Introduction

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Inhibitors of voltage-gated sodium channels are widely used clinically. Blockers of sodium channels in the heart are potent antiarrhythmics, whereas the inhibition of neuronal sodium channels is useful for local anesthesia and treatment of epilepsy (Hondeghem and Katzung, 1984; Catterall, 1987; Butterworth and Strichartz, 1990; Ragsdale et al., 1991; Caron and Libersa, 1997). These overlapping actions bear risks and benefits. On the one hand, local anesthetics inadvertently injected into a blood vessel can cause severe cardiac arrhythmias. On the other hand, some antiarrhythmics, including mexiletine, penetrate the blood-brain barrier and have interesting neuroprotective properties (e.g., Stys and Lesiuk, 1996).

Voltage-gated brain sodium channels are heteromultimeric proteins consisting of a principal -subunit of 260 kDa, as well as a ₁-subunit of 36 kDa and a ₂-subunit of 33 kDa (Catterall, 1992). Both - and ₁-subunits also are expressed in cardiac myocytes (Qu et al., 1995a). The -subunit consists of four homologous transmembrane domains (I-IV), each containing six transmembrane -helical segments, called S1 through S6 (Catterall, 1992; Fozzard and Hanck, 1996). The principal electrophysiological functions are mediated by the -subunit; the -subunits have only minor effects when the channels are heterologously expressed in mammalian cells (Isom et al., 1995). The type IIA sodium channel -subunit is a principal isoform expressed in the brain (Gordon et al., 1987; Beckh et al., 1989), and its heterologous expression in mammalian cells yields sodium currents with physiological and pharmacological properties that are similar to those observed in rat brain neurons (Ragsdale et al., 1991; West et al., 1992). The rh1 sodium channel -subunit is the primary isoform expressed in the heart (Rogart et al., 1989; Kallen et al., 1990), and expression of this isoform in Xenopus oocytes or mammalian cells yields channels with physiological and pharmacological properties characteristic of heart sodium channels (Cribbs et al., 1990; White et al., 1991; Qu et al., 1994, 1995a).

Mutations of amino acid residues F1764 and Y1771 in transmembrane segment IVS6 of the rbIIA sodium channel to alanine substantially reduced block by the local anesthetic etidocaine (Ragsdale et al., 1994) and by a range of local anesthetic, anticonvulsant, and antiarrhythmic drugs (Ragsdale et al., 1996; Wang et al., 1998; Wright et al., 1998), defining a local anesthetic receptor site in this region of the channel structure. Mutation of F1762 in the rh1 sodium channel, which is homologous to rbF1764, to alanine also resulted in loss of block of the rh1 channel by the quaternary lidocaine analog QX314 (Qu et al., 1995b). Mexiletine is a class I antiarrhythmic that inhibits both brain and heart sodium channels in vitro and in vivo (Stys and Lesiuk, 1996; Wang et al., 1997). In the experiments described here, we compared the effects of R-mexiletine on cloned rat brain and heart sodium channel -subunits heterologously expressed in a common cellular background, and we examined the effects of mutations in the local anesthetic receptor site on mexiletine block of these two channel types. Our results show that heart sodium channels have an intrinsically higher affinity for block by R-mexiletine than do brain channels when expressed in the same cellular background. Surprisingly, although mutations of the homologous phenylalanine residue in brain and heart channels (rhF1762A/rbF1764A) reduced block by R-mexiletine and abolished the difference in affinity between the two channel isoforms, the effect was primarily on drug association rate for rbIIA and on drug dissociation rate for rh1. Thus, although this phenylalanine residue is essential for mexiletine block of both heart and brain sodium channel isoforms and is responsible for the difference in mexiletine affinity between them, its role in drug binding is fundamentally different in the two channel backgrounds. This finding suggests that there are important differences in the interaction of local anesthetics and related compounds with brain and cardiac sodium channels. These differences may be valuable in the development of novel, subtype-selective antiarrhythmic drugs.

	Materials and Methods

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Cell Maintenance and Transient Transfection for Electrophysiological Recording. tsA-201 cells, which are a subclone of HEK293 cells expressing simian virus 40 t-antigen, were a kind gift from Dr. Robert Dubridge (Cell Genesys, Foster City, CA). Cells were maintained in Dulbecco's modified Eagle's medium/F12 media (Gibco Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 25 U/ml penicillin and 25 µg/ml streptomycin (Sigma, St. Louis, MO.). An EcoRV fragment containing mutants F1764A and Y1771A of RIIA in pVA2580 (Ragsdale et al., 1996) was subcloned into pCDM8 containing the remainder of the rIIA sodium channel -subunit, as described (Linford et al., 1998). rH1 (Rogart et al., 1989) and rH1 mutant F1762A in pCDM8 were described previously (Qu et al., 1995b). tsA-201 cells were transiently transfected with wild-type (WT) or mutant -subunits and a vector encoding the human CD8 cell surface protein (EBO-pCD-leu2; American Type Culture, Rockville, MD) for cell recognition, as described (Margolskee et al., 1993). Successfully transfected cells were labeled to recognize them for recording using anti-CD8-coated polystyrene microspheres (Dynabeads M-450 CD8; Dynal, Great Neck, NY), as described (Jurman et al., 1994).

Electrophysiological Recording. Sodium currents were recorded from transiently transfected tsA-201 cells in the whole-cell voltage-clamp configuration (Hamill et al., 1981) at 22°C, as described (Qu et al., 1996). The extracellular solution contained 140 mM NaCl, 5 mM CsCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 10 mM glucose, and 10 mM HEPES (pH 7.4, adjusted with NaOH). The intracellular solution contained 90 mM CsF, 50 mM CsCl, 10 mM CsEGTA, 10 mM NaF, 2 mM MgCl₂, and 10 mM HEPES (pH 7.4, adjusted with CsOH). Recording pipettes had resistances of 0.8 to 1.8 M when filled with intracellular solution. Mexiletine is a racemate of S(±)- and R()-enantiomers, which have differential effects on sodium channels (De Luca et al., 1995). In this study, only the ()-enantiomer (R-mexiletine) was used to avoid differential effects of enantiomers on the sodium channel mutants. Purity of the R-enantiomer of mexiletine was >98%. The compound was synthesized at the Department of Pharmaceutical Chemistry at Boehringer Ingelheim KG (Ingelheim, Germany). R-mexiletine was dissolved in extracellular solution at the highest concentration to be used in an experiment, and diluted with extracellular solution to each of the other concentrations. The cells were bathed with the effluent of a gravity-driven "sewer-pipe" perfusion system consisting of a series of parallel tubes with each tube containing either control solution or a solution containing R-mexiletine. Solutions were changed by translating the array of tubes so that the tube containing the appropriate concentration was bathing the cell. The entire Petri dish was perfused continuously with control extracellular solution. Solution changes were complete within 2 s. Currents were recorded using an Axopatch 200B patch clamp amplifier (Axon Instruments, Foster City, CA). Voltage-clamp commands were delivered and currents recorded using PClamp 6 controlling a Digidata 1200 interface (Axon Instruments). Whole-cell capacitance was compensated using the internal voltage-clamp circuitry and ~80% of series resistance was compensated. Residual linear leakage and capacitance were subtracted using a P/4 protocol when appropriate (Bezanilla and Armstrong, 1977). Data analysis and curve fitting were performed using Sigma Plot (SPSS, Chicago, IL) or Prism (GraphPad Software, San Diego, CA). All data points are the means of three to six experiments, and grouped data are reported as ±S.D.

	Results

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Mutations F1764A and F1771A Inhibit Tonic and Frequency-Dependent Block of Brain Sodium Channels by R-Mexiletine. The principal characteristics of sodium channel block by R-mexiletine are illustrated by the current traces in Fig. 1 (inset). tsA-201 cells expressing rbIIA sodium channel -subunits were voltage-clamped at a holding potential of 100 mV, and a control current trace was recorded in response to a 10-ms depolarization to 0 mV. The cell was then exposed to R-mexiletine (100 µM) in the absence of pulses, followed by a 5 Hz train of 100 pulses (10 ms duration) to 0 mV. Approximately 20% of the current was tonically blocked in the first test pulse at this concentration, and an additional 25% block was observed at the steady state during the 5-Hz train (Fig. 1A, inset). The frequency-dependent component of block reached steady state rapidly, as illustrated for 300 µM R-mexiletine in Fig. 1A. Similar experiments were performed in different R-mexiletine concentrations, yielding concentration-response curves with IC₅₀ values of 305 and 165 µM for tonic and frequency-dependent block, respectively (Fig. 1B, WT).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Mutations in the IVS6 region of brain-type sodium channel -subunits influence tonic and frequency-dependent block by mexiletine. A, a single pulse to 0 mV was applied in the absence of drug. Then R-mexiletine was washed into the bath for 30 s to 3 min without pulsing. Control experiments showed that equilibrium R-mexiletine block was reached within seconds. Finally, a 5-Hz train of 100 depolarizations (10-ms duration) to 0 mV was applied to voltage-clamped tsA-201 cells that had been transfected with rat brain sodium channel -subunits. Inset, current traces evoked by the single pulse in drug-free solution (control) and by pulse 1 and pulse 100 of the train in the presence of 100 µM R-mexiletine. Scale bars: 2 ms, 2 nA. A, the additional frequency-dependent block observed during 5-Hz trains of pulses in the presence of 300 µM R-mexiletine for WT (), F1764A (), and Y1771A () channels. B, the inhibition in response to pulse 1 is referred to as tonic block, and to pulse 100 as frequency-dependent block. Concentration-response curves are plotted for tonic (, , ) and frequency-dependent (, , ) block of rbWT (, ), rb F1764A (, ), and rbY1771A (, ). The lines are fits to the data for each construct with IC50 values of 305 and 148 µM for tonic and frequency-dependent block of rbWT channels, 614 and 410 µM for rbF1764A, and 718 and 617 µM for Y1771A. Solid lines, tonic block; dotted lines, frequency-dependent block.

Effects of Mutations F1764A and Y1771A on Drug Binding to the Resting and Inactivated States of Brain Sodium Channels. The tonic block of sodium current observed at a holding potential of 100 mV reflects a combination of binding to resting and inactivated sodium channels, because drug binding favors the inactivated state. Therefore, to estimate the affinity for block of resting channels, we determined the IC₅₀ value for block of sodium channels at increasingly negative membrane potentials where the contribution of the inactivated state would be progressively reduced. Limiting values for the IC₅₀ were reached at 140 mV (data not shown¹), corresponding to block of the resting state with an IC₅₀ value of 547 ± 107 µM for WT channels, 816 ± 224 µM for F1764A, and 642 ± 75 µM for Y1771A (Fig. 2, solid lines). Thus, no significant effect of mutations on resting affinity was observed.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Mutations rbF1764A and rbY1771A reduce block of resting and inactivated sodium channels. The inhibition of inactivated sodium channels was investigated using a 1-s depolarizing prepulse to 40 mV (60 mV for rbY1771A) to inactivate most channels and allow R-mexiletine to bind to the inactivated channels. The membrane potential then was returned to the holding potential of 100 mV (120 mV for Y1771A) for 10 ms to allow drug-free channels to recover from inactivation. A test pulse to 0 mV then was applied. R-mexiletine block of peak test pulse current is plotted as a function of R-mexiletine concentration for WT (), rbF1764A (), and rbY1771A () channels. The dotted lines are fits of a logistic equation to the data with IC50 values of 20.7 µM (WT), 156 µM (rbF1764A), and 309 µM (rbY1771A), with slopes of 0.9, 1.5, and 1.3, respectively. Resting channel block (solid lines) IC50 values were 533 µM (, WT), 813 µM (, F1764A), and 655 µM (, Y1771A), with slopes of 1.2, 1.2, and 1.4, respectively. Varying slope values are expected from allosteric models of drug binding in which binding is distributed among multiple states with varying affinities.

Block of WT and F1762A Mutant Rat Heart Sodium Channels by R-Mexiletine. We studied the block of cloned and expressed rh1 sodium channel -subunits by R-mexiletine to compare the potency and mechanism of block to brain channels. Tonic and frequency-dependent block were initially examined during a train of 100 depolarizations using a protocol analogous to that used in Fig. 1 (Fig. 3A). For these experiments, the holding and test potentials were 20 mV negative to those used for brain channels to compensate for the more negative activation and inactivation properties of the rat heart channel (Fozzard and Hanck, 1996; T.W. and T.S., unpublished observations). As with brain channels, frequency-dependent block was observed at 5 Hz for WT and was much impaired for mutant rhF1762A (Fig. 3A). Concentration-response curves (Fig. 3B) obtained using this protocol gave IC₅₀ values of 165 and 52 µM for tonic (closed circles) and frequency-dependent (open circles) block of WT rh1 channels, and IC₅₀ values of 748 and 619 µM for rhF1762A.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Tonic and frequency-dependent inhibition of WT and mutant rat heart sodium channels. A (inset), current recorded during test pulses to 20 mV in control and during the 1st and 100th pulses of a 5-Hz train in the presence of 100 µM R-mexiletine in a cell expressing rhWT channels. Scale bars: 2 ms, 5 nA. A, the pulsewise development of frequency-dependent block using 5-Hz stimulation in the presence of 300 µM R-mexiletine was measured for WT () and rhF1762A () sodium channels. The protocol was the same as in Fig. 1, except that holding and test potentials were 120 mV and 20 mV, respectively, to account for the more negative voltage dependence of the heart channel. B, Concentration-response curves for tonic (, ) and frequency-dependent (, ) block of rhWT (, ) and rhF1762A (, ) by R-mexiletine determined from pulse 1 and pulse 100 of trains as described in the legend to Fig. 1. The solid lines are fits of a logistic equation, with IC50 values for rhWT of 165 and 52 µM for tonic and phasic block, respectively, and for rhF1762A of 748 and 618 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Mutant rhF1762A reduces block of resting and inactivated sodium channels. The inhibition of inactivated sodium channels was investigated using a 1-s depolarizing prepulse to 60 mV. The membrane potential then was returned to the holding potential of 120 mV for 10 ms, and a test pulse to 20 mV was applied. The stimulus sequence was applied once in the presence of each drug concentration. R-mexiletine block of peak test pulse current is plotted as a function of R-mexiletine concentration for rhWT () and rhF1762A (). The dotted lines are fits of a logistic equation to the data for inactivated channels, with IC50 values of 9.4 µM (rhWT) and 149 µM (rhF1762A) and slope values of 0.93 (rhWT) and 1.0 (rhF1762A). To measure block of resting channels, the indicated concentrations of R-mexiletine were applied to rhWT or rhF1762A channels at a holding potential of 160 mV, and block was measured during a test depolarization to 20 mV. The concentration-response curve for rhWT () and rhF1762A () reaches a limiting affinity at this membrane potential, as determined from parallel measurements at 100 mV, 120 mV, 140 mV, and 160 mV. Fit of logistic equations to the mean data (solid lines) for WT are IC50 = 325 µM, slope = 1.2, and for rhF1762A are IC50 = 1389 µM, slope = 1.3.

Comparison of Affinity and Kinetic Mechanism of Block of Brain and Cardiac Sodium Channels by Mexiletine. WT heart sodium channels were more sensitive to frequency-dependent block at high frequencies than were WT brain sodium channels (Fig. 5A). Similarly, the IC₅₀ values for the inhibition of resting and inactivated WT heart channels were approximately 2-fold lower than those for WT brain channels (Fig. 5B). In contrast, the concentrations for half-maximum block of inactivated rhF1762A and rbF1764A were similar (151 versus 157 µM; Fig. 5B), indicating that differences in interaction between bound mexiletine and F1764/F1762 are responsible for the difference in overall affinity for mexiletine block of the two channels.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Comparison of mexiletine block of brain and heart sodium channels. A, cells expressing rbWT or rhWT sodium channels were stimulated with trains of 15 depolarizations (10-ms duration) to 0 mV (brain, filled bars) or 20 mV (heart, open bars) at the indicated frequencies in the presence of 100 µM mexiletine. The fraction of unblocked current was determined as the peak current evoked by pulse 15 as a fraction of the peak current evoked by pulse 1 (the tonically blocked channel) for each stimulus frequency. The fraction of blocked current (1-fraction of unblocked current) was plotted as a function of stimulus frequency. Frequency-dependent block in control solutions was <5% at the frequencies tested. B, comparison of IC50 values for block of resting channels (filled bars) and of inactivated channels (open bars), for the indicated sodium channel types. C, hyperpolarizing shifts of steady-state inactivation curves in response to R-mexiletine. Steady-state inactivation of sodium channels was investigated using 1-s prepulses to a variable potential, followed by a test pulse to 0 mV (for rb channels) or 20 mV (for rh channels). The control curve was completed and curves then were obtained in progressively increasing R-mexiletine concentrations. The shift in the voltage of half-inactivation at a given concentration was greater for WT heart channels () than for WT brain channels (). In contrast, in rhF1762A () and rbF1764A channels (), a given concentration of R-mexiletine induced comparable shifts.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Kinetics of onset and recovery from use-dependent block for WT and mutant channels. A, the time course of onset of R-mexiletine block during depolarization was studied using a 0-mV prepulse of variable duration followed by a test pulse to the same potential. A 45-ms interval was present between prepulse and test pulse to allow drug-free channels to recover from fast inactivation. Peak test pulse current in the presence 300 µM R-mexiletine was normalized to control values obtained using the same protocol and plotted versus prepulse duration. The resulting data points were fit with the sum of two exponentials with WT time constants () of 1.0 ms and 25 ms with the fraction slow = 0.5; for F1764A,  = 11 and 1140 ms, with fraction slow = 0.89. This slower time constant represents a minimum value attributable to the failure to reach steady state at the longest depolarization used (1000 ms). The baseline of the fits was subtracted from both the data and the fit for each construct. The resulting values then were normalized with respect to the first data points to compare the kinetics of development of block for rbWT () and rbF1764A (). B, onset of R-mexiletine block of rhWT () and rhF1762A () channels studied as in A, except that prepulses and test pulses were to 20 mV. The effect of mutant rbF1764A on the onset of block was much stronger than was the effect of the equivalent heart mutant, rhF1762A. C, inhibition by 300 µM mexiletine was generated by applying a 10-Hz conditioning train of 10 pulses to 0 mV. The membrane potential then was returned to 100 mV, and a test pulse to 0 mV was applied after a recovery interval of variable duration. The rates of recovery for rbWT () and rbF1764A () were generated by plotting the peak test pulse current normalized to its value after 5000-ms recovery as a function of recovery interval. The data were fitted by double-exponential functions, where the faster time constant reflected the recovery from inactivation of unblocked channels, and the slower time reflected the recovery from block by mexiletine. The fast component of recovery was subtracted from the data, and the normalized slow component of recovery representing drug dissociation was plotted. The mean values obtained from the fits were for WT (mean ± S.E.): fraction fast (FF) = 0.36 ± 0.02 ms,  fast (f) = 15 ± 2 ms,  slow (s) = 530 ± 15 ms; for F1764A: FF = 0.91 ± .03, f = 3.3 ± 0.4 ms, s = 540 ± 157 ms. Recovery time courses for rhWT () and rhF1762A () sodium channels using the protocol in of C, except that conditioning and test pulses were to 20 mV and the holding and recovery potentials were 120 mV. The mean values obtained from the fits were for rhWT: FF = 0.32 ± 0.02, f = 26 ± 8 ms, s = 671 ± 71 ms; for rhF1762A: FF = 0.63 ± 0.05, f = 6 ± 0.5 ms, s = 170 ± 7 ms.

	Discussion

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R-Mexiletine Block of Native and Heterologously Expressed Heart Sodium Channels Is Similar. R-mexiletine has been described as a fast-onset, frequency-dependent blocker of heart sodium channels. Using rat ventricular myocytes, Yatani and Akaike (1985) found an IC₅₀ value of 28 µM for tonic inhibition. Those data were obtained using a holding potential of 80 mV, which caused ~50% channel inactivation under their experimental conditions. In the same preparation, Ono et al. (1994) reported half-maximum inhibition of depolarized channels to be 15 µM. Heterologous expression of WT human heart sodium channels resulted in steady-state block of inactivated channels with an IC₅₀ value of 15 µM (Wang et al., 1997). These data are consistent with the IC₅₀ value of 9.4 µM that we obtained for R-mexiletine block of expressed rat heart sodium channels under depolarized conditions in the present study. Thus, the data on rat heart sodium channel -subunits presented here are in good agreement with those obtained previously. Although these results were obtained with channels formed by sodium channel -subunits alone, coexpression of sodium channel ₁- and ₂-subunits in mammalian cells cause only minor quantitative effects on channel function. Thus, we expect block of WT and mutant channels formed by the full complement of subunits to be qualitatively similar to that determined here with -subunits alone.

Higher Intrinsic Affinity for Block of Heart Versus Brain Sodium Channels. The best estimate of affinity for resting channels is to determine block of peak current using increasingly negative holding potentials. Block by R-mexiletine reaches a limiting affinity that is not reduced as the holding potential is made more negative. This limiting IC₅₀ value is 547 µM in the brain channel and 325 µM in the heart channel. Thus, when elicited from the fully resting state, the heart sodium channel is ~1.7 times more sensitive to block than is the brain channel. Likewise, our estimates for block of inactivated channels obtained using a depolarizing prepulse indicates approximately 2-fold higher sensitivity of inactivated heart channels to block by R-mexiletine. These findings contrast with a recent report comparing lidocaine sensitivity for resting brain and heart sodium channels, which indicated that there is little intrinsic difference in sensitivity of the isoforms to block of resting channels by lidocaine (but see Wang et al., 1996; Wright et al., 1997). Preferential block of heart sodium channels likely contributes to the selective therapeutic effect of mexiletine on cardiac arrhythmias.

Interactions with rbF1764/rhF1762 Are Responsible for the Difference in Affinity for Brain and Heart Sodium Channels. The greater affinity of heart sodium channels for block by R-mexiletine was largely abolished by the mutations F1764A and F1762A. The affinity of the inactivated states of these two mutants was not markedly different (K_I = 157 and 151 µM, respectively), and IC₅₀ values at different potentials also were similar for these two mutants. These results suggest that interactions with this key phenylalanine could account for the higher affinity of heart sodium channels for R-mexiletine.

Homologous Mutations F1764A and F1762A Affect R-Mexiletine Block in Different Ways. In spite of the similarities in steady-state block of the mutant brain and heart channels, the mutations at F1764/1762 disrupted R-mexiletine block in strikingly different ways. Mutation F1764A of the brain channel had a pronounced effect on the rate of association of the drug with the channel, but there was little effect of the mutation on drug dissociation. In contrast, mutation of the analogous amino acid in the heart sodium channel accelerated drug dissociation with little effect on the rate of drug binding. The simplest interpretation of these findings is that in the brain channel, F1764 facilitates rapid drug binding. In contrast, in the WT heart channel, F1762 stabilizes the drug in its binding site but plays little role in association. Thus, these findings suggest that the conserved F1764/1762 residues in transmembrane segment IVS6 play fundamentally different roles in mexiletine binding in the brain and heart sodium channel backgrounds.

Common and Divergent Effects of IVS6 Mutations on Block of Brain Sodium Channels by R-Mexiletine and Other Local Anesthetic/Antiarrhythmic Compounds. Each point mutation in transmembrane segment IVS6 that was known to reduce block by other local anesthetic, anticonvulsant, and antiarrhythmic drugs also reduced frequency-dependent R-mexiletine block of rbIIA sodium channels (Ragsdale et al., 1994; Qu et al., 1995b). Frequency- and voltage-dependent block of brain sodium channels by etidocaine is reduced in mutants F1764A and Y1771A, and F1764 is more important for binding than is Y1771 (Ragsdale et al., 1994). Although these two mutations also reduced the affinity for R-mexiletine, Y1771A reduced R-mexiletine block more profoundly than did F1764A. Similarly, Ragsdale et al. (1996) found that effects of a range of sodium channel inhibitors on mutants F1764A and Y1771A were all reduced compared with WT channels, but the rank order of potencies for frequency-dependent block was different for the compounds tested. Block by lidocaine was reduced more by mutant F1764A than by mutant Y1771A, in contrast to our results with mexiletine, for which binding is more affected in Y1771A. Thus, sodium channel modulators of subtly different chemical structure may interact in different ways with the local anesthetic receptor site in segment IVS6 of the sodium channel.