Introduction

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Since the pioneering work of Numa's group (Noda et al., 1984), a wide variety of cloned sequences of Na⁺ channels have been obtained from different organs in the same species as well as from identical organs among different species. As a new family of Na⁺ channels, Akopian et al. (1996) have been able to isolate and clone a new type of tetrodotoxin-insensitive (TTX-i) Na⁺ channel from dorsal root ganglion (DRG) neurons. In TTX-i Na⁺ channels of rat DRG cells, 65% of an open reading frame encoding a 1957-amino-acid protein was shown to be identical with that of the rat cardiac TTX-i Na⁺ channel. TTX-i Na⁺ channels have been reported to retain unique pharmacological properties that are qualitatively as well as quantitatively different from those of TTX-sensitive (TTX-s) Na⁺ channels. It has been shown that versutoxin and robustoxin purified from the venoms of Australian funnel-web spiders have no effect on TTX-i Na⁺ channels but produce a dose-dependent slowing or removal of sodium channel inactivation and a reduction in peak I_Na in TTX-s Na⁺ channels (Nicholson et al., 1994, 1998). TTX-s Na⁺ channels are also more susceptible to local anesthetics than TTX-i Na⁺ channels (Scholz et al., 1998b): IC₅₀ for the tonic block of TTX-i Na⁺ channels by lidocaine was 210 µM, whereas TTX-s Na⁺ channels showed an IC₅₀ value five times lower at 42 µM. On the contrary, Scholz et al. (1998a) have shown that halothane suppresses fast and slow TTX-i Na⁺ channels with IC₅₀ values of 5.4 and 7.4 mM, whereas it suppresses TTX-s Na⁺ channels with a slightly higher IC₅₀ value of 12.1 mM. Ethanol at the concentration of 200 mM suppresses maximal available TTX-i Na⁺ channels by 18% and TTX-s Na⁺ channels by 7% (Wu and Kendig, 1998).

Some of the biological toxins that act selectively on sodium channels have been used extensively as useful tools for analyzing the characteristics of Na⁺ channel-gating processes. Batrachotoxin (BTX), grayanotoxin (GTX), veratridine, and aconitine, classified as toxins binding to site 2 (Catterall, 1980), are endowed with some characteristics in common: 1) they bind to the sodium channel in its open state, 2) the modified sodium channel loses the inactivation process, and 3) the activation voltage of the modified sodium channel is shifted in the direction of hyperpolarization (Khodorov, 1985; Narahashi and Herman, 1992).

Thus, it is worth examining how different Na⁺ channel isoforms respond to GTX analogs to obtain information on the molecular structure of the gating mechanism of Na⁺ channel. In this study, we compared the pharmacological action of GTX analogs on TTX-s and TTX-i Na⁺ channels in DRG cells and heart Na⁺ channels of the frog, and have thereby obtained novel information about the difference in GTX actions among these Na⁺ channel isoforms and the interaction of GTX analogs with their binding sites on the intracellular aspect of Na⁺ channels.

	Materials and Methods

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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.

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.

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View larger version (19K): [in this window] [in a new window]   Fig. 1.   Structure of GTX-I and -dihydro-GTX-II.

Ionic Current Recordings. Whole-cell patch pipettes with a resistance of less than 2 M 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).

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 IC₅₀ value 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

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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): ₁ = 1.4 ± 0.5 s, ₂ = 18.6 ± 1.8 s (n = 3) for GTX-I and ₁ = 0.5 ± 0.2 s, ₂ = 5.4 ± 0.8 s (n = 3) for -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.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Time course for development and decay of modified currents through Na+ channels. The internal solution contained 100 µM GTX-I. A, change in sustained currents after applying conditioning pulses are presented as the function of numbers of conditioning depolarizing pulses (). Conditioning pulses to 0 mV of 5 ms in duration from holding potential of 120 mV with interpulse interval of 13 ms.  at the very beginning of record represents sustained current at 120 s after the termination of conditioning pulses. B, ventricular cells were subjected to 1000 repetitive depolarizing pulses having the same qualities of conditioning pulses as in A. The resultant sustained currents in the response to test pulse to 80 mV were plotted against various time intervals after conditioning pulses.

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 -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 -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.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effect of 300 µM -dihydro-GTX-II on macroscopic Na+ currents recorded from DRG cells and ventricular myocytes. A-C, family of Na+ currents associated with step depolarizations from 80 to +20 mV with a 20 mV step from a membrane potential of 140 mV is depicted under control conditions (without prepulses; upper row) and after GTX-evoked modification (with repetitive conditioning pulses; middle row). Current-voltage relationships are shown for control peak Na+ currents () and for steady-state (modified) currents evoked by conditioning repetitive pulses in GTX ()(bottom row). The dashed lines drawn were calculated by the GHK equation (see under Dose-Response Relationship for GTX-Evoked Modification). A, currents through TTX-i Na+ channels from DRG cells. B, currents through TTX-s Na+ channels in DRG cells. C, currents through Na+ channels of ventricular myocytes. Pulse protocol for GTX-evoked channel modification is shown as inset (C; bottom row); the conditioning parameters, x, y, and n, are 8, 6, and 150 for A; 8, 3, and 300 for B; and 13, 5, and 1000 for C. Repetitive conditioning pulses for GTX-evoked channel modification always preceded each test pulse. Step test pulses (10 mV) of 160-ms duration were applied after holding the membranes at 140 mV for 10 ms. Interval between each combination of conditioning and test pulses ranged from 5 to 120 s.

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 constantthe ratio of permeability of control versus that of modified Na⁺ channelis 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 -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% (-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:

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Dose-response curves plotting normalized slope conductance of GTX-modified Na+ channels against the concentration of GTXs. Solid line for DRG TTX-i Na+ channels are curves of best fit to experimental data with EC50 = 31 µM (A), and EC50 = 54 µM for GTX-I  dihydro-GTX-II (B), respectively. Calculated maximal normalized slope conductance of TTX-i is 0.23 for GTX-I and 0.37 for -dihydro-GTX-II, respectively. The dashed lines were drawn by eye. Data, in most instances, were taken from three to six cells.

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.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Determination of single-channel conductance for control and modified Na+ channels in a ventricular myocyte. A, sequential current records for a patch-clamped Na+ channel in response to a voltage ramp, generated according to the protocol illustrated (inset); currents through the unmodified channels are shown in left column and those through the modified Na+ channels in the right column. In B, two records in which a single unmodified channel opened during rising phase of voltage ramp () were superimposed; a straight line was fitted to the data. In C, a record of the modified current, obtained during the falling phase of ramp pulse (), is displayed graphically along with the line of best fit. Slope of fitted lines directly gives the single channel conductance.

Effect of GTX Analogs on the P_o. Treatment with 100 µM -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.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Analysis of single-channel currents modified by -dihydro-GTX-II obtained in neuronal (DRG) TTX-i Na+ channel (A, B, and C) or ventricular Na+ channel (D, E, and F). A and D, representative single-channel current records from DRG TTX-i (A) and ventricular (D) cells. Symbol c (in A and D) marks the closed channel (zero current) level. B and E, open time histograms from single-channel recordings, including segments shown in A and D, were fitted by a single exponential function with indicated time constants. C and F, closed time histograms from single-channel recordings, including segments shown in A and D, were fitted either by a single exponential or by the sum of two exponentials, as appropriate, with indicated time constants. Membrane potentials in each cell-attached experiment were 0.7 ± 1.4 mV (n = 3) for DRG cells and 2.7 ± 2.0 mV (n = 3) for ventricular cells using glass microelectrodes.

	Discussion

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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 -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 -dihydro-GTX-II and GTX-I, the induced changes in single channel conductance and P_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 -dihydro-GTX-II.

Generally, the whole-cell conductance (G) is expressed as N_total × P_o × g, where N_total is the number of functioning Na⁺ channels in a cell, and g 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 as N_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) is N_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 for N_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 -dihydro-GTX-II. There is a large difference in potency among the GTX analogs used. For example, -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, -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 -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 -dihydro-GTX-II presents a hydroxyl group. The more hydrophilic hydroxyl group (or its smaller molecular radius) could promote more rapid dissociation of -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.