Introduction

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The voltage-gated Na⁺ channels are membrane proteins responsible for the generation of action potentials in excitable membranes. In mammalian cells, the Na⁺ channel family contains one large  subunit and one or two smaller  auxiliary subunits (1, 2) (Catterall, 2000). The -subunit clone consists of about 2000 amino acids and, when expressed alone in mammalian cells, exhibits functional Na⁺ currents with gating kinetics comparable with those of the native Na⁺ channels. The -subunit primary sequence contains four homologous domains (D1-D4), each with six transmembrane segments (S1-S6) (Fig. 1A). Current structural models suggest that the Na⁺ channel is organized as a pseudotetramer with S6 segments possibly lining the internal vestibule of the pore.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   A, the transmembrane organization of the Na+ channel -subunit. The arrows indicate the putative BTX binding site and the putative LA binding site at segment D1-S6, D3-S6, and D4-S6. The role of D2-S6 in BTX and LA action is unknown as labeled by a question mark. P designates the pore region within the S5-S6 extracellular linker. B, amino acid sequences of S6 transmembrane segments in domain D1-D4. Residues critical for BTX action are in bold letters. Residues critical for LA binding are underlined. Notice that a total of seven common residues within D1-S6, D3-S6, and D4-S6 are critical for both BTX and LA action.

Local anesthetics (LAs) block voltage-gated Na⁺ channels in a complicated state-dependent manner. LAs seem to have higher affinities toward open and inactivated states (Strichartz, 1973; Courtney, 1975). In contrast, a steroidal alkaloid toxin, batrachotoxin (BTX), isolated from poison-dart frogs, inhibits both Na⁺ channel fast and slow inactivation and shifts the activation threshold to the hyperpolarizing direction (Khodorov, 1978). Consequently, the Na⁺ channel opens readily with BTX present, even at the resting membrane potential. The receptors for LAs and BTX have been mapped within S6 segments of domains D1, D3, and D4 with several common residues important for binding of both LAs and BTX (Fig. 1B; Ragsdale et al., 1994; Wang and Wang, 1998, 1999; Linford et al., 1998; Vedantham and Cannon, 2000; Wang et al., 2000). Furthermore, a quaternary derivative of lidocaine, QX-314, can access the LA receptor of the neuronal Na⁺ channels only via internal application (Strichartz, 1973). These findings together imply that S6 segments are closely aligned to form the internal vestibule of the Na⁺ permeation pathway. Detailed mapping of S6 segments may therefore reveal the architecture of this pore region.

The fact that LAs can accelerate the dissociation rate of BTX from its receptor (Postma and Catterall, 1984) suggests that both LAs and BTX can bind simultaneously. Consistent with this result, LAs block single, BTX-modified Na⁺ channels incorporated in lipid bilayers, apparently in a one LA to one BTX-modified channel relationship (Moczydlowski et al., 1986; Wang, 1988). The previous explanation for these observations is that BTX allosterically affects the binding affinity of LAs toward their receptor in Na⁺ channels and vice versa. However, recent studies based on site-directed mutagenesis now revise this view. It was suggested that different surfaces of common residues are important for BTX and LA binding (Linford et al., 1998) or, alternatively, that common residues are involved in BTX and LA binding in a state-dependent manner (Wang et al., 2000). Thus, studies of the involvement of S6 segments in BTX and LA actions will be essential for understanding the physiological consequences of these ligands.

In this report, we address two unsettled questions. First, we asked whether segment D2-S6 is involved in BTX action. We created a series of lysine mutants within this region and measured the mutants' BTX sensitivity. Two mutants became completely resistant to BTX modification. Second, we asked whether these BTX-resistant mutant channels also become resistant to LA. Surprisingly, they remained relatively sensitive to bupivacaine block.

	Materials and Methods

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Site-Directed Mutagenesis. Point mutations of a µ1 Na⁺ channel clone (Trimmer et al., 1989) in a pcDNA1/Amp expression vector were performed as described previously (Nau et al., 1999) by means of the Transformer Site-Directed Mutagenesis Kit (CLONTECH, Palo Alto, CA). A mutagenesis primer and a restriction primer were used to generate the desired mutant. The potential mutants were selected and confirmed by DNA sequencing at the mutated site with appropriate primers.

Transient Transfection. The culture of HEK293t cells and their transient transfection were performed as described previously (Cannon and Strittmatter, 1993). Cells were first grown to 50% confluence in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (HyClone, Logan, UT), 1% penicillin and streptomycin solution (Sigma, St. Louis, MO), 3 mM taurine, and 25 mM HEPES (Life Technologies). Transfection of these cells with µ1 (10 µg) and reporter plasmid CD8-pih3 m (1 µg) was accomplished by a calcium phosphate precipitation method in a Ti25 flask. Cells were replated 15 h after transfection, maintained at 37°C in a 5% CO₂ incubator, and used for experiments after 1 to 4 days, generally. However, mutant µ1-V787K usually did not express sufficient currents until day 4. Transfection-positive cells were identified by immunobeads (CD8-Dynabeads; Dynal, Lake Success, NY).

Whole-Cell Voltage Clamp. The whole-cell configuration of a patch-clamp technique (Hamill et al., 1981) was used to record Na⁺ currents in cells coated with CD8 immunobeads. Experiments were performed at room temperature (23 ± 2°C). Glass electrodes contained 100 mM NaF, 30 mM NaCl, 10 mM EGTA, and 10 mM HEPES adjusted to pH 7.2 with CsOH. The electrodes had a tip resistance of 0.5 to 1.0 M; access resistance was generally  2 to 3 M. With series resistance compensation of 60 to 90%, the voltage error at +50 mV was < 4 mV on average. Series resistance errors with this magnitude are generally tolerable because quantitative measurements of current kinetics and drug block are insignificantly affected by such errors (Bean, 1992). The bath solution contained 65 mM NaCl, 85 mM choline chloride, 2 mM CaCl₂, and 10 mM HEPES adjusted to pH 7.4 with tetramethyl hydroxide. These ionic conditions resulted in smaller Na⁺ currents at voltages from 60 to +10 mV, which in turn minimized the series resistance artifact in the conductance-voltage measurement. Stock solution of bupivacaine was prepared at 100 mM in aqueous solution and stored at 20°C until needed. BTX was prepared at 0.5 mM in dimethyl sulfoxide and stored at 4°C. Bupivacaine was purchased from Sigma, and BTX was a generous gift of Dr. John Daly (National Institutes of Health, Bethesda, MD). To conserve the use of BTX, we included this toxin in the pipette solution at 5 µM final concentration when needed. This toxin concentration was previously used to demonstrate the BTX-resistant phenotype in poison-dart frogs (Daly et al., 1980) and was high enough to modify >90% of available wild-type Na⁺ channels under appropriate conditions. Whole-cell currents were recorded with Axopatch 200B, filtered at 5 kHz, and collected by pClamp software (Axon Instruments, Foster City, CA). In some experiments, currents were recorded by EPC-7. After gigaohm seal formation and establishment of whole-cell voltage clamp, the cells were dialyzed for ~20 min before data were acquired. Most of the capacitance and leakage current were canceled by the patch-clamp circuitry and further subtracted by the P/4 method. An unpaired Student's t test was used to evaluate estimated parameters (mean ± SEM or fitted values ± SE of the fit); p values of < 0.05 were considered statistically significant.

	Results

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Expression and Gating of D2-S6 Mutant Channels. We created lysine mutants in the D2-S6 region of µ1-G783 to µ1-L795 residues. Four of these 13 mutants expressed sufficient Na⁺ currents in transfected HEK293t cells for further experiments; these mutants are N784K, L785K, V787K, and L788K (mutants of G783, L786, N789, L790, F791, L792, A793, L794, and L795 expressed poorly, generally <0.5 nA). The current-voltage families were recorded and the peak conductance/voltage curves were constructed (Fig. 2A, right and left, respectively). The activation parameters of these mutant channels were obtained by a curve-fitting program and are included in Table 1. The N784K and V787K mutants display V_0.5 values similar to those of wild-type (~35 mV), whereas L785K and L788K mutants show a significant shift of 5.2 mV and +19.4 mV, respectively (p < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   A, voltage dependence of activation. Mutant and wild-type Na+ currents were elicited by 4-ms test pulses ranging from 100 mV to +50 mV. Current traces were superimposed (top left). Pulses were delivered in 10-mV increments from a holding potential of 140 mV. Membrane conductance (GNa) was calculated from the equation GNa = INa/(Em  ENa), where INa is the peak current elicited by the test pulse, Em is the amplitude of the test pulse, and ENa is the reversal potential of the Na+ current. Data were normalized with respect to the maximal GNa (top right) and fitted to a Boltzmann equation: 1 / [1 + exp (V0.5  V / ka)]. The average midpoint voltage (V0.5) and slope factor (ka) for mutant and wild-type channels are shown in Table 1. B, steady-state fast inactivation (h). Na+ currents were evoked by a 3.6-ms test pulse to +50 mV after 100-ms conditioning pulses (EC) ranging between 150 mV and 25 mV. The EC were delivered in 5-mV increments from a holding potential of 140 mV. Pulses were administered at 10-s intervals. Current traces were superimposed (bottom left). Peak currents were measured at +50 mV, normalized with respect to the value at EC = 150 mV, plotted against the EC, and fitted to a Boltzmann equation (bottom right). The midpoint voltage for fast inactivation (h0.5) and the slope factor (kh) are shown in Table 1.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Prolonged depolarization causes slow inactivation of µ1-787K mutant channels. Cell membranes were kept at a holding potential of 140 mV and received 10-s conditioning pulses ranging in amplitude from 180 mV to 50 mV. A 100-ms interval to 140 mV preceded the 4.4-ms test pulse to +30 mV (see inset). Pulses were delivered at 40-s intervals. Representative current traces were superimposed for comparison (A). Peak currents were normalized to the current evoked by the conditioning pulse of 180 mV, plotted against conditioning voltage (B), and fitted to a Boltzmann equation [(A1  A2)/(1 + epx ((EC  s0.5) / ks)) + A2]. The midpoint voltage for inactivation (s) and the slope factor (k) for each channel are shown in Table 1. Notice that µ1-787K shows a profoundly enhanced slow-inactivation phenotype. C, repetitive pulses also reduce the peak current in the µ1-787K mutant (, n = 3) but not in the wild-type (, n = 4). A 24-ms pulse at +50 mV was applied at 2 Hz for a total of 100 pulses. The peak currents were measured, normalized with respect to the value of the first pulse, and plotted against the pulse number.

BTX-Resistant Phenotype of N784K and L788K. Despite profound gating changes, µ1-V787K mutant channels remained sensitive to BTX. When 5 µM BTX was included in the pipette solution, up to 1000 repetitive pulses produced BTX-modified Na⁺ currents that are not inactivated (Fig. 4C as in wild-type, 4A). The peak current amplitude of trace 1000P is about half the size of trace 30P, a result caused by the enhanced use-dependent inhibition of Na⁺ current during repetitive pulses. Similar to µ1-V787K, µ1-L785K mutant channels remained sensitive to BTX modification as shown in Fig. 4B. Thus, binding of BTX occurs with mutant channels despite the lysine substitution at these two positions.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   BTX-sensitive phenotypes of µ1-L785K and µ1-V787K mutants and wild-type. Repetitive pulses at +50 mV for 24-ms at 2 Hz as described in Fig. 3C were applied to cells transfected with wild-type channels (A), µ1-L785K (B), and µ1-V787K (C) mutant channels. The pipette solution contained 5-µM BTX. Outward Na+ currents were shown at 30, 100, 300, 600, and 1000 pulses, and the current traces were superimposed for comparison. Notice the increase in noninactivating currents and in tail current amplitude after repetitive pulses. Cells were dialyzed by internal solution for 10 to 20 min with infrequent test pulses to determine the peak current amplitude until it reached steady state. Five to six cells of each channel type were used.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   BTX-resistant phenotype of µ1-N784K and µ1-L788K mutant channels. A pulse protocol identical to that in Fig. 4 was applied to cells transfected with µ1-784K (A, left) and µ1-788K (B, left). Notice the minimal amount of noninactivating currents and tail current after repetitive pulses. In contrast, µ1-N784C and µ1-L788A mutant channels exhibit a BTX-sensitive phenotype (A and B, right). The pipette solution contained 5-µM BTX. Outward Na+ currents were superimposed for comparison. Five to six cells for each mutant were used. Peak currents in N784K and N784C mutants are significantly reduced during repetitive pulses.

Bupivacaine Sensitivity of Mutants 784K, 785K, 787K, and 788K. To compare the bupivacaine sensitivity of mutant channels with that of wild-type channels, we used a pulse protocol (detailed in Fig. 3A) to measure the voltage-dependent binding of bupivacaine from 180 mV to 50 mV. A conditioning pulse at various voltages was first applied for 10 s to allow the drug binding to reach the steady state, followed by an interpulse of 140 mV for 100 ms, which allowed drug-free inactivated channels to return to their resting state. The bupivacaine-bound channels recover rather slowly with a time constant of 2 to 4 s at 140 mV (Nau et al., 1999). Finally, a test pulse of +30 mV was applied to activate the drug-free resting channels. Figure 6 shows the results for the wild-type channels using this pulse protocol in the absence of 100 µM bupivacaine (Fig. 3B, dashed line). In the presence of 100 µM, at conditioning voltages of 180 to 140 mV, the bupivacaine block was constant about 45% (Fig. 6, ). This low-affinity bupivacaine block corresponds to the block of the resting state of Na⁺ channels. The bupivacaine block increased progressively from voltage 130 to 80 mV until it reached a constant level of ~95% from 70 to 50 mV. This high-affinity bupivacaine block is probably caused by the block of the inactivated state of Na⁺ channels. Previous studies have shown that BTX-resistant mutant channels in general have reduced inactivated affinity toward LAs (Nau et al., 1999; Wang and Wang, 1999; Wang et al., 2000). In contrast to this general rule, both 784K and 788K BTX-resistant mutant channels still display comparably high (inactivated) affinity toward bupivacaine. Figure 6 ( and , respectively) shows the voltage-dependent binding of bupivacaine block in these mutant channels. The s values are listed in Table 1 along with the wild-type value.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Steady-state block of wild-type and mutant Na+ channels by 100 µM bupivacaine. The same pulse protocol as described in Fig. 3A was used in the absence of drug (dashed line for wild-type), and the protocol was repeated after attaining tonic inhibition by 100 µM bupivacaine. Peak currents for each channel were normalized with respect to the control value at the conditioning voltage of 180 mV, plotted against conditioning voltage, and fitted to a Boltzmann equation. The s and k for the control and drug-bound channels are shown in Table 1 along with h0.5 and kh values.

Dose-Response Curve for K_R and K_I Measurements of Mutant Channels. Knowing the voltage range for the measurement of resting affinity (K_R) and inactivated affinity (K_I) in these lysine mutants, we then proceeded to determine their K_R and K_I values directly at various bupivacaine concentrations at either 70 mV or 160 mV conditioning voltage (Fig. 7). The K_R and K_I values were estimated by curve fitting and are listed in Table 2. Hill coefficient was near unity (p = 1.0 to 1.3) for wild-type and mutant channels, suggesting that there is only one LA binding site within the -subunit Na⁺ channel protein. Our results from dose-response curves further suggest that there are no direct interactions between bupivacaine and residues 784, 785, and 788 within the D2-S6 region. The K_I value for 787K cannot be determined because of its enhanced inactivation gating at 70 mV (Fig. 3, A and B), nor can the K_R value for µ1-V787K be estimated accurately. Although the estimated K_R value for µ1-V787K at 160 mV seems significantly lower than that of the wild-type (Fig. 7B; Table 2), this phenomenon could be caused by inactivated channels at 160 mV conditioning pulse (Fig. 3B).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   State-dependent block of µ1 wild-type and mutant Na+ channels by bupivacaine. The same pulse protocol as described in Fig. 3A was used with conditioning pulses to 70 mV for block of inactivated channels (A) and 160 mV for block of resting channels (B). Pulses were delivered at 30-s intervals. The peak current for each drug concentration was measured, normalized to the peak current in the absence of drug, and plotted against drug concentration. The data were fitted to the Hill equation; the IC50 values and the Hill coefficient (p) are listed in Table 2.

	Discussion

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There are two major findings of this report. First, two residues (µ1-N784K and µ1-L788K) within segment D2-S6 of -subunit Na⁺ channels are critical for BTX action but not the two adjacent residues (µ1-L785K and µ1-V787K). Substitution with lysine in either the µ1-L784 or the µ1-V788 position renders the channels completely resistant to 5 µM BTX. Second, although these two specific residues are critical for BTX action, they remain sensitive to bupivacaine block. Similar bupivacaine sensitivities are found in the two mutants µ1-L785K and µ1-V787K. Therefore, no evidence of a strong or a direct contact is found between the D2-S6 residues and LAs.

Implications about BTX Action. Thus far we have identified a cluster of residues at the S6 segments of all four domains that seem to be important for the action of BTX (Fig. 1B). These residues are located near the middle region (position 13 to 21; Fig. 1B) of the S6 transmembrane segment, which contains a total of ~28 amino acids and presumably forms an -helical structure (Lipkind and Fozzard, 2000). The physiological consequences of BTX poisoning are paralysis, convulsion, and death of the animal. BTX inhibits both fast and slow inactivation of the wild-type Na⁺ channel and shifts the voltage dependence of the activation process toward the hyperpolarizing direction, which in turn permits the channel to open persistently even at resting membrane potentials. It is probable that structural perturbations of D2-S6 segment via a lysine point mutation occur either distantly or locally in µ1-N784K and µ1-L788K mutants, which in turn results in the BTX-resistant phenotype. Precisely how the side chain and the -carbon backbone structural perturbations cause the BTX-resistant phenotype in µ1-N784K and µ1-L788K but not µ1-N784C, L788A, L785K, and V787K mutants remains to be studied.

Implications about LA and Insecticide Actions. Although D1-S6, D3-S6, and D4-S6 segments were previously shown to interact with LAs, no such strong interactions have been detected so far between the D2-S6 segment and the LA drugs. Except in D2-S6, all BTX-resistant Na⁺ channels display a greatly reduced binding affinity toward LAs (Fig. 1B). In general, a reduction in affinity of ~10- to 20-fold was found for the inactivated state in these channels (Nau et al., 1999; Wang and Wang, 1999; Wang et al., 2000). In contrast, the LA binding affinity of inactivated BTX-resistant Na⁺ channels with mutations in D2-S6 segments is quantitatively less affected, ~2.6-fold or less (K_I values of 4.5 µM for wild-type and 11.8 µM for L788K mutant channels). This reduction could be caused by a long-range effect of the positive charge from the lysine residue. Together, these results suggest that the residues within D2-S6 are not as near the bound LA molecule in situ as those within the three other S6 segments. Therefore, the D2-S6 segment represents a unique domain that is critical for BTX but not for LA action. The fact that the LA drugs are smaller molecules than the toxin BTX may be the physical reason why they interact with D1-S6, D3-S6, and D4-S6 but not D2-S6, even in the inactivated state.