An Analysis of the Variations in Potency of Grayanotoxin Analogs in Modifying Frog Sodium Channels of Differing Subtypes

Materials and Methods

Cell Preparations.

Frogs (Rana catesbeiana) were sacrificed by decapitation and the spinal cord was destroyed. Single ventricular myocytes were taken from the hearts using essentially the same technique as described previously (Seyama and Yamaoka, 1988). Briefly, the heart was mounted on a Langendorff apparatus and perfused retrogradely via the aorta with a Ca²⁺-free solution containing Yakult collagenase (0.025 mg/ml; Yakult, Tokyo, Japan), Wako collagenase (0.35 mg/ml; Wako Pure Chemical Industries Ltd, Osaka, Japan), type III trypsin (0.06 mg/ml; Sigma Ltd., St. Louis, MO), and crystallized BSA (0.6 mg/ml; Seikagaku Corporation, Tokyo, Japan) for 20 min at 32°C. The dispersed cells were kept in a solution containing reduced (200 μM) Ca²⁺ for 30 min and then centrifuged for 1 min at 65g. After eliminating the cell debris, the collected cells were maintained in Leibowitz's L-15 medium (Gibco Laboratories, Gaithersburg, MD) until experimental use.

DRG were dissected from bullfrogs. The ganglia were minced and then shaken for 60 to 90 min at room temperature in cell dispersion medium containing 2 mg/ml collagenase (type IA; Sigma), 0.3 mg/ml trypsin (type IX; Sigma) and 0.2 mg/ml deoxyribonuclease I (Sigma). The cell dispersion solution contained 120 mM NaCl, 10 mM glucose, 10 mM HEPES, 2.5 mM KCl, 0.01 mM CaCl₂, and pH was adjusted to 7.2 with NaOH. The dispersed cells were centrifuged for 1 min at 65g, and the collected cells were stored in modified L-15 medium: 36 ml of 1 mM CaCl₂ solution and 7.15 ml of fetal bovine serum were added to 100 ml of L-15 medium (Campbell, 1992).

Solutions and Chemicals.

For measuring whole-cell Na⁺ current, the composition of the external solution was 90 mM NaCl, 15 mM tetraethylammonium chloride, 9 mM MgCl₂, 1 mM CaCl₂, 0.005 mM LaCl₃, and 10 mM HEPES. The pH of the external solution was adjusted to 7.2 with NaOH. The internal solution consisted of 60 mM CsF, 40 mM CsCl, 20 mM NaF, 5 mM EGTA, and 5 mM HEPES. The pH of the internal solution was adjusted to 7.0 with CsOH.

To assess the effects of GTX on whole-cell Na⁺currents, GTX analogs were added to the pipette solution, because GTX is known to act intracellularly (Seyama et al., 1988). Pipette solutions for cell-attached single-channel recordings contained 250 mM NaCl, 0.3 mM CaCl₂, 1 mM MgCl₂·6H₂O, 0.01 mM LaCl₃·7H₂O, 5 mM HEPES, and 0.001 mM TTX. The pH was adjusted to 7.2 with NaOH. Bath solutions for DRG cells contained 120 mM KCl, 75 mM CsCl, 1 mM CaCl₂·7H₂O, 2 mM MgCl₂·6H₂O, and 5 mM HEPES, and that for ventricular myocytes contained 115 mM KCl, 3 mM MgSO₄·7H₂O, and 5 mM HEPES. The pH of both bath solutions was adjusted to 7.2 with KOH.

The structural formulae of the GTX analogs used in this study are shown in Fig. 1. Both GTX analogs were kindly provided by Professor Emeritus J. Iwasa of Okayama University, Faculty of Agriculture, Okayama, Japan. All stock solutions of GTX analogs were dissolved in dimethyl sulfoxide at a concentration of 10⁻¹ or 10⁻² M.

Because GTX molecules are permeable through cell membranes and the pipettes containing GTX act as a point source, one may be concerned how evenly GTX was distributed in the intracellular phase and whether the diffusion of GTX out of the membrane to the external phase lowered the concentration of toxin in the internal side. We resolved this concern by showing that GTX-modification occurred to the same extent whether GTX of the same concentration was present on both sides or only on the internal side. To begin with, we collected necessary data for constructing current-voltage curves with and without conditioning pulses using a patch electrode containing 100 μM GTX-I in ventricular myocytes. Successively, GTX-I of the same concentration was additionally introduced into the external side, and the same kinds of data from the same preparation were collected after soaking the preparation for 20 min. The resultant normalized slope conductances (see under Dose-Response Relationship for GTX-Evoked Modification) from four series of experiments were estimated to be 0.044 ± 0.008 for internal and 0.047 ± 0.009 for both sides applications (n = 4). Because there was no statistical significance in the difference between the two values, it is reasonable to assume that the cell membranes functioned as an effective diffusion barrier.

Ionic Current Recordings.

Whole-cell patch pipettes with a resistance of less than 2 MΩ were used for obtaining optimum voltage control. Whole-cell currents (filtered at 5 KHz) were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Routine series-resistance compensation was performed to values more than 80% to minimize voltage-clamp errors. Recordings were started 5 min after establishing a conventional whole-cell recording configuration. Single Na⁺ channel currents were measured using the cell-attached variation of the patch clamp technique (Hamill et al., 1981). Single-channel currents were filtered at 5 KHz except those in Fig. 6. In Fig. 6, 1 KHz filtering was set on recording data and further digital filtering at a cut-off frequency of 400 Hz was employed. The whole-cell membrane currents were digitized at a sampling frequency of 20 to 100 kHz with a 12-bit analog-to-digital converter (TL-1 DMA interface; Axon Instruments), controlled by pClamp software (Axon Instruments); digitized currents were stored on diskettes. In single-channel recordings, the sampling rate ranged from 5 KHz to 100 KHz. Data are presented as mean ± 1 SD along with the number of observations made (n). In the whole-cell configuration, the background current was subtracted from the total current recorded to obtain the actual Na⁺ current. The background current records required were recorded in 0.3 μM TTX solutions for ventricular myocytes (Yamaoka, 1987), and in solutions containing 1.0 μM TTX plus 1 mM Cd²⁺ for DRG cells. All experiments were conducted at room temperature (23–26°C).

Single Channel Analysis.

Open channel probability (P_o,GTX) and dwell time histograms for GTX-modified Na⁺ channels (at −60 mV) were obtained from the segment in a record having no overlapping openings for at least 10 s (Fig. 6, A and D).P_o,GTX and the histograms were calculated using idealized record analysis (pClamp software).

The procedure for obtaining open channel probability under control conditions (at the time of peak Na⁺ current;P_o,control) was as follows. First, we averaged 100 to 150 traces at −10 mV (multichannel recordings) to get an ensemble record. Next, we inspected these 100 to 150 traces and selected the trace containing the maximum number of simultaneously open channels, referring to maximal individual ionic current. We estimated the number of channels in the patch (N) by dividing the peak of maximal individual ionic current by the single channel current. Separately, we also divided the peak of ensemble current by the single channel current to obtain NP_o. Then, we obtained P_o,control by dividingNP_o by N. Therefore,P_o,control indicates the open probability of a single channel at the peak of ensemble record.P_o,control, thus estimated, was found to be 0.40 ± 0.08 (n = 5) for DRG TTX-i Na⁺ channels and 0.32 ± 0.06 (n = 5) for ventricular Na⁺channels. In ventricular myocytes, the maximum individual ionic currents used in this calculation were less than 7.4 pA, which corresponds to the summation of four single-channel unit currents. Regarding DRG TTX-i Na⁺ channels, the maximum individual ionic current in most cases was less than 4.8 pA, which corresponds to the summation of four single-channel unit currents. The estimated values are thought to approximate to the true value ofP_o for a single channel, because a small number of channels within a membrane patch leads to a higher likelihood that all channels in a patch will open simultaneously at least once in a large series of trials. The values ofP_o,control obtained in this study are in agreement with values reported by Aldrich et al. (1983). Capacitative and leakage currents were subtracted from the records using blank traces without channel openings.

Separation of TTX-i and TTX-s Na⁺ Currents in Isolated DRG Cells.

DRG cells have been reported to contain two types of Na⁺ channels, TTX-s and TTX-i Na⁺ channels. Because Hille (1968) has demonstrated that 0.3 μM TTX blocks the Na⁺channels of the frog node of Ranvier, the sensitivity of the Na⁺ channels of DRG cells to 0.3 μM TTX was examined. The amplitude of the Na⁺ current (under voltage clamp conditions) was decreased to 20 to 70% of the control value. The residual Na⁺ current remained unchanged, even when the concentration of TTX was increased to 1.0 μM. This finding indicates that 0.3 μM TTX is sufficient in concentration to completely block TTX-s Na⁺channels. Residual Na⁺ current with 0.3 μM TTX treatment can be suppressed by 0.3 mM Cd²⁺(Narahashi et al., 1994; Akopian et al., 1996). In separate experiments, the IC₅₀ value for Cd²⁺ in suppressing the Na⁺current was estimated to be 8.1 ± 2.0 μM (n = 3) from the dose-response curve for Cd²⁺ in the presence of 0.3 μM TTX. To obtain the IC₅₀value for TTX-sensitive Na⁺ channel, we increased the external concentration of Cd²⁺ up to 10 mM from 0.3 mM. The resultant dose-response curves yielded an IC₅₀ value of 10.0 ± 2.2 mM (n = 7). There is a big difference in the sensitivity to Cd²⁺ between TTX-i and TTX-s Na⁺ channels, enough to separate one from the other pharmacologically. From these observations, we define Na⁺ channels activated in the presence of 1.0 μM TTX as TTX-i Na⁺ channels and those seen in the presence of 0.3 or 1 mM Cd²⁺ as TTX-s Na⁺ channel.

Results

Whole-Cell Recordings

Time Course of GTX Modification of Na⁺ Channels.

As previously reported, a prerequisite for GTX-induced modification of Na⁺ channels in squid axons is a sustained depolarization to a membrane potential more positive than −80 mV (Yakehiro et al., 1997). However, in the present experiments, the application of a single depolarizing prepulse was not effective in modifying cardiac or DRG cell Na⁺ channels. We therefore examined the time course of GTX-modification and recovery from modification. Figure 2 shows a typical example of the time course of GTX-I (100 μM) modification for ventricular Na⁺ channels. Because GTX shifts the activation curve of Na⁺ channels to the hyperpolarizing direction, GTX-induced modification was recognized as a sustained inward Na⁺ current, in response to the test pulse to −80 mV from a holding potential of −120 mV after repetitive conditioning pulses. As the numbers of conditioning pulses became larger, a sustained inward current flowing at the end of the pulse to −80 mV gradually increased. A quick development of sustained Na⁺ current soon attained a steady state when the number of conditioning pulses was more than 100. To induce full modification of DRG TTX-s and TTX-i Na⁺ channels, approximately 100 repetitive conditioning pulses were also required. Because GTX modification is reversible and recovery from modification is independent of GTX concentration (Yakehiro et al., 1997), the time course of recovery from modification in isolated ventricular myocytes at a membrane potential of −120 mV was determined by plotting sustained currents during the test pulse to −80 mV against various intervals after 1000 (GTX-I) conditioning pulses to 0 mV. The time course of current decay could be described as the sum of two exponentials (Fig. 2B): τ₁ = 1.4 ± 0.5 s, τ₂ = 18.6 ± 1.8 s (n = 3) for GTX-I and τ₁ = 0.5 ± 0.2 s, τ₂ = 5.4 ± 0.8 s (n = 3) for α-dihydro-GTX-II. Because the conditioning interpulse interval was much briefer than the time constants, the release of GTX analogs should not substantially affect the rate of GTX-induced modification of Na⁺channels.

GTX-Evoked Modification of Na⁺ Channels in DRG Cells and Ventricular Myocytes.

After blocking TTX-s Na⁺ channels using 1.0 μM TTX, the residual (TTX-i) current was modified by the application of 150 conditioning pulses in the presence of 300 μM α-dihydro-GTX-II. As shown in the middle panel of Fig. 3A, the peak currents recorded from GTX-modified channels are suppressed in amplitude relative to the control current, and sustained currents appear at large negative membrane potentials, thus shifting the activation voltage in the hyperpolarizing direction by as much as 50 mV. After blocking the TTX-i channels by external Cd²⁺ (1 mM), the residual TTX-s Na⁺ current was modified similarly by 300 conditioning pulses above in the presence of 300 μM α-dihydro-GTX-II (Fig. 3B). The current-voltage relationship again reveals a shift of the activation curve in the hyperpolarizing direction (Fig. 3B). In isolated ventriculomyocytes, 1000 conditioning pulses from a holding potential of −80 mV modified the cardiac Na⁺ current in a manner qualitatively similar to that in the other kinds of Na⁺ channels studied (Fig. 3C). In Fig. 3, it is not clear whether GTX-induced modification of Na⁺ channels reaches saturation level or not. To obtain more accurate data for modification, we conducted a single-channel experiment as presented in a later section.

Dose-Response Relationship for GTX-Evoked Modification.

Because the number of Na⁺ channels expressed on a cell varies, it is necessary for GTX-modified Na⁺channel to be normalized to a representative factor of normal Na⁺ channel in the active state. We chose the maximum permeability constant of the Goldman-Hodgkin-Katz (GHK) equation (Goldman, 1943; Hodgkin and Katz, 1949) for this purpose, because the current-voltage relationship for Na⁺channels, particularly modified ones, show a concave curve. The degree of GTX-evoked modification of Na⁺ channels at various concentrations of GTX in the pipette was estimated as the permeability constant for sustained (i.e., modified) Na⁺ currents after conditioning prepulses, referred to the maximum permeability constant for the unmodified peak Na⁺ current without conditioning pulses. The maximum permeability for both control and GTX-modified Na⁺ channels was computed by fitting the GHK equation to the current-voltage relationship at a range from +10 mV to +30 mV for control, and from −20 mV to +10 mV for GTX-modified Na⁺ channels. In GHK equation:I is the current amplitude, A is the permeability constant, E is the voltage, F is Faraday's constant, R is the gas constant, T is absolute temperature, and [Na⁺]_i and [Na⁺]_o are internal and external Na⁺ concentrations, respectively.A is the only variable term, and the other terms remain constant regardless of the states of Na⁺ channel. Thus, the normalized permeability constant—the ratio of permeability of control versus that of modified Na⁺channel—is equal to the normalized slope conductance. The dose-response curve for GTX-evoked modification of the Na⁺ channels was constructed by plotting the normalized slope conductance against GTX concentration. As shown in Fig. 4, TTX-i Na⁺channels in DRG cells were the most sensitive to both GTX-I and α-dihydro-GTX-II. At the highest concentration employed (300 μM), the conductance of GTX-modified Na⁺ channels reached as much as 21% (GTX-I) and 30% (α-dihydro-GTX-II) of that in control channels. The data were fitted with a sigmoidal curve, assuming 1:1 drug-receptor stoichiometry, as given by the equation:

normalized slope conductance =Y_max/(1 + EC₅₀/[GTX]_i),

where Y_max indicates the relative maximal permeability constant for GTX-modified Na⁺ channels, EC₅₀ denotes the half-maximal concentration, and [GTX]_irepresents the intracellular GTX concentration. The EC₅₀ was estimated to be 31 μM for GTX-I and 54 μM for α-dihydro-GTX-II, respectively.

TTX-s Na⁺ channels in DRG cells were far less sensitive to both GTX-I and α-dihydro-GTX-II than were TTX-i channels. Even at the highest concentration tested (300 μM), the normalized slope conductance reached only 43% (GTX-I) or 18% (α-dihydro-GTX-II) of the corresponding value for TTX-i Na⁺ channels at the same toxin concentration. In addition, both GTX-I and α-dihydro-GTX-II were far less effective in modifying cardiac Na⁺ channels than TTX-i Na⁺ channels in DRG cells. In cardiomyocytes, the normalized slope conductance for the modified current in the presence of α-dihydro-GTX-II (300 μM) was 0.03 [i.e., about half of the corresponding value (0.08) in GTX-I at the same concentration]. It seems reasonable to conclude that the pharmacological action of α-dihydro-GTX-II in modifying DRG TTX-s channels and ventricular Na⁺ channels was much weaker than that of GTX-I.

Single-Channel Recordings

Reduced Single-Channel Conductance of GTX-Modified Na⁺Channels.

Because whole-cell recordings showed that GTX analogs are much more potent in modifying TTX-i Na⁺channels from DRG cells than Na⁺ channels from cardiomyocytes (see Fig. 3), we measured currents through single ion channels in an effort to compare the properties (single-channel conductance, probability of channel opening) of the modified Na⁺ channels in DRG cells (TTX-i channels) and cardiomyocytes.

Before the formation of cell-attached patches, ventricular myocytes were preincubated in an external solution containing 100 μM GTX for 20 min, because GTX has been shown to have access to its binding site only after passing through the cell membrane (Seyama et al., 1988). Because the open time for GTX-modified ion channels is on the order of several tens of milliseconds and that for unmodified channels lasts as long as several milliseconds in selected records, we were able to obtain the single-channel current-voltage relationship directly by applying a ramp clamp. The ramp clamps used had a rising and falling slope of 8 V/s (voltage range, −120 to +40 mV) and were applied at a frequency of 1 Hz. Twenty consecutive recordings are displayed in Fig.5A. During the first 10 episodes, single-channel currents were observed from time to time during the rising phase of the voltage ramp, but not during the falling phase, because of the full inactivation of the Na⁺channels (Hodgkin and Huxley, 1952), which were not yet modified. Evidence of channel modification can be seen at the eleventh episode: a single channel opening was followed by a sustained inward current, and this slow sustained current continued during the falling phase of the ramp. Successive ramp depolarizations (episodes 11 to 19) produced a gradual change in inward current corresponding to the macroscopic sustained inward current observed under a whole-cell voltage clamp after a series of conditioning prepulses (Fig. 3). In analyzing the data obtained, records of inactivating single channel currents were taken to represent the current flowing through unmodified Na⁺ channels, giving a value of 34 pS for single-channel conductance. In separate experiments, the conventional step pulse method for single-channel conductance also gave a value of 32 pS (data not shown). The noninactivating currents of prolonged duration were analyzed separately and represented currents through GTX-modified channels. The resultant current-voltage curves for the slow sustained current were concave in shape (Fig. 5C). A straight line was fitted to the data between −80 mV and −20 mV, and the single-channel conductance was determined from the slope. In this example, the single channel conductance of ventricular Na⁺ channels was decreased from 34 pS to 11 pS after α-dihydro-GTX-II modification; Duch et al. (1992) reported a similar decrease in single channel conductance. All data for single Na⁺ channel conductance before and after GTX modification are summarized in Table 1.

Effect of GTX Analogs on the P_o.

Treatment with 100 μM α-dihydro-GTX-II induced a characteristic, long-lasting opening of single Na⁺ channels (Fig.6, A and D). Another noticeable finding is the presence of a clear subconductance state in Fig. 6A. This topic will be discussed in detail in a subsequent communication.

As shown in Fig. 6, B and E, the distribution of open times could be fitted by a single exponential function with a time constant, τ_o, of 286 ms (neuronal TTX-i channel) and 218 ms (ventricular Na⁺ channels). Comparison of open time histograms constructed for Na⁺ channels in DRG cells and in ventricular myocytes revealed no qualitative difference (Table 2). The closed time histogram for TTX-i Na⁺ channels in DRG cells is best described as the sum of two exponential components, indicating that there are two closed states (Fig. 6C). By contrast, the closed time histogram for Na⁺ channels in ventricular myocytes was well fitted by a single exponential function (Fig. 6F). Mean dwell times for GTX-modified Na⁺ channels are summarized in Table 2. Because the channel openings did not overlap one another, P_o,GTX was determined by dividing the total time spent in the open state by the pulse length.P_o,control values for TTX-i Na⁺ channels in DRG cells (0.40) and those for the ventricular Na⁺ channel (0.32; see underMaterials and Methods), were in the same range.P_o,GTX for ventricular and DRG TTX-i Na⁺ channels, modified by both GTX analogs, were similar to each other regardless of the GTX analog tested (Table 2). The resemblance in P_o,GTX for these Na⁺ channels is ascribed to similar single channel kinetics (τ_o in B and C, and τ_c in C and F are close to each other in Fig.6). Thus, it is thought that changes in P_ocannot explain the marked difference in GTX potency recorded in the whole-cell configuration that we observed.

Discussion

The present study has revealed the characteristics of GTX-evoked modification of Na⁺ channels and differences in the efficiency of GTX-evoked modification among three Na⁺ channel subtypes. First, GTX modifies the Na⁺ channels only when repetitive conditioning depolarizing pulses precedes a test pulse. Second, the Na⁺ channels modified by GTX can generate a slow sustained current that begins to flow at a membrane potential of −90 mV. Third, in the whole-cell variation of the patch clamp technique, the degree of GTX-evoked modification is measured as the relative maximum permeability for modified Na⁺ channels referred to that for Na⁺ channels without conditioning pulses. TTX-i Na⁺ channels (DRG cells) were the most sensitive to both GTX analogs. Other TTX-s and ventricular Na⁺ channels proved to be far less sensitive to GTX analogs. The most striking difference in the response of Na⁺ channels to GTX analogs was observed between the neuronal TTX-i and ventricular Na⁺channels, using α-dihydro-GTX-II to modify the channels. Fourth, to investigate the factors responsible for the observed difference in the response of DRG cells and cardiomyocytes to α-dihydro-GTX-II and GTX-I, the induced changes in single channel conductance andP_o was examined by the patch-clamp method. As summarized in Tables 1 and 2, there is virtually no difference in either parameter, after preparations were subjected to GTX-I or α-dihydro-GTX-II.

Generally, the whole-cell conductance (G) is expressed asN_total × P_o ×g, where N_total is the number of functioning Na⁺ channels in a cell, andg is the single-channel conductance. For the reader's convenience, the additional subscript attached to these symbols represents the experimental condition. The maximum conductance at the time of peak Na⁺ current in control (G_control) is expressed asN_total ×P_o,control ×g_control, and the whole-cell conductance of the Na⁺ channels under conditions in which all the channels are modified by GTX (G_GTX) isN_total× P_o,GTX × g_GTX. Thus, the relative maximum conductance,G_GTX/G_control, is calculated as (P_o,GTX ×g_GTX)/(P_o,control× g_control), where the values forN_total in the numerator and denominator were cancelled out. Because there is a large discrepancy between the calculated and the observed values of maximum normalized slope conductance (in Table 3) and all the parameters used to calculate the theoretical values were, with the exception of the N_total parameter, determined experimentally, it is reasonable to suppose that our starting assumption that all the Na⁺ channels were modified by GTX is no longer valid. Thus, a major factor responsible for the wide variation in the response to the two GTX analogs in the whole-cell recording is suggested to be the number of Na⁺ channels modified by GTX analogs.

From these calculations, the modified fraction of Na⁺ channels in neuronal TTX-i and ventricular Na⁺ channels has been estimated to be 42% and 21%, respectively, in 300 μM GTX-I, and 40% and 4%, respectively, in 300 μM α-dihydro-GTX-II. There is a large difference in potency among the GTX analogs used. For example, α-dihydro-GTX-II modified only 4% of Na⁺ channels in ventricular myocytes, whereas GTX-I modified 42% of TTX-i Na⁺ channels in DRG cells. The most likely explanation is that, once bound, these two biologically active GTX analogs had a similar capacity to modify Na⁺ channels, but the ease with which GTX analogs can bind and unbind at the site of action could differ. In support of this explanation, α-dihydro-GTX-II molecules unbind three times faster than GTX-I molecules from ventricular Na⁺channels. One could speculate that the marked difference in dissociation rates between GTX-I and α-dihydro-GTX-II is caused by the different molecular species occupying the C-14R position (Tsuji et al., 1991): GTX-I presents an acetyl group and α-dihydro-GTX-II presents a hydroxyl group. The more hydrophilic hydroxyl group (or its smaller molecular radius) could promote more rapid dissociation of α-dihydro-GTX-II from its binding site.

It has been shown that the primary structure of rat DRG TTX-i Na⁺ channels is akin to that of rat cardiac Na⁺ channels (Akopian et al., 1996), and the response to biological toxins, such as TTX, venoms from Australian funnel-web spiders (Nicholson et al., 1994), and μ-conotoxin GIIIA is much stronger in TTX-s Na⁺ channels than that in TTX-i Na⁺ channels or cardiac Na⁺ channels (Moczydlowski et al., 1986). On the contrary, both GTX analogs give rise to more pharmacologically conspicuous response in DRG TTX-i Na⁺ channels compared with those in DRG TTX-s and cardiac Na⁺channels. Because Yamaoka (1987) showed that frog ventricular Na⁺ channels are suppressed by TTX with an IC₅₀ value of 27 nM, which is 2 orders of magnitude lower than the IC₅₀ value of several micromolar for the mammalian ventricular Na⁺channels, frog ventricular Na⁺ channels are pharmacologically close to the group of TTX-s Na⁺ channels.