 channel is exceptionally well suited for the behavior of its activation gating system to be described by the construction of the limiting conductivity function using the Almers method. The accuracy of Z_eff evaluation in this channel is not influenced either by the behavior of the inactivation system or the decrease in channel density due to the slow kinetics of both processes. However, the Almers method cannot be applied to the classical sodium Na_V1.1 channel because its fast inactivation process strongly increases the error of Z_eff evaluation [4].

Unique structural and kinetic features of the Na_V1.8 channel make it possible to register weak nonlinearities manifested only at the most negative values of the membrane potential, E. In this case, the application of the Almers method allowed us to obtain the Z_eff values by investigating exemplary neurons. A more detailed discourse on our patch-clamp experiment methodology was presented in recent publications [3,4].

Dissociated sensory neurons obtained with the short-term cell culture technique were used in the experiments. Dorsal root ganglia (DRG) were isolated from the L5-S1 region of the spinal cord of newborn Wistar rats and placed in Hank’s solution. Enzymatic treatment was carried out for 2 to 5 min at 37 °C [24] using a solution composed of 1 mL Hank’s solution, 1 mL Eagle’s medium, 2 mg/mL type 1A collagenase, 1 mg/mL pronase E, and 1 mM HEPES Na, pH = 7.4. Washing and subsequent culturing were performed in Eagle’s medium with the addition of fetal bovine serum (FBS, 10 %), glucose (0.6 %), gentamicin (40 U/mL), and glutamine (2 mM). Mechanical dissociation by pipetting was carried out to obtain isolated neurons, which were then cultured in collagen-coated 40-mm Petri dishes. After 1–2 h of culturing, visually unimpaired cells were chosen for further experiments.

The following solutions were used to investigate the slow sodium Na_V1.8 currents. The extracellular solution: 70 mM choline chloride, 65 mM NaCl, 10 mM HEPES Na, 2 mM CaCl₂, 2 mM MgCl₂, and 0.1 µM tetrodotoxin, pH = 7.4. The intracellular solution: 100 mM CsF, 40 mM CsCl, 10 mM NaCl, 10 mM HEPES Na, and 2 mM MgCl₂, pH = 7.2. The pH values were adjusted with HCl. The experiments were terminated when the responses of other slower, tetrodotoxin-resistant sodium channels were visually detected in recordings of ionic currents. Single neurons were put into the experimental bath (volume 200 μL) using a micropipette. The external solution in the bath was refreshed using passive flow under gravity. The control sodium currents were recorded 10 min after a giga-ohm seal between the tip of the microelectrode and the neuron membrane formed. After that, the studied agent was added, and the sodium currents were recorded once again 15 min after the agent application.

The “whole-cell recording” configuration of the patch-clamp method was implemented with the help of a hardware–software setup that comprised a patch-clamp L/M-EPC 7 amplifier, digital–analog and analog–digital converters, and a computer with a custom software package for the automated running of experiments developed in our laboratory. Data processing was aimed at constructing the logarithmic voltage sensitivity, L(E), function using the Almers method [10] and further obtaining the value of effective charge (Z_eff, in electric charge units) transferred by the Na_V1.8 channel activation gating system.

The series resistance (R_S), which determines both the dynamic and stationary errors of the method, was evaluated automatically during the experiment [22]. It should be maintained under 3 MOhm. In this case, the stationary error can be neglected if I_Na^max does not exceed 1 nA. The membrane capacitance (C_m) was also evaluated in the course of the experiment, which allowed us to automatically subtract the capacitive (I_C) and leakage (I_L) currents. The peak current–voltage characteristics were further constructed, making it possible to obtain the chord conductance (G_Na) of the Na_V1.8 channel and the Z_eff value.

The following protocol of voltage Impulses was applied. The first impulse was equal to the resting potential of −60 mV. The step of holding potential, usually equal to −110 mV, was generated after. A set of sequential test impulses of 50 ms duration with an increment of 5 mV was used further to record the family of sodium currents in the voltage range from −60 to 45 mV. Registration of the amplitude (peak) values of the currents generated in response to each voltage step allowed us to build the peak current–voltage curve.

The DRG neuron membrane contained another slow sodium channel subtype, Na_V1.9, which produces a tetrodotoxin-resistant current with wide overlap between activation and steady-state inactivation, and appears to modulate resting potential and to amplify small depolarizations [25]. This overlap makes it impossible to apply the Almers method for the quantitative evaluation of Z_eff, so the method applicability was tested in every single experiment. Only the neurons that demonstrated the electrophysiological behavior described in detail earlier [4], i.e., the position of the current–voltage function extremum, E ≈ 0 (Figure 1c,d), were used to study the effects of the attacking molecules.

The Z_eff values were estimated using the limiting slope procedure [10]. The ratio of the number of open Na_V1.8 channels (N_o) to the number of closed channels (N_c) is calculated as:

where G_Na^max and G_Na(E) are the maximal value and the voltage dependence of the chord conductance, respectively. G_Na(E) can be obtained in patch-clamp experiments as:

where I_peak is the amplitude value of the sodium current and E_Na is the reversal potential for sodium ions. G_Na(E) is a monotonous function approaching its maximum G_Na^max at positive E. The limiting slope procedure can be applied, according to the Almers theory, as:

where k is the Boltzmann constant, T is the absolute temperature, C is a constant, and e₀ is the electron charge.

The Almers method has the following practical application. When the membrane potential, E, approaches minus infinity (E → −∞), Z_eff can be estimated from the slope of the asymptote passing through the first experimental points determined by the very negative values of E, since Boltzmann’s principle is applicable at these potentials. The Almers limiting slope procedure makes it possible to experimentally evaluate Z_eff by constructing the voltage dependence of the logarithmic voltage sensitivity function, L(E):

The asymptote passing through the very first points of the L(E) function obtained at the most negative E allows us to calculate Z_eff, which is linearly proportional to the tangent of the asymptote slope.