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Evidence for Drug Binding Along the Activation Pathway

Sho-Ya Wang¹, Jane Mitchell³, Edward Moczydlowski², and Ging Kuo Wang³

¹ Department of Biology, State University of New York at Albany, Albany, NY 12222
² Department of Biology, Clarkson University, Potsdam, NY 13699
³ Department of Anesthesia, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115

Address correspondence to Ging Kuo Wang, Dept. of Anesthesia, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Fax: 617730-2801; email:wang{at}zeus.bwh.harvard.edu

	ABSTRACT

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According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na⁺ channels with higher affinities. However, an alternative view suggests that activation of Na⁺ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na⁺ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC₅₀) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 µM (n = 5) and 81.7 ± 10.6 µM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC₅₀ values for resting-channel block at –140 mV were >12-fold higher than those for open-channel block. With 300 µM benzocaine, rapid time-dependent block ( 0.8 ms) of inactivation-deficient Na⁺ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at –30 mV. The peak current at –30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na⁺ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.

Key Words: sodium channel • fast inactivation • persistent sodium currents • local anesthetics • modulated receptor hypothesis

	INTRODUCTION

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The in vivo targets of local anesthetics (LAs) are voltage-gated Na⁺ channels responsible for the generation of action potentials in excitable membranes (Hille, 2001). Molecular cloning has thus far identified nine Na⁺ channel -subunit isoforms in mammals. The -subunit isoforms contain four repeated domains (D1–D4), each with six transmembrane domains (S1–S6) and can be functionally expressed in frog oocytes or in mammalian expression systems (Catterall, 2000). LAs interact with residues in the middle of D4S6, D3S6, and D1S6 segments, probably situated within the inner cavity of the Na⁺ permeation pathway (Ragsdale et al., 1994; Wang et al., 1998, 2000; Yarov-Yarovoy et al., 2001, 2002).

The block of Na⁺ currents by lidocaine and benzocaine has been studied extensively. Lidocaine contains a tertiary amine group, which can be protonated in the aqueous solution. In contrast, benzocaine is a neutral LA. Lidocaine not only elicits a tonic block of Na⁺ currents when infrequently stimulated but also produces an additional use-dependent block during repetitive pulses (Hille, 2001). On the other hand, benzocaine fails to produce any use-dependent block. As a neutral drug, benzocaine may escape too rapidly during the interpulse to accumulate such a block. However, both drugs significantly shift the steady-state inactivation of Na⁺ channels to the hyperpolarizing direction as if LAs stabilize the inactivated state. Hille (1977) proposed a modulated receptor hypothesis to account for the complicated block of LAs. His hypothesis states that the LA receptor changes its conformation during state transitions and that the fast-inactivated state has a higher LA affinity than does the resting state. A similar hypothesis (Hondeghem and Katzung 1977, 1984) suggested that a class 1 antiarrhythmic agent, lidocaine, may additionally block open cardiac Na⁺ channels with a higher affinity.

To date, the proof of the high-affinity open-channel block by LAs remains controversial. Several pieces of evidence indicate that the open-channel block by lidocaine plays a minimal role in the use-dependent block. For example, both Cahalan (1978) and Yeh (1978) reported that QX-314, a quaternary ammonium derivative of lidocaine, failed to block inactivation-deficient Na⁺ currents in pronase-treated squid axons. Their results supported the conclusion that the inactivated state plays a dominant role in the use-dependent block of Na⁺ currents by LAs. Bean et al. (1983) later found that lidocaine binds strongly to the inactivated state of cardiac Na⁺ channels with an estimated dissociation constant of 10 µM, a value within the therapeutic plasma concentration (6.4–21.3 µM). Recent experiments with inactivation-deficient mutant Na⁺ channels (IFM/QQQ) also show that the inactivated state has a higher LA affinity than does the open state (Bennett et al., 1995; Grant et al., 2000; O'Leary and Chahine, 2002).

Wang et al. (1987), however, reported that QX-314 retains its potency on inactivation-deficient Na⁺ currents in squid axons treated with chloramine-T and suggested that the open-channel block plays a significant role in the use-dependent block. Vedantham and Cannon (1999) found that the lidocaine-induced slowing of Na⁺ channel repriming was not the result of a slowing of recovery of the fast-inactivation gate. Their finding indicates that use-dependent block does not involve an accumulation of fast-inactivated channels. Moreover, various class 1 antiarrhythmic agents, such as lidocaine, flecainide, amiodarone, and mexiletine, potently block persistent late Na⁺ currents either derived from genetic diseases or under pathological conditions (Ju et al., 1992; An et al., 1996; Wang et al., 1997, 1999; Nagatomo et al., 2000; Maltsev et al., 2001).

To investigate the possible role of the activation pathway in LA block by benzocaine and lidocaine, we chose to study LA actions on stably transfected HEK293 cells expressing inactivation-deficient rat skeletal muscle Na⁺ channels, rNav1.4-L435W/L437C/A438W (WCW mutant; Wang et al., 2003a). The point mutations of WCW mutant channels are at the positions 19, 21, and 22 of the D1S6 COOH terminus. The advantage of WCW mutant Na⁺ channels is that, unlike the IFM/QQQ mutant cardiac Na⁺ channels (Grant et al., 2000), they are well expressed in HEK293 cells. A large persistent late Na⁺ current in HEK293 cells allows direct examinations of the open-channel block by LAs. The disadvantage of this approach is that any mutations or chemical/enzymatic treatments in theory could alter indirectly or directly the structure of the lidocaine/benzocaine receptor. Such receptor alteration could also occur in studies of late Na⁺ currents under pathological conditions or by S6 mutations that cause genetic diseases (Takahashi and Cannon, 2001). Despite this uncertainty, our result suggests an important therapeutic role of the open-channel block by lidocaine in vivo. To our surprise, we found that benzocaine blocks the open channel potently at +30 mV as well as the closed intermediate states at –30 mV.

	MATERIALS AND METHODS

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HEK293 Cells with Stably Transfected rNav1.4-L435W/L437C/A438W cDNA
Mutagenesis of rNav1.4-L435W/L437C/A438W cDNA in the pcDNA3 vector was performed as described previously (Wang et al., 2003a). Cultured HEK293 cells were maintained at 37°C in a 5% CO₂ incubator in DMEM (Life Technologies) containing 10% FBS (HyClone), and 1% penicillin and streptomycin solution (Sigma-Aldrich). The HEK293 cells were transfected with the mutant clone in pcDNA3 vector (10 µg) by a calcium phosphate precipitation method. Transfected HEK293 cells were treated with 1 mg/ml G-418 (Invitrogen) in 100-mm culture dishes, and individual G-418 resistant colonies were isolated using glass cylinders (i.d. = 6 mm) 2 wk after transfection as described previously (Wang et al., 2004a). One colony was frozen, later reestablished, and maintained in Ti-25 flasks without the addition of G-418 for all studies described here.

Electrophysiology and Data Acquisition
The whole-cell configuration of a patch-clamp technique (Hamill et al., 1981) was used to study Na⁺ currents in HEK293 cells at room temperature (22 ± 2°C). Electrode resistance ranged from 0.5 to 1.0 M. Command voltages were elicited with pCLAMP8 software and delivered by Axopatch 200B (Axon Instrument) or by EPC-7 (List Electronics). Cells were held at –140 mV and dialyzed for 10–15 min before current recording. Most of the capacitance and leak currents were cancelled with a patch-clamp device and by P/–4 subtraction. Liquid junction potential was not corrected. Peak currents at +30 mV were 2–20 nA for the majority of cells. Access resistance was 1–2 M under the whole-cell configuration; series resistance compensation of >85% typically resulted in voltage errors of 4 mV at +50 mV. Most dose–response studies were performed at +30 mV for the outward Na⁺ currents. Such recordings allowed us to avoid the complication of series resistance artifacts and to minimize inward Na⁺ ion loading (Cota and Armstrong, 1989). Curve fitting was performed by Microcal Origin. An unpaired Student's t test was used to evaluate estimated parameters (mean ± SEM or fitted value ± SE of the fit); P values of <0.05 were considered statistically significant.

Solutions and Chemicals
Lidocaine-HCl and benzocaine were purchased from Sigma-Aldrich. Drugs were dissolved in DMSO at 100 mM as stock solutions and stored at 4°C. Final drug concentrations were made by serial dilution. The highest DMSO concentration in the bath solution (1%) had little effect on Na⁺ currents. Cells were perfused with an extracellular solution containing (in mM) 65 NaCl, 85 choline-Cl, 2 CaCl₂, and 10 HEPES (titrated with tetramethylammonium-OH to pH 7.4). In some experiments, we used the Na⁺-free extracellular solution by replacing 65 mM NaCl with 65 mM choline-Cl. The pipette (intracellular) solution consisted of (in mM) 100 NaF, 30 NaCl, 10 EGTA, and 10 HEPES (titrated with cesium-OH to pH 7.2).

	RESULTS

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A Hek293 Cell Line Expressing Robust rNav1.4-WCW Inactivation-deficient Na⁺ Currents
Fig. 1 A shows the Na⁺ currents at various membrane potentials from a cell stably transfected with rNav1.4-WCW mutant channels. The inactivation-deficient Na⁺ currents were activated at –50 or –60 mV. The peak currents were measured and the peak conductance/voltage relationship was then plotted (Fig. 1 B). The result shows that the activation parameters (midpoint voltage, V_0.5 = –31.7 ± 1.2 mV, and slope factor, k = 9.6 ± 1.0 mV, n = 6) for the WCW mutant channels are similar to those of wild-type counterparts (P > 0.05; V_0.5 = –32.0 ± 0.9 mV; k = 8.7 ± 0.8 mV; Wang et al., 2003a). The level of expression of this WCW-transfected cell line was relatively high with 407 ± 32 pA/pF at +50 mV (n = 31), compared with 250 pA/pF in transiently transfected cells expressing the wild-type rNav1.4 channels (Nau et al., 2003). The high level of Na⁺ channel expression in this cell line remained stable for several months.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Activation of rNav1.4-WCW mutant Na+ channels. (A) Currents were evoked by 50-ms test pulses from –100 to +50 mV in 10-mV increments. (B) Normalized membrane conductance (gm) was plotted against the voltage. gm was calculated from the equation gm = INa/(Em – ENa), where INa is the peak current, Em is the amplitude of the voltage step, and ENa is the estimated reversal potential of the Na+ current. Plots were fitted with a Boltzmann function (1/[1 + exp((V0.5 – V)/k)]). The average midpoint voltage (V0.5) and slope (k) were –31.7 ± 1.2 mV and 9.6 ± 1.0 mV, respectively (n = 6). Holding potential was set at –140 mV.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Conventional steady-state inactivation measurement (h) of rNav1.4-WCW mutant Na+ channels. (A) Currents were evoked by a 5-ms test pulse (Vtest) at +30 mV. Test pulses were preceded by 100-ms conditioning pulses (Vcon), increasing in 5-mV increments from –160 to –15 mV (inset). (B) Normalized availability of rNav1.4-WCW mutant channels plotted as a function of the conditioning pulse voltage. The plot was fitted with a Boltzmann function. The average midpoint (V0.5) and slope (k) were –36.2 ± 0.9 mV and 8.7 ± 0.6 mV, respectively (n = 7). Holding potential was set at –140 mV.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Block of rNav1.4-WCW mutant currents by lidocaine. (A) Representative current traces are shown at various lidocaine concentrations. Cells were depolarized by a 50-ms test pulse at +30 mV. Pulses were delivered at 60-s intervals. (B) Peak and maintained late currents at the end of the test pulse as shown in A were measured at various lidocaine concentrations. Data were normalized to the control saline response and fitted with the Hill equation. The IC50 value for the peak current was 314 ± 25 µM (Hill coefficient, 0.8 ± 0.1) (, n = 5). For the maintained late current the IC50 was 20.9 ± 3.3 µM (0.9 ± 0.1) (, n = 5).

Concentration- and Time-dependent Block of Inactivation-deficient Na⁺ Currents by Benzocaine
Previous reports all indicate that benzocaine effectively stabilizes the inactivated sodium channels (Hille, 2001). To investigate the possible open-channel block by this drug, we applied benzocaine to inactivation-deficient mutant channels. The superimposed WCW mutant Na⁺ currents are shown without and with various concentrations of benzocaine (Fig. 4 A; 10 µM to 1 mM). Beside concentration-dependent block, we unexpectedly found that benzocaine also elicited a time-dependent block of mutant currents similar to the phenotype produced by lidocaine (Fig. 3 A). Benzocaine was evidently capable of blocking the open state of mutant channels.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Block of rNav1.4-WCW Na+ currents by benzocaine. (A) Representative current traces are shown at various benzocaine concentrations. Cells were depolarized by a 50-ms test pulse at +30 mV. Pulses were delivered at 60-s intervals. (B) Peak currents and maintained late currents at the end of the test pulse as shown in A were measured at various benzocaine concentrations. Data were normalized to the control saline response and fitted with the Hill equation. The IC50 value for the peak current was 1.16 ± 0.05 mM (Hill coefficient, 0.9 ± 0.1) (, n = 5). For the maintained current, the IC50 was 81.7 ± 10.7 µM (0.9 ± 0.1) (, n = 5).

Recovery from the Open-channel Block
We measured the recovery time course from the open-channel block by lidocaine and benzocaine. Most of mutant channels are noninactivating after a 30-mV pulse for 50 ms (Fig. 5 A, circle) and can be reactivated rapidly. With 100 µM lidocaine, the recovery time course at holding potential (–140 mV) followed a two-exponential function (square). The slow component (40.2%) had a time constant of 292 ± 19 ms (n = 5), whereas the fast component had a time constant of <5 ms. This slow recovery time constant for lidocaine block is slower than that found for the inactivated rNav1.4 wild-type Na⁺ channels (179 ± 17 ms; Wright et al., 1997; P < 0.05). With 300 µM benzocaine, most of mutant channels recovered quickly with a fast time constant of <5 ms (triangle) while a small component (9%) recovered with a time constant of 220 ms.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Recovery time courses from the open-channel block and additional use-dependent block by lidocaine and benzocaine. (A) Recovery time courses without (•) and with the open-channel block by lidocaine () and benzocaine () at the holding potential are plotted against normalized currents. The pulse protocol is shown in inset. The fitted values and number of experiments are given in the text. (B) Additional use-dependent block by lidocaine (top) but not by benzocaine (middle) is found in the second pulse. The pulse protocol is shown at the bottom. Peak currents before and after LAs are normalized and the mean values are given in the text.

Minimal Effects of External Na⁺ Ions on Lidocaine or Benzocaine Block
Barber et al. (1992) reported that lidocaine block was not affected by external Na⁺ ion concentrations ranging from 20 to 150 mM. In our experiments, the concentration of Na⁺ ions in the external solution was 65 mM. We therefore tested whether complete removal of Na⁺ ions from the external solution has any effects on lidocaine and benzocaine block of mutant currents. Without external Na⁺ ions, the IC₅₀ values for the resting- and open-channel block are 324 ± 26 µM (with Hill coefficient of 0.97 ± 0.07, n = 5; Fig. 6 A, square) and 17.6 ± 3.2 µM (0.63 ± 0.07, n = 5; triangle) for lidocaine and 1.15 ± 0.07 mM (0.92 ± 0.04, n = 5; Fig. 6 B, square) and 107 ± 6 µM (0.88 ± 0.04, n = 5; triangle) for benzocaine. These IC₅₀ values are not significantly different from those obtained in the presence of external Na⁺ ions at 65 mM (Figs. 3 and 4; P > 0.05). We conclude that external Na⁺ ions have minimal effects on the block of mutant currents by benzocaine or by lidocaine.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Block of rNav1.4-WCW Na+ currents by lidocaine or by benzocaine without external Na+ ions. Cells were depolarized by a 50-ms test pulse at +30 mV. Pulses were delivered at 60-s intervals. Holding potential was set at –140 mV. (A) Dose–response curve of lidocaine is shown without external Na+ ions. Peak currents from the beginning of the test pulse and maintained late currents at the end of the pulse were measured as described in Fig. 3. Data were normalized to the control, plotted against lidocaine concentration, and fitted with the Hill equation. The IC50 value for the peak current was 324 ± 26 µM (Hill coefficient, 0.97 ± 0.07) (, n = 5). The IC50 for the maintained late current was 17.6 ± 3.2 µM (0.63 ± 0.07) (, n = 5). (B) Dose–response curve of benzocaine is shown without external Na+ ions. Peak and maintained late currents were measured as described in Fig. 4, normalized, plotted against benzocaine concentration, and fitted with the Hill equation. The IC50 for the peak current was 1.45 ± 0.07 mM (0.86 ± 0.03) (, n = 5). The IC50 for the maintained late current was 107 ± 6 µM (0.88 ± 0.04) (, n = 5).

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Current–voltage relationship in the presence of 300 µM benzocaine. (A) A representative family of Na+ currents were evoked by 50-ms test pulses increasing in 10-mV increments from –100 to +50 mV (pulse protocol shown in inset) in the presence of 300 µM benzocaine. Holding potential was set at –140 mV. Control data similar to those illustrated in Fig. 1 A in the absence of drug were not shown. (B) Peak currents for control () and 300 µM benzocaine () were plotted against membrane voltage. (C) Both peak (, n = 4) and persistent (, n = 4) late currents at the end of the test pulse as shown in A were measured, normalized to peak currents measured from the same cell in control saline (Idrug/Icontrol), and plotted against membrane voltage.

View larger version (17K): [in this window] [in a new window]   FIGURE 8. Current–voltage relationship in the presence of 100 µM lidocaine. (A) A representative family of Na+ currents were evoked by 50-ms test pulses increasing in 10-mV increments from –100 to +50 mV (inset) in the presence of 100 µM lidocaine. Control data similar to those illustrated in Fig. 1 A without drug were not shown. (B) Peak currents for control () and 100 µM lidocaine () were plotted against membrane voltage. (C) Both peak (, n = 5) and persistent (, n = 5) late currents as shown in A were measured, normalized to peak currents measured from the same cell in control saline (Idrug/Icontrol), and plotted against membrane voltage.

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Conventional h measurement of rNav1.4-WCW Na+ channels with LAs presence. (A) Superimposed current traces were evoked by a 5-ms test pulse to +30 mV in the presence 300 µM benzocaine. Test pulses were preceded by 100-ms conditioning pulses, ranging from –160 to –15 mV in 5-mV increments (inset). Notice that Na+ currents were activated at the conditioning pulse >–50 mV. (B) Peak currents at the test pulse of +30 mV were measured, normalized, and plotted as a function of the conditioning voltage. The plot was fitted with a Boltzmann function (1/[1 + exp((V0.5 – V)/k)]). The average midpoint (V0.5) and slope (k) for the control were –36.0 ± 1.77 mV and 8.2 ± 1.2 mV, respectively (, n = 5), and for the cells treated with benzocaine were –70.5 ± 0.4 mV and 11.3 ± 0.3 mV, respectively (, n = 5). (C) Currents were evoked as described (A) in the presence of 300 µM lidocaine. (D) Normalized Na+ current availability, plotted as in B, and fitted with a Boltzmann function. The average midpoint (V0.5) and slope (k) for the control were –35.3 ±1.5 mV and 7.3 ± 1.0 mV, respectively (, n = 6), and for cells treated with lidocaine were –62.3 ± 0.3 mV and 8.2 ± 0.3 mV, respectively (, n = 6). All cells were perfused with external solution containing no Na+ ions.

	DISCUSSION

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Robust Expression of WCW Mutant Na⁺ Channels in HEK293 Cells
The high expression of rNav1.4-WCW mutant muscle Na⁺ channels in stably transfected HEK293 cells is unusual, since a previous report showed rather poor expression of inactivation-deficient hNav1.5-IFM/QQQ mutant cardiac Na⁺ channels in HEK293 cells (Grant et al., 2000). Evidently, the inactivation-deficient phenotype alone cannot explain the limited expression of IFM/QQQ cardiac mutant channels in HEK293 cells. Regardless the reasons for this robust expression, our cell line will be useful for the study of the open-channel block by LAs and by class 1 antiarrhythmic agents. In addition, this cell line can be used for the investigation of the possible involvement of intermediate closed states in drug binding during channel activation as shown in this report. Such a cell line should also be suitable for the high throughput screening of novel open-channel blockers (Sanguinetti and Bennett, 2003; Wang et al., 2004a).

Open-channel Block of WCW Mutant Channels by Lidocaine and Benzocaine
The time-dependent inhibition of WCW mutant currents by lidocaine, benzocaine, and class 1 antiarrhythmic agents (Wang et al., 2003b, 2004b) was taken as evidence for the open-channel block. This phenotype is consistent with the location of the LA receptor within the pore-forming S6 segments. Such a phenotype was found in the block of K⁺ channels by internal TEA ions and various homologues (Armstrong, 1971). The dose–response curves show that this open-channel block by benzocaine and lidocaine is nearly as potent as the inactivated-channel block (Meeder and Ulbricht, 1987; Wright et al., 1997). In addition, lidocaine, but not benzocaine, elicited an additional use-dependent block (Fig. 5 B) because of the slow recovery of the open-channel block by lidocaine. Our findings imply that the open-channel block by lidocaine may also play an important role in vivo, particularly during ectopic high-frequency firings when Na⁺ channels open repetitively (>20 Hz; Devor et al., 1992). Furthermore, the IC₅₀ value for the open-channel block by lidocaine is near the therapeutic plasma concentration range for its antiarrhythmic action (Roden, 2001) and for its treatment of neuropathic pain (Boas et al., 1982), implying that ectopic hyperexcitability and/or persistent late Na⁺ currents found under pathological conditions are susceptible for LA open-channel block, as also recently demonstrated for LQT-3 syndromes (An et al., 1996; Wang et al., 1997; Nagatomo et al., 2000).

The results described above, however, remain incompatible with the observations that show (a) the lack of open-channel block by QX-314 in pronase-treated squid axon and (b) the absence of the high-affinity block by LAs in IFM/QQQ mutant Na⁺ channels (see INTRODUCTION). One interpretation is that WCW mutations at D1-S6 fortuitously modify the LA receptor, which is different from that in normal wild-type Na⁺ channels. The argument against this view is that LAs also potently block the open state of normal Na⁺ channels treated with chloramine-T (Ulbricht and Stoye-Herzog, 1984; Wang et al., 1987, 2004b). Alternatively, the discrepancy may be due to differences in expression systems, mutation sites, and/or pulse protocols. This discrepancy is yet to be resolved.

Lack of Na⁺ Ion Effects in Lidocaine and Benzocaine Block
External Na⁺ ions significantly antagonize binding of QX-314, a quaternary ammonium derivative of lidocaine (Cahalan and Almers, 1979). This antagonism was due to repulsion between the Na⁺ ion and the positive charge carried by QX-314 within the pore region. As anticipated, we did not observe antagonism between external Na⁺ ions and bound neutral benzocaine (Fig. 6 B). However, since lidocaine can also carry a positive charge when protonated, the lack of antagonism between the external Na⁺ ions and lidocaine block is unexpected (Fig. 6 A). Barber et al. (1992) reported a similar negative result by external Na⁺ ions for lidocaine block of normal cardiac Na⁺ channels. The reason for this phenomenon is unclear. One possibility is that H⁺ dissociated from lidocaine when Na⁺ ions are approaching the LA receptor. This could not occur for QX-314 because it carries a permanent charge. Yeh and Tanguy (1985) showed that unlike QX-314, lidocaine in its neutral form leaves the closed channel rapidly through the hydrophobic pathway. This hydrophobic pathway could also in part provide unprotonated lidocaine with an LA block that is not antagonized by external Na⁺ ions. This possibility is discussed next.

Direct Intermediate Closed-channel Block of WCW Mutant Channels by LAs
At +30 mV, the time-dependent block by 300 µM benzocaine is rapid, with a time constant of 0.8 ms, whereas at –30 mV, there is no evidence of a time-dependent benzocaine block. Closer examinations of benzocaine block revealed a slow activation time course at –30 mV as compared with the rapid time course of benzocaine block at +30 mV (Fig. 7). Could block of Na⁺ channels by benzocaine occur during transitions to the intermediate closed states before Na⁺ currents reach their peak (i.e., first latency distribution; Aldrich et al., 1983)? If so, the peak current at –30 mV should be reduced more than that at +30 mV. A much greater reduction in peak currents by benzocaine was indeed observed at –30 mV (Fig. 7, B and C). By the same token, this result suggests that the rapid open-channel block by neutral benzocaine at +30 mV does not require that benzocaine come exclusively via the hydrophilic pathway.

The results for lidocaine were less clear-cut than those for benzocaine but the trend with respect to intermediate closed-channel block appears the same (Fig. 8). This ambiguity could be due to (a) differences in size and hydrophobicity of lidocaine and benzocaine, (b) the protonation/deprotonation of lidocaine within the aqueous pore, or (c) differences in open times of mutant channels at various voltages. These factors will likely affect the kinetics of the access/exit of the drug via hydrophobic and hydrophilic pathways (Hille, 1977).

Vedantham and Cannon (1999) recently hypothesized that transitions to intermediate closed states, rather than transitions to inactivated states as stated in the classic modulated receptor hypothesis, play a crucial role in the shift of the h curve by lidocaine. Assuming that intermediate closed state interacts with benzocaine or lidocaine, a significant block by LAs should develop during the 100-ms conditioning pulse because of the significant increase in the availability of intermediate closed states along the activation pathway (e.g., at –70 mV conditioning pulse). Our experimental data from inactivation-deficient Na⁺ channels (Fig. 9) strongly support such a hypothesis for LA block by benzocaine and lidocaine.

It is worth noting that we make no distinction between the high-affinity LA binding among closed or closed/inactivated channels. Perhaps fast inactivation in normal Na⁺ channels masks the high-affinity LA interactions with intermediate closed and the final open states. Slow-inactivated Na⁺ channels may also display high-affinity binding with LAs as residual slow inactivation is evident for the open and intermediate preopen mutant Na⁺ channels (Figs. 1 and 2). Transient intermediate "activated" states will in time enter the absorbing inactivated state (Aldrich et al., 1983). Inactivated Na⁺ channels are likely to bind to lidocaine with a high affinity albeit more slowly (Wright et al., 1997) than open Na⁺ channels. A similarly high-affinity LA receptor should be present in the intermediate closed, intermediate closed/inactivated, open, and open/inactivated channels. If so, the use-dependent action of LAs during depolarization does not require inactivated states but may simply be explained by higher affinity for intermediate closed and open states (Vedantham and Cannon, 1999).

In molecular terms, the formation of a high-affinity LA receptor during activation gating could well be due to the rearrangement of S6 segments. This is feasible considering that S6 segments are structurally analogous to the pore-forming inner helices of K⁺ channels (Yarov-Yarovoy et al., 2002). These inner helices contain a gating hinge (glycine) that bends 30° in K⁺ channels upon activation (Jiang et al., 2002). In the straight configuration, four inner helices form a bundle, closing the channel near its intracellular surface, whereas in the bent configuration, the inner helices splay open, creating a wide pathway. Thus, Na⁺ channel activation could easily modulate the configuration of the LA receptor situated at pore-forming S6 segments. In contrast, the movement of the intracellular fast-inactivation gate (IFM motif) is not involved in the formation of a high-affinity lidocaine receptor as inferred by Vedantham and Cannon (1999). Nonetheless, fast inactivation may play an important but indirect role for the LA binding since (a) the activation and inactivation gating processes are coupled and (b) the inactivation gate, if docked within the pore region near the LA receptor (Yarov-Yarovoy et al., 2002; Wang et al., 2003b), will likely limit the LA access/exit via the hydrophilic pathway.

	ACKNOWLEDGMENTS

 
This work was supported by National Institutes of Health (GM48090 and HL66076).

Olaf S. Andersen served as editor.

Submitted: 18 June 2004
Accepted: 13 October 2004

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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