Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels

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1 Keywords: 0199 Journal of Physiology (2000), 524.1, pp Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels Zhenhui Chen *, Boon-Hooi Ong, Nicholas G. Kambouris *, Eduardo Marb an, Gordon F. Tomaselli and Jeffrey R. Balser Departments of Anesthesiology and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, Institute of Molecular Cardiobiology and *Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (Received 7 October 1999; accepted after revision 13 December 1999 ) 1. Local anaesthetics such as lidocaine (lignocaine) interact with sodium channels in a manner that is exquisitely sensitive to the voltage-dependent conformational state of the ion channel. When depolarized in the presence of lidocaine, sodium channels assume a long-lived quiescent state. Although studies over the last decade have localized the lidocaine receptor to the inner aspect of the aqueous pore, the mechanistic basis of depolarization-induced usedependent lidocaine block remains uncertain. 2. Recent studies have shown that lowering the extracellular Na concentration ([Na ]ï) and mutations in the sodium channel outer P-loop modulate occupancy of a quiescent slow inactivated state with intermediate kinetics (termed IM) that involves structural rearrangementsintheouterpore. 3. Site-directed mutagenesis and ion-replacement experiments were performed using voltageclamped Xenopus oocytes and cultured (HEK-293) cells expressing wild-type and mutant rat skeletal muscle (ì1) sodium channels. 4. Our results show that lowering [Na ]ï potentiates use-dependent lidocaine block. The effect of [Na ]ï is maintained despite a III IV linker mutation that partially disrupts fast inactivation (F1304Q). In contrast, the effect of lowering [Na ]ï on lidocaine block is reduced by a P-loop mutation (W402A) that limits occupancy of IM. 5. Our findings are consistent with a simple allosteric model where lidocaine binding induces channels to occupy a native slow inactivated state that is inhibited by [Na ]ï. Lidocaine (lignocaine) and its analogues, compounds that suppress Na current (INa), are used as local anaesthetics and as therapies for cardiac arrhythmias and epilepsy. When depolarized, Na channels only transiently conduct ions due to rapidly developing (fast) inactivation (Hodgkin & Huxley, 1952), a conformational change involving amino acid residues in the III IV interdomain linker (St uhmer et al. 1989; West et al. 1992) and vicinal sites near the inner surface of the channel (McPhee et al. 1994, 1995, 1998; Smith & Goldin, 1997; Lerche et al. 1997; Filatov et al. 1998; Tang et al. 1998). While drug-free Na channels quickly recover from fast inactivation between depolarizing stimuli, lidocaine delays such recovery, inducing a stable non-conducting state that results in cumulative reduction in INa with brief, repetitive stimuli (so-called use-dependent block ) (Courtney, 1975). Lidocaine binds to amino acids positioned on the intracellular side of the putative selectivity filter (Ragsdale et al. 1994, 1996; Sunami et al. 1997), near (and perhaps overlapping) the fast-inactivation gating structures (Bennett et al. 1995). A high-affinity binding interaction between lidocaine and the fast inactivated conformational state is a structural hypothesis for use-dependent local anaesthetic block supported by compelling, but circumstantial, experimental evidence (Hille, 1977; Hondeghem & Katzung, 1977; Cahalan, 1978; Yeh, 1978; Bean et al. 1983; Balser et al. 1996b). Surprisingly, a recent study found that amino acid residues intimately involved in fast inactivation recover to their pre-inactivated conformation rapidly, even while conduction is fully suppressed by lidocaine (Vedantham & Cannon, 1999), casting doubt on the prevailing notion that lidocaine arrests Na channels in the fast inactivated conformational state. In rare circumstances such as prolonged depolarization, Na channels may assume slow inactivated conformations from which recovery is considerably delayed (Adelman & Palti, 1969; Chandler & Meves, 1970; Rudy, 1978). One such slow inactivated state with intermediate kinetics (Cummins & Sigworth, 1996; Hayward et al. 1996) is influenced by externally positioned residues in the outer

2 38 Z. Chen and others J. Physiol pore region (ì1-w402) (Balser et al. 1996a; Kambouris et al. 1998; Benitah et al. 1999). Earlier studies (Cahalan & Almers, 1979) suggested that external Na opposed the use-dependent action of charged (quaternary) lidocaine derivatives through a competitive knock-off mechanism. However, recent studies have shown that external Na also prevents Na channels from entering slow inactivated states (Townsend & Horn, 1997), including an inactivated state of intermediate stability (denoted IM) (Benitah et al. 1999). Moreover, earlier studies proposed that local anaesthetic action involves a gating conformational change linked to a stable inactivated state (Khodorov et al. 1976; Zilberter et al. 1991), and we recently showed that an outer pore (P-loop) mutation that opposes IM also reduces use-dependent lidocaine action (Kambouris et al. 1998). Here we examine the possibility that this gating effect of external Na, inhibition of IM, plays a role in Na antagonism of use-dependent lidocaine action. Our data show that augmenting slow inactivation by lowering the extracellular Na concentration ([Na ]ï) sensitizes the Na channel to usedependent lidocaine action, even when fast inactivation is disabled (ì1-f1304q). Moreover, the effects of lowering [Na ]ï on slow inactivation and lidocaine action are decreased by the P-loop mutation that opposes IM (W402A). Our findings are consistent with a simple allosteric model where lidocaine binding induces channels to occupy a native slow inactivated state that is inhibited by [Na ]ï. METHODS Molecular biology Site-directed mutagenesis of the ì1 Na channel á subunit (Trimmer et al. 1989) was performed on a full-length cdna clone encoding the wild-type ì1 channel using a PCR-based method (Weiner et al. 1993). Both sense and antisense oligonucleotides (30 34mer) containing the desired mutation(s) were used as primers for amplification with Pfu DNA polymerase (Stratagene). The PCR product was treated with DpnI toselectforthemutant plasmid and then transformed into E. coli for subsequent isolation and purification using standard protocols. Both modifications (W402A, F1403Q) were confirmed by di-deoxy sequencing of the region of the á subunit containing the mutation and by electrophysiological characterization of, at least, two independent mutagenic clones (Lawrence et al. 1996; Kambouris et al. 1998). In vitro mrna transcription was performed using the SP6 promoter region and commercially available reagents (Gibco, Gaithersburg, MD, USA). Ion channel expression All procedures surrounding the care and use of Xenopus laevis conformed with our institution animal care committee guidelines. Oocytes were harvested from human chorionic gonadotropinprimed adult females (Nasco, Fort Atkinson, WI, USA) after 30 min of submersion anaesthesia in an aqueous solution containing 0 17 % 3_aminobenzoic acid ethyl ester (methanesulphonate salt, Sigma). Following survival surgery, the animals were allowed to recover in shallow water to maintain head out position until awake ( 30 min). Oocytes were isolated by digestion in a 2 mg ml solution of collagenase (Type IA; Sigma) and stored in a modified Barth s solution containing (mò): NaCl, 88; KCl, 1; NaHCO, 2 4; Tris base, 15; Ca(NO )µ.4hµo, 0 3; CaClµ.6HµO, 0 41; MgSOÚ.7HµO, 0 82; sodium pyruvate, 5; theophylline, 0 5; supplemented with penicillin, 100 U ml ; streptomycin, 100 ìg ml ; fungizone, 250 ng ml and gentamicin, 50 ìg ml. Oocytes were injected with 50 nl of a ìg ìl solution of crna transcribed from wildtype or mutant cdna. In all oocyte experiments, an equimolar ratio of similarly transcribed rat brain â1 subunit RNA was coinjected with the á subunit crna. For expression in human embryonic kidney (HEK-293) cells, the wild-type ì1 á subunit was subcloned into the vector HindIII XbaI site of the vector GFPIRS for bicistronic expression of the channel protein and GFP reporter as previously described (Johns et al. 1997). Cells were transfected with this plasmid using lipofectamine (Gibco), and were cultured in modified Eagle s medium (MEM; Gibco) supplemented with 10 % fetal bovine serum and 1 % penicillin streptomycin in a 5 % COµ incubator at 37 C for 1 3 days. The HEK cells were not transfected with the Na channel â1 subunit. Cells exhibiting green fluorescence were chosen for electrophysiological analysis. Electrophysiology and data analysis Whole-cell Na current (INa) was recorded from Xenopus oocytes using a two-electrode voltage clamp, OC-725B (Warner Instrument Corp., Hamden, CT, USA), 1 3 days after crna injection. Currents were measured using electrodes of 1 3 MÙ resistance when filled with 3 Ò KCl. Currents were recorded in frog Ringer (ND-96) solution containing (mò): NaCl, 96; KCl, 2; MgClµ, 1; Hepes, 5; ph 7 6, and [Na ]ï was reduced to 25 or 10 mò by equimolar replacement with N-methyl-ª_glucamine-chloride (NMGCl). INa was recorded from HEK-293 cells using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). Electrode resistances were 1 3 MÙ when filled with a pipette solution containing (mò): NaF, 140; NaCl, 10; EGTA, 5; Hepes, 10 (ph 7 40). Series resistance was 85 % compensated. The 150 mò Na bath solution contained (mmol l ): NaCl, 150; KCl, 4 5; CaClµ, 1 5; MgClµ, 1; Hepes, 10 (titrated to ph 7 40 with NaOH). [Na ]ï was reduced to 10 mò by equimolar replacement with NMGCl. In both oocyte and HEK cell experiments, junction potentials were minimized during bath solution changes by using a 3 Ò KCl agar bridge. Inactivation gating kinetics and use-dependent block were assessed using voltage-clamp protocols described in the text and figure legends. In all cases, lidocaine HCl (2 % preservative free; Abbott Laboratories, North Chicago, IL, USA) was added to the bathing solution at concentrations indicated in the text. In experiments where lidocaine was used, a recovery period at 100 mv of at least 20 s was allowed prior to each set of measurements in order to avoid cumulative use-dependent block. All electrophysiological recordings were made at room temperature (20 22 C). Multi-exponential functions were fitted to the data using non-linear least-squares methods (Microcal Origin version 4.10, Microcal Software Inc., Northampton, MA, USA) to determine the amplitudes and time constants associated with development or recovery from slow inactivation or use-dependent block. Pooled data are expressed as the means ± s.e.m., and statistical comparisons were made using one-way ANOVA (Origin) with P û 0 05 indicating significance. A Markov gating scheme (Fig. 5) was evaluated using numerical integration methods tailored for stiff sets of differential equations (Hindmarsh, 1983; Petzold, 1983) and linked to custom-written software (Balser et al. 1990). Model parameters were minimized by fitting data from four conditions simultaneously (highïlow [Na ]ï, ± lidocaine) using a simplexbased global fitting method described previously (Tomaselli et al. 1995) and as discussed in the legend of Fig. 5.

3 J. Physiol Lidocaine and slow inactivation 39 Table 1. Kinetic parameters for development of IM 96 mò [Na ]ï 24 mò [Na ]ï 10 mò [Na ]ï Lidocaine A ô (ms) A ô (ms) A ô (ms) ± ± ± ± ± 0 05 * 452 ± ìò 0 39 ± ± ± 0 03 * 201 ± 34 * 0 83 ± 0 02 * 86 ± 9 * The function y = y0 + Aexp( tïô) was fitted to the data in Fig. 1, panels B and C, and the values (means ± s.e.m.) for A and ô are shown. *Value differed significantly from data measured under the same conditions in 96 mò [Na ]ï. RESULTS Effects of lowering [Na ]ï on use-dependent lidocaine block During brief depolarizations, ì1 Na channels recover from inactivation rapidly in physiological (96 mò) [Na ]ïwhenthe â1 subunit is coexpressed (Cannon et al. 1993; Chang et al. 1996). Figure 1A shows pairs of Na currents elicited at the beginning of a 1 s depolarization (P1), and a second current (superimposed) recorded at the same membrane potential (P2) after a brief (20 ms) recovery interval at 100mV. Under these conditions, fast inactivated channels recover in<10ms; hence, any reduction in INa during the P2 pulse reflects slow inactivation (IM). In 96 mò [Na ]ï, (Fig. 1A, left top) peak INa during the P2 pulse is only slightly decreased relative to P1 (92 ± 2 %, n = 7), confirming that little IM occurs normally. However, lowering [Na ]ï to 10 mò (Fig. 1A, right top) increased entry into IM (P2: 65±6% of P1, n = 8). While reducing [Na ]ï decreased the magnitude of INa, the current was within an acceptable range for voltage control in either [Na ]ï. In Fig. 1B, the duration of the P1 pulse was varied from 10 ms to 1 s to examine the rate at which IM develops. Summary data (shown for 10, 24, and 96 mò [Na ]ï) are well-fitted by a single exponential function (continuous lines, fit parameters in Table 1), suggesting occupancy of a single slow inactivated state. The development of a single slow inactivated state with intermediate kinetics under these conditions (with â1 subunit coexpression) is consistent with previous results in oocytes (Featherstone et al. 1996; Kambouris et al. 1998). However, depolarizations of similar duration in mammalian expression systems (Hayward et al. 1997) or longer depolarizations in oocytes (minutes; Todt et al. 1999) recruit additional slow kinetic components with longer time constants for recovery. Previous studies suggest that these more stable inactivated states do not influence lidocaine block (Kambouris et al. 1998). Figure 1A (lower, left) shows that in 96 mò [Na ]ï, 50 ìò lidocaine caused a reduction in P2 peak INa (65±6%ofP1, n = 7) that was similar in magnitude to lowering [Na ]ï to 10 mò in drug-free solutions. Lowering [Na ]ï to 10 mò while in lidocaine (Fig. 1A, bottom, right) dramatically augmented use-dependent block (P2: 8 5 ± 2 %, n = 10). Notably, the lidocaine-associated block was entirely dependent on the preceding depolarization (i.e. was use dependent). Even 100 ìò lidocaine did not block the current elicited by the first pulse (P1) after a long hyperpolarization in either 10 mò [Na ]ï (peak INa 99 ± 11 % of control, n = 12) or 96 mò [Na ]ï (99 ± 9 %, n = 10). Figure 1C shows the time course for development of use-dependent lidocaine block as the length of the P1 pulse was extended from 10 ms to 1 s (note ordinate difference from Fig. 1B). The results in drug-free conditions (from Fig. 1B) are shown as dotted lines. As before, the time-dependent loss of availability was well fitted by a single exponential (continuous line). Both the rate and extent of use-dependent block were augmented by lowering [Na ]ï from 96 to 10 mò. The time constant for block development decreased from 389±39ms in 96 mò [Na ]ï to 86 ± 9 ms in 10 mò [Na ]ï (P < 10 É, Table 1). If inducing slow inactivation plays a major role in usedependent lidocaine block, then recovery from usedependent block should recapitulate recovery from IM. Moreover, increasing the lidocaine concentration should increase the extent of use-dependent block, but should not alter the rate at which channels recover availability. Figure 1E shows the fraction of channels available to open (P2ÏP1) as the recovery interval at 100 mv is lengthened following a brief depolarization to 20 mv. A single exponential was fitted to fractional recovery data spanning an 8 to 5000 ms time range (continuous lines). In lidocainefree solutions, the time constant for recovery from IM was 335±87ms (10 mò [Na ]ï, Table 2), consistent with earlier results linking varying [Na ]ï (Benitah et al. 1999) and outer pore mutations (Kambouris et al. 1998) to IM. In 50 and 100 ìò lidocaine, the total number of channels exhibiting slow recovery progressively increased (i.e. the y intercept decreased), but the time constant for slow recovery was unchanged (see Table 2). Hence, the rate of recovery from lidocaine block was concentration independent and was kinetically indistinguishable from recovery from IM. Lidocaine induces a hyperpolarizing shift in the voltage dependence of INa availability (Bean et al. 1983), implying lidocaine-bound channels occupy a stabilized inactivated state. We therefore examined how [Na ]ï influences voltage-

4 40 Z. Chen and others J. Physiol dependent availability of INa. The time constant for development of IM was consistently û 500 ms (Table 1); hence, 5 s prepulses (Fig. 1D, inset) were used to achieve steady-state occupancy of IM while minimizing recruitment of even more stable (ultra-slow) inactivated states seen with longer ( 1 min) depolarizations (Kambouris et al. 1998; Todt et al. 1999). Figure 1D shows that lowering [Na ]ï induced a 7 mv hyperpolarizing shift in steady-state Figure 1. The effects of [Na ]ï on slow inactivation and use-dependent lidocaine block A, whole-cell currents recorded from Xenopus oocytes in the absence (top) and presence (bottom) of 50 ìò lidocaine. Oocytes were bathed in solutions containing either 96 mò (left) or 10 mò [Na ]ï (right). The superimposed current pairs were recorded at the beginning of a pulse of 1000ms duration to 40 mv (holding potential 100 mv, bar indicates zero current level) and then during a second pulse to 40 mv which followed a 20 ms of recovery period at 100 mv. B, symbols indicate peak INa (means ± s.e.m.) during the P2 pulse relative to the P1 pulse with varying P1 pulse durations (protocol inset). Oocytes were bathed in either 96 mò [Na ]ï (n = 7), 24 mò [Na ]ï (n = 13), or 10 mò [Na ]ï (n = 8). Continuous lines are single exponential fits (y = y0 + Aexp( tïô)) to the mean data; kinetic parameters derived from fitting data measured in individual oocytes are summarized in Table 1. C, the symbols show data obtained in an identical manner in solutions containing 50 ìò lidocaine and either 96 mò [Na ]ï (n = 10), 24 mò [Na ]ï (n = 7), or 10 mò [Na ]ï (n = 8). Continuous lines show single exponential fits to the mean data (parameters summarized in Table 1). Dotted lines show the results in drug-free conditions (from panel B) for comparison. D, voltage-dependent availability is plotted for oocytes bathed in 10 mò [Na ]ï (n = 9) or 96 mò [Na ]ï (n = 5). The continuous lines are Boltzman equations (1 + exp(v V½Ïä)) fitted to the mean data. Summary values for the mid-point (V½) and slope factor (ä) were, respectively, 62 0 ± 1 5 and 3 4 ± 0 2 mv in 10 mò [Na ]ï, and 55 4 ± 0 7 and 3 5 ± 0 2 mv in 96 mò [Na ]ï. V½ values in 96 and 10 mò [Na ]ï differed significantly (P = 0 01). E, slow time-dependent recovery of availability following a 50 ms depolarization to 20 mv is plotted over an ms range. Recovery components more rapid than 8 ms (fast inactivation, deactivation) are not considered. Data are from oocytes bathed in 10 mò [Na ]ï (n = 10), 96 mò [Na ]ï ìò lidocaine (n = 12), 10 mò [Na ]ï + 50 ìò lidocaine (n =7) and 10mÒ [Na ]ï ìò lidocaine (n = 6). Continuous lines are single exponential fits (y = y0 + A(1 exp( tïô))) to the mean data; kinetic parameters are given in Table 2.

5 J. Physiol Lidocaine and slow inactivation 41 availability, consistent with the notion that external Naº destabilizes IM (Benitah et al. 1999). In this case, if usedependent lidocaine block requires stable occupancy of IM, then raising [Na ]ï should speed recovery from lidocaine block. Figure 1E shows that raising [Na ]ï to 96 mò not only reduced the extent of slow inactivation (filled vs. open circles in 100 ìò lidocaine), but also speeded the time constant for recovery from block (Table 2:252 ± 12 ms vs. 368±58ms, P = 0 02). Effect of lowering [Na ]ï on lidocaine block in a fastinactivation mutant The kinetic overlap between fast inactivation and entry into IM in low [Na ]ï makes it difficult to rule out the possibility that the inhibitory effect of [Na ]ï on lidocaine block is due to an indirect effect on fast inactivation. We therefore examined the interaction between [Na ]ï and lidocaine block in mutant ì1 channels with impaired fast inactivation (F1304Q) (West et al. 1992; Lawrence et al. 1996). Figure 2A shows slowly decaying F1304Q currents during a 200 ms depolarization in 96 mò (left) or 10 mò [Na ]ï (right) before and during exposure to lidocaine (200 ìò). In drug-free conditions, the slow decay of F1304Q current has been linked to slow inactivation (Townsend & Horn, 1997). The rate of this slow decay is increased either by lowering [Na ]ï (Fig. 2A, see also Townsend & Horn, 1997) or by lidocaine (Fig. 2A and Table 2. Kinetic parameters for recovery from IM Lidocaine [Na ]ï A ô (ms) 0 10mÒ 0 09 ± ± ìò 10 mò 0 38 ± 0 06 * 360 ± ìò 10 mò 0 47 ± 0 04 * 368 ± ìò 96 mò 0 29 ± 0 02 * 252 ± 12 ** The function y = y0 + A(1 exp( tïô)) was fitted to the data in Fig. 1, panel E. Fitted parameters (means ± s.e.m.) are shown. Lidocaine increased the fraction of slowly recovering channels (* P < versus 0 ìò lidocaine, 10 mò [Na ]ï). In 10 mò [Na ]ï, lidocaine did not change the time constant for slow recovery (ô), although in 100 ìò lidocaine raising [Na ]ï from 10 to 96 mò significantly decreased ô (**P = 0 02). Balser et al. 1996b). Moreover, lowering the [Na ]ï to 10 mò potentiated the effect of lidocaine, markedly speeding F1304Q current decay (Fig. 2A, right). In Fig. 2B these [Na ]ï-dependent differences in lidocaine action are summarized by plotting the fraction of peak current remaining 200 ms into the depolarizing pulse (f200). Lowering [Na ]ï from 96 to 10 mò hastened the current decay of F1304Q produced by either 50 or 200 ìò lidocaine (P < 0 001). Hence, in conditions where IM is facilitated (by Figure 2. Interaction between [Na ]ï and lidocaine block in F1304Q channels A, whole-oocyte currents elicited at 20 mv (holding potential 100 mv, bar indicates zero current level) before and during lidocaine exposure (200 ìò) in either 96 mò [Na ]ï (left) or 10 mò [Na ]ï (right). Lidocaine enhanced the decay of whole-cell current to a greater extent when [Na ]ï was reduced. B, summary data plotting the fractional reduction in current by 200 ms (f200) at 20mV in 96 mò [Na ]ï (control n = 8, 50 ìò lidocaine n = 8, 200 ìò lidocaine n =8) or 10mÒ [Na ]ï (control n =7, 50ìÒ lidocaine n = 4, 200 ìò lidocaine n = 5). At each lidocaine concentration (0, 50, 200 ìò), current decay was hastened by lowering [Na ]ï from 96 to 10 mò (*P < 0 001).

6 42 Z. Chen and others J. Physiol low [Na ]ï) and fast inactivation is destabilized, low concentrations of lidocaine rapidly induce the formation of a stable non-conducting state when the membrane is depolarized. Lidocaine block in a mutant channel with impaired slow inactivation Previous work has shown that mutation of a P-loop tryptophan (ì1-w402) reduces an intermediate kinetic component of slow inactivation (IM) (Balser et al. 1996a). Alanine replacement (W402A) primarily attenuates the IM state (in preference to more long-lived slow inactivated states), and also reduces use-dependent lidocaine block (Kambouris et al. 1998). However, this correlation between reduction of IM and reduced lidocaine block could be fortuitous: the mutation could modify slow inactivation gating and the lidocaine receptor (directly or indirectly) through independent mechanisms. Nonetheless, if augmentation of lidocaine block by low [Na ]ï was attenuated by the W402A mutation, the hypothesis of a mechanistic linkage between IM occupancy and lidocaine block would be strengthened. Figure 3 shows the time course for development of slow inactivation (open symbols) and use-dependent lidocaine block (filled symbols) in W402A channels bathed in 10 mò [Na ]o. The continuous lines are single exponential fits to these data. The comparable exponential fits to the wild-type data (from Fig. 1B and C) are shown by the dashed lines. In drug-free conditions, the IM state developed more slowly (ô = 1870 ms) and less fully (A = 0 19) in the W402A channel relative to the wild-type channel (Table 1, comparable parameters for wild-type were: ô = 452 ms, A = 0 38). In 50 ìò lidocaine, the rate (ô = 274 ms) and extent (A = 0 38) of use-dependent block was similarly reduced compared to wild type (Table 1, ô = 86 ms, A = 0 83). Hence, the W402A mutation reduced the slow inactivation normally induced by lowering [Na ]o andatthe same time inhibited the development of use-dependent lidocaine block in low [Na ]o. These findings suggest that external Na and the P-loop mutation both modulate usedependent lidocaine action through effects on the IM state. Electrochemical gradient and use-dependent block These findings point toward a mechanistic link between occupancy of the IM state and use-dependent lidocaine action. However, all the currents measured in the oocyte experiments were inward (Fig. 1A) and lidocaine block consistently increased in parallel with a reduced electrochemical gradient for Na (Fig. 1C). We therefore reexamined the effect of lowering [Na ]ï on lidocaine block in ì1 Na channels expressed in HEK-293 cells using the whole-cell voltage-clamp method (see Methods). Using this approach, we could raise [Na]é to 150 mò, and thereby induce outward currents in low [Na ]ï at typical depolarized membrane potentials. Figure 4A shows INa measured before (left) and during (right) exposure to 10 ìò lidocaine in either 10 mò [Na ]ï (top) or 150 mò [Na ]ï (bottom). In each panel, the superimposed Na currents were elicited by an 800 ms depolarizing pulse to 20 mv and a subsequent brief (50 ms) pulse to the same membrane potential, separated by a 20 ms interval at 100 mv to allow recovery from fast inactivation (protocol inset, Fig. 4A). Hence, the fractional decrement in peak INa between the superimposed currents (pulse 2Ïpulse 1) reflects slow inactivation or usedependent lidocaine block. Although the macroscopic currents were somewhat smaller in cells bathed in low [Na ]ï due to gating effects, the absolute driving force for Na flux Figure 3. Lidocaine block of a mutant channel (W402A) with impaired slow inactivation The time course for development of slow inactivation (9, n = 6) and use-dependent lidocaine block (8, n = 6) is shown for W402A channels bathed in 10 mò [Na ]o. The continuous lines are single exponential fits to these data (similar to Fig. 1, parameters given in text). Comparable results in wild-type channels at thesame[na ]ï(fromfig. 1B and C) are shown by the dashed lines. Compared to wild type, the W402A mutation markedly reduced the extent of slow inactivation in 10 mò [Na ]o, and also significantly reduced the development of use-dependent lidocaine (lido) block in 10mÒ [Na ]o.

7 J. Physiol Lidocaine and slow inactivation 43 through the open channel at 20 mv was 28 mv greater in 10 mò [Na ]ï (+48 mv vs. 20 mv). The left panels (drug free) indicate that slow inactivation was still 15 % greater in low [Na ]ï despite the change in direction of current flow or the increased electrochemical gradient (summarized data Fig. 4B, left). Use-dependent current reduction in lidocaine was 23% greater in low [Na ]ï (right panels, Fig. 4A; summary data Fig. 4B, right). Lidocaine block increased under conditions where slow inactivation was enhanced even though the currents in low [Na ]ï were outward and the electrochemical gradient driving INa was increased. DISCUSSION In a previous paper (Kambouris et al. 1998) we reported that alanine mutation of a P-loop tryptophan (W402A), external to both the selectivity (DEKA) ring (Heinemann et al. 1992) and the putative local anaesthetic receptor in domain IV S6 (Ragsdale et al. 1994), unexpectedly reduced use-dependent lidocaine block. Since the W402A mutation also reduced IM, the results suggested the possibility that this component of slow inactivation gating and usedependent lidocaine action are mechanistically linked. However, although these two effects of the mutation (gating Figure 4. Na channel á subunits expressed in HEK-293 cells A, Na currents were recorded from HEK cells prior to (left) and during lidocaine exposure (right, 10 ìò) in either 10 mò (top) or 150 mò [Na ]ï (bottom). The pipette [Na]é was 150 mò, so that INa at 20 mv was outward in 10 mò [Na ]ï (Vclamp ENa = +48 mv), but inward in 150mÒ [Na ]ï (Vclamp ENa = 20 mv). In each case, the first (larger) INa was elicited by an 800 ms depolarizing pulse to 20 mv, while the second (smaller) INa was recorded from a brief (50 ms)stepto 20mVthatfolloweda 20 ms recovery interval at 100 mv (see inset voltage clamp protocol). B, the bar graph plots the fractional change in peak INa between the superimposed currents in panel A (pulse 2Ïpulse 1) for each of the four bath conditions. The number of cells recorded in each condition are shown in parentheses. * Significant differencefromthematchedconditionin150mò [Na ]ï (P < 0 05).

8 44 Z. Chen and others J. Physiol and pharmacological) shared similar kinetic features, we could not exclude the possibility that the mutation modified IM and the local anaesthetic receptor through distinct mechanisms. Our rationale for the present study was to explore this potential linkage using a different, nonmutagenic approach. Our results show that lowering [Na ]ï both augments entry into the IM state and potentiates usedependent block by lidocaine (Fig. 1). With a view to bridging this result to our prior mutagenesis work (Kambouris et al. 1998), we show (Fig. 3) that W402A reduced the effect of lowering [Na ]ï on both slow inactivation gating and lidocaine block, suggesting that Figure 5. A model for lidocaine block that invokes [Na ]ï-dependent slow inactivation A, in this two-affinity modulated receptor model, IM represents the slow inactivated state for which lidocaine has high affinity, while NIM represents all other states (closed, open, fast inactivated) for which lidocaine has lower affinity. The forward rate constant controlling slow inactivation in drug-free conditions (á) was allowed to differ in 96 mò vs. 10 mò [Na ]ï, and the forward rate constant for slow inactivation with lidocaine bound (á') was determined by á and the lidocaine off-rates (k2off, k1off) from detailed balances (eqn (1)). The lidocaine kon was fixed at 10Ì m s (see text). The remaining rate constants (â, k2off, k1off) were derived from fitting the model to the data, but were constrained to be the same in high or low [Na ]ï. In the model, both lidocaine-bound states (Lido NIM, IM Lido) were considered to be non-conducting, so that NIM is the only state where channels are available to open and conduct Na. B, the data shown were replotted from Fig. 1B and C. The ordinate (PµÏP1) represents the probability that channels are not slow inactivated, and are therefore available to open. Using a numerical integration method (Methods), the model was evaluated at each time point (abscissa, Fig. 5B) and the calculated probability of occupying the NIM state was compared directly to the PµÏP1 data value. At t = 0, the probability of occupying the NIM state was set to 1 0 (PµÏP1 = 1 0). The model was fitted to the data in four conditions (highïlow [Na ]ï, ± lidocaine) simultaneously using a simplex-based global fitting approach (Nelder & Mead, 1965) that updates the parameters to minimize the sum-of-squares error. Continuous lines show the non-linear least-squares fit of the model to the mean 10 mò and 96 mò [Na ]ï data. Fitted parameters for the rate constants controlling slow inactivation were: á in 96 mò [Na ]ï = s, á in 10 mò [Na ]ï = 0 92 s, and â = 1 41 s. Fitted values for lidocaine k1off and k2off were 127 ² 10Å s and 386 s, giving Kd values for the NIM and IM states of 1 27 mò and 3 86 ìò, respectively.

9 J. Physiol Lidocaine and slow inactivation 45 [Na ]ï and the P-loop mutation both modify lidocaine block through effects on the IM state. In addition, we find that these effects of [Na ]ï on lidocaine action are not abolished by mutagenic disruption of fast inactivation (Fig. 2). An allosteric model for lidocaine-induced use dependence We considered whether the opposing effects of Naº and lidocaine on use-dependent channel availability could be rationalized by a modulated receptor scheme (Fig. 5A). For simplicity, all gated conformational states other than the slow inactivated state (IM) were grouped together as available states for Na conduction (NIM). Included among the NIM states is the fast inactivated state, recognizing that (1) fast-inactivation gating structures recover rapidly despite use-dependent lidocaine block (Vedantham & Cannon, 1999) and (2) under the conditions of our protocol (Fig. 1) fast inactivated channels were allowed sufficient time to recover, and were therefore available to open. Discrete block of unitary current cannot be observed with lidocaine (Grant et al. 1989), so the true on-rate for lidocaine binding remains uncertain. Hence, we arbitrarily set the onrate for lidocaine binding at a fixed upper limit approaching diffusion (10Ì m s ), and allowed the unbinding rates to determine the stabilities of the two (NIM and IM) drugbound complexes. We further assumed that channels in the IM state have the higher affinity for lidocaine (i.e. a lower koff). We allowed the forward rate constant for slow inactivation (á) to differ in 10 and 96 mò mò Naº, but assumed (in the simplest case) the rate constant controlling recovery from slow inactivation (â) was not influenced by [Na ]ï or drug binding (Fig. 1E), and was therefore constrained to be identical for drug-free (top of Fig. 5A) and drug-bound (bottom of Fig. 5A) channels. Based on these definitions and detailed balances, the forward rate constant for slow inactivation with lidocaine bound (á') is explicitly defined as a function of á and the off-rates for lidocaine binding (k1off, k2off)asfollows: á'=ák1offïk2off. (1) Since we assume lidocaine binds with greater affinity to the IM state (i.e. k2off < k1off),therateofslowinactivation with lidocaine bound must exceed the rate under drug-free conditions (á' > á). In this sense, the lidocaine action parallels that of allosteric effector molecules (Monod et al. 1965), where drug binding allosterically promotes an intrinsic channel conformation (i.e. slow inactivation). Figure 5B shows model fits (continuous lines) to the 10 mò and 96 mò [Na ]ï data from Fig. 1B and C. The fitted rate constants and binding affinities (see legend) were all constrained to be identical in 10 and 96 mò [Na ]ï, with the exception of á which decreased 15-fold as [Na ]ï was raised, consistent with slowed development of IM. By speeding entry of drug-bound channels into the IM state, the model simulates the marked increase in the rate at which lidocaine block develops when [Na ]ï is reduced. The model converged on Kd values for the NIM and IM states (1 27 mò and 3 86 ìò, respectively) that resemble previously determined experimental Kd values for tonic and inactivated-state lidocaine block of skeletal muscle Na channels (Nuss et al. 1995). Processes other than IM may influence use-dependent Na channel block Sodium channel ì1 á subunits expressed in oocytes exhibit an unusual degree of slow (mode 2) gating and slow inactivation when the â1 subunit is omitted (Zhou et al. 1991). In the oocyte experiments (Figs 1 3), the â1 subunit was coexpressed in order to minimize these phenomena, and thereby more closely recapitulate the in vivo gating phenotype. However, use-dependent lidocaine block of Na channels expressed in oocytes is attenuated by â1 coexpression, a phenomenon that may result from â1 effects on slow gating (Balser et al. 1996c) or from a direct â1 interaction with the lidocaine receptor (Makielski et al. 1996). Thus, our intepretation of the lidocaine [Na ]ï interaction in oocytes could be confounded if there are changes in á â1 coupling as [Na ]ï changes. However, gating of the ì1 Na channel á subunit in cultured mammalian cells (in contrast to oocytes) is almost entirely rapid, regardless of whether the â1 subunit is coexpressed (Ukomadu et al. 1992). The experiments in Fig. 4were performed without â1 subunit coexpression and recapitulated the results in oocytes, suggesting that these findings are not contingent on the expression system or variable á â1 coupling. Although our results cannot exclude the possibility that Naº inhibits use-dependent lidocaine action through a competitive or otherwise gating-independent mechanism, several factors support a role for slow inactivation. The wild-type Na channel occupies the open state (and conducts Na ions) for only a few milliseconds (coincident with peak INa), while use-dependent lidocaine block develops over hundreds of milliseconds (Table 1, Fig. 1C), during a time period when channels are inactivated and non-conducting. Moreover, lowering the[na ]ï to 10 mò still produced no first-pulse (so-called tonic ) block in 100 ìò lidocaine (see Results, paragraph 2), a concentration that induces substantial usedependent block under these conditions (Fig. 1C). At the same time, lowering [Na ]ï enhanced IM entry and lidocaineuse dependence, while both effects were reduced by a mutant (W402A) that inhibits the same slow inactivation gating process (Fig. 3). Finally, use-dependent lidocaine block tracks the degree of slow inactivation in a manner independent of the electrochemical gradient for Na (Fig. 4). Nonetheless, it remains possible that a competitive interaction between Na and lidocaine influences the results to some degree, especially if the two species interact at a site where Na occupancy correlates poorly with current magnitude. Further, while Naº probably inhibits slow inactivation through occupying a site external to the selectivity filter

10 46 Z. Chen and others J. Physiol (Townsend & Horn, 1997), it is still conceivable that Naº and the W402A mutation modify slow inactivation and usedependent lidocaine action via independent, albeit parallel mechanisms. A competitive interaction between Na and lidocaine in the permeation pathway could have influenced the F1304Q results (Fig. 2) given the much longer singlechannel open time and high frequency of re-openings for this ì1 mutant (Lawrence et al. 1996). This could partly explain why lowering [Na ]ï seemed to potentiate lidocaine suppression of F1304Q INa to a greater extent than wild type. It is well known that cardiac (hh1) INa is more sensitive to use-dependent lidocaine action than skeletal muscle INa (Nuss et al. 1995; Wang et al. 1996). In light of recent reports that find slow inactivation is more prominent in the skeletal muscle isoform (Richmond et al. 1998), a positive interaction between slow inactivation and lidocaine sensitivity may seem paradoxical. However, the component of slow inactivation that is amplified in skeletal muscle has a kinetic signature (ô = 5 20 s) far slower than the intermediate IM state discussed here (ô < 500 ms). Additionally, we have recently shown that hh1 channels recover from IM states more slowly than ì1 channels, and this may partly account for the enhanced sensitivity of the cardiac isoform to usedependent lidocaine action (Nuss et al. 2000). Other differences between classic slow inactivation and the IM state warrant discussion in this context. Most studies find that slow inactivation competes with fast inactivation (Rudy, 1978; Featherstone et al. 1996), whereas our prior studies suggest that fast inactivation and the IM state are positively coupled. After relatively brief periods of depolarization (< 100 ms) sufficient to induce IM but not classic slow inactivation, ì1 channels carrying the III IV linker IFM QQQ mutation exhibited less slow inactivation than wild type (Balser et al. 1996a). In addition, following a 1 s depolarization, partly destabilizing fast inactivation (ì1 F1304Q) hastened recovery from an intermediate (ô 500 ms) component of inactivation, but slowed recovery from a more stable (ô 5 s) inactivated state (Nuss et al. 1996). Taken together, these results support the notion that fast inactivation and IM are positively coupled, while fast inactivation and classic slow inactivation are inversely coupled. The mechanistic role of fast inactivation in lidocaine action remains unresolved. Accessibility of the III IV linker to covalent modification is reduced by inactivation, but accessibility recovers rapidly with hyperpolarization, and this recovery is not slowed by use-dependent lidocaine block (Vedantham & Cannon, 1999). While this result would suggest use-dependent lidocaine action does not involve fastinactivation gate trapping, mutations that severely disrupt fast inactivation (i.e. III IV linker IFM QQQ) markedly attenuate use-dependent block of peak INa (Bennett et al. 1995). At the same time, lidocaine markedly accelerates the decay of whole-cell current through F1304Q channels during sustained depolarization (Balser et al. 1996b), and this effect is potentiated by reducing [Na ]ï (Fig. 2). Wholecell current decay through F1304Q channels in drug-free conditions is multiexponential, and prior studies suggest this represents entry into multiple inactivated states. Specifically, whole-cell (Nuss et al. 1996) and single-channel analyses (Lawrence et al. 1996; Townsend & Horn, 1997) suggest that the early, more rapid decay phase represents entry into a destabilized fast inactivated state, while the slower decay that follows represents partitioning into more stable, slow inactivated states. Hence, prior studies have suggested that lidocaine may influence fast inactivation gating in III IV linker mutants by repairing the destabilized gating process to some degree (Balser et al. 1996b; Fanet al. 1996). Therefore, the salutary effect of lowering [Na ]ï on lidocaine action in Fig. 2 could be influenced by concurrent lidocaine effects on fast and slow inactivation gating. We previously found that the W402A mutation reduced the rate of entry into the IM state during depolarization (Kambouris et al. 1998). However, the mutation did not shift the steady-state availability curve to depolarized potentials (Kambouris et al. 1998), contrary to the expectation for an inactivation-destabilizing intervention (and in contrast to raising [Na ]ï in Fig. 1D). It is important to note that Fig. 1D assesses the equilibrium between inactivated states and closed states during modest (sub-threshold) depolarization, but does not consider inactivated-state stability at more depolarized membrane potentials sufficient to open the channel. Figure 1B and prior data (Kambouris et al. 1998) indicate that either raising [Na ]ï or the W402A mutation decrease the rate of entry into IM at depolarized membrane potentials sufficiently to activate channels. W402A antagonizes the effects of reducing [Na ]ï on both IM and usedependent development of lidocaine block (Fig. 3), suggesting these two interventions share common mechanisms at depolarized membrane potentials. At hyperpolarized potentials, in Xenopus oocytes with the â1 subunit coexpressed, the W402A mutation seemed to have little effect on the time constants for recovery from slow inactivation, consistent with the absent shift in steady-state availability (Kambouris et al. 1998). Conversely, raising [Na ]ï increases the rate of recovery from slow inactivation at hyperpolarized voltages (Townsend & Horn, 1997), and our data (Fig. 1D) show that Naº shifts the steady-state availability curve at subthreshold voltages in a positive direction. Both findings suggest IM is destabilized by Naº at negative voltages, and Fig. 1E shows that the rate of recovery from use-dependent lidocaine block at 100 mv is also hastened when [Na ]ï is raised. The evidence that mutating residues accessible to the pore modifies slow inactivation (Balser et al. 1996a; Todt et al. 1999) suggests a relationship between the Na permeation pathway and the IM gating mechanism. An analogous slow inactivation process in K channels (so-called C-type inactivation) involves conformational rearrangements in the outer pore, and is inhibited by raising the extracellular

11 J. Physiol Lidocaine and slow inactivation 47 concentration of K (Lopez-Barneo et al. 1993; Liu et al. 1996). Moreover, use-dependent block of Shaker channels by intracellular pore blockers involves induced C-type inactivation (Baukrowitz & Yellen, 1996). Hence, our results may suggest some general similarities between use-dependent drug action in Na channels and K channels. We caution, however, that these findings should not be generalized to suggest occupancy of IM fully explains the use-dependent current suppression by all local anaesthetics. In contrast to lidocaine, which exhibits a preference for inactivated channels, high-affinity binding to the open channel may play a greater role in use-dependent block by disopyramide (Barber et al. 1992) as well as quinidine and flecainide (Ragsdale et al. 1996). Hence, future studies will determine the relative role of the IM state in the action of other Na channel blocking compounds with diverse use-dependent kinetic properties. 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