Gp-7812

The Position of the Fast-Inactivation Gate during Lidocaine Block of Vasanth Vedantham* and Stephen C. Cannon*‡§ From the *Program in Neuroscience, Division of Medical Sciences and ‡Department of Neurobiology, Harvard Medical School, Boston,Massachusetts 02115; and §Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02214 Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slow-ing the rate of Naϩ channel repriming after depolarizations. It has been proposed that the fast-inactivation mech-anism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action hasbeen difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For thatreason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substitutedat phenylalanine 1304 in the rat adult skeletal muscle sodium channel ␣ subunit (rSkM1) with [2-(trimethylam-monium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of thefast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence ofsite 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. Moresignificantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from aslowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumula-tion of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activa-tion pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.
local anesthetic • SkM1 • antiarrhythmic • patch clamp • methanethiosulfonate defective Na channels are more resistant to some ofthe effects of local anesthetics than are normal chan- The gating of voltage-sensitive Naϩ channels deter- nels (Cahalan, 1978; Wang et al., 1987; Yeh and Tan- mines the time course of the rising phase of the action potential and the length of the refractory period in However, a number of questions remain. Is there co- nerve, skeletal muscle, and heart. As a result, Naϩ chan- operativity, negative or positive, between lidocaine and nels are the targets of several classes of drugs that mod- the fast-inactivation gate? Does use-dependent block in- ulate electrical excitability, including antiarrhythmics, volve an accumulation of fast-inactivated sodium chan- local anesthetics, antimyotonics, and anticonvulsants.
nels? Is there a direct, mutually stabilizing interaction Among these, lidocaine and related local anesthetics between lidocaine binding and closure of the fast-inac- have received a great deal of experimental attention tivation gate? Answers to these questions have been dif- because of their striking effects on Naϩ channels: they ficult to obtain, primarily because gating transitions induce a voltage-dependent inhibition of the peak cur- that occur in drug-bound channels do not involve rent upon infrequent stimulation (tonic block), and changes in ionic current, and are thus electrophysio- they dramatically slow repriming of sodium channels logically silent. Indeed, neither the ionic current nor after depolarizations (use-dependent block), thereby the gating current provides direct information about preventing the repetitive discharges that occur in car- the position of the fast-inactivation gate during local diac arrhythmia, epilepsy, and myotonia (Butterworth To circumvent this difficulty, we have employed a con- Several experimental findings implicate a role for the formational marker for the position of the fast-inactiva- Naϩ channel fast inactivation mechanism in generating tion gate, the reactivity of a cysteine substituted for phe- these effects: depolarization favors local anesthetic nylalanine 1304 in the rat adult skeletal muscle sodium binding, many local anesthetics shift the steady state channel ␣ subunit with the thiol-modifying reagent availability (hϱ) curve in the hyperpolarizing direction [2-(trimethylammonium)ethyl]methanethiosulfonate (Bean et al., 1983; Hille, 1977), and fast-inactivation– (MTS-ET).1 Site 1304 lies in the sodium channel III–IV Address correspondence to Dr. Stephen Cannon, EDR413A, Massa-chusetts General Hospital, Boston, MA 02214. Fax: 617-726-3926; 1Abbreviation used in this paper: MTS-ET, [2-(trimethylammo- J. Gen. Physiol. The Rockefeller University Press • 0022-1295/99/01/7/10 $2.00 interdomain and plays a crucial role in fast inactivation seal formed without suction were included in the data set even if (West et al., 1992). In a previous study, we have demon- they did not last long enough for a switching test (such patches strated that the reaction rate of the substituted cysteine invariably exhibited rapid exchange kinetics).
with MTS-ET follows closely the voltage dependence ofsteady state fast inactivation (Vedantham and Cannon, 1998). This has enabled us to use this reaction rate as a Curve fitting was performed off line using a custom AxoBasic measure of the fraction of channels whose fast-inactiva- analysis program (Axon Instruments, Inc.) or SigmaPlot (Jandel tion gates are shut under conditions of particular interest.
Scientific Co.). Steady state fast inactivation, hϱ, and the voltage In this study, we have determined the position of the dependence of the modification rate were fitted to Boltzmanncurves with maximum values, I fast-inactivation gate in channels that are bound to the local anesthetic drug lidocaine under several experi- where V1/2 is the voltage at half maximum, and k is the slope fac- mental conditions. We found that lidocaine does not tor. Error bars indicate means Ϯ SEM.
compete with closure of the fast-inactivation gate; on For modification experiments, the fraction modified after a the contrary, the fraction of blocked channels that are given pulse of MTS-ET was calculated by averaging the value ofthe Naϩ current between 3 and 3.5 ms after depolarization to fast inactivated increases with depolarization and with Ϫ20 mV. For each experiment, the fraction modified (F) versus drug concentration. More surprisingly, our data show cumulative exposure time (texp) were fit to a monoexponential: that recovery from fast inactivation precedes recovery of the ionic current in drug-bound channels and is just ciprocal of the reaction rate, Fo is the mean value of the current as fast as recovery in the absence of drug, demonstrat- between 3 and 3.5 ms before any exposure has occurred, andI , the maximum value of the mean current between 3 and 3.5 ing that use-dependent block does not involve an accu- ms, was a free parameter in the fit.
mulation of fast-inactivated channels. Based on thesefindings, we propose a model in which lidocaine bind-ing affinity is modulated by gating transitions along the activation pathway, without a direct interaction be-tween lidocaine binding and fast inactivation.
Modification of F1304C by MTS-ET in the Presence All experiments were conducted in excised inside-out patches pulled from Xenopus oocytes coinjected with Expression of Naϩ Channels rat adult skeletal muscle sodium channel ␣ subunitF1304C and human ␤1 subunit RNA. Fig. 1 A shows The construction of cDNAs encoding F1304C and human Naϩ macroscopic current traces elicited by depolarization channel ␤1 subunit in pGEMHE is described in Vedantham and from Ϫ120 to Ϫ20 mV before and after a 5-s applica- Cannon (1998). RNA for F1304C and ␤1 subunit were all gener-ated by in vitro translation of linearized plasmids (Message tion of 8 ␮M MTS-ET to the intracellular side. As re- Machine kit; Ambion Inc.). Xenopus oocytes were harvested and ported previously for this channel (Vedantham and coinjected with F1304C ϩ human ␤1 RNA as described in Cannon, 1998) and for the rat brain IIA homologue Chen and Cannon (1995). Before electrophysiological record- (Kellenberger et al., 1996), MTS-ET modification ing, oocytes were incubated for 2–3 d at 18ЊC in ND-96 (96 mM causes an increase in the peak current and a dramatic NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES,pH 7.6) supplemented with pyruvate (2.5 mM) and gentamicin disruption of fast inactivation, consistent with the im- portance of F1304 (F1489 in the brain IIA channel) forfast inactivation (West et al., 1992).
As in the absence of lidocaine, MTS-ET increases the peak current and disrupts fast inactivation in the pres- Recording conditions and solution exchange were as described ence of 1.0 mM lidocaine (Fig 1 B). However, block by in Vedantham and Cannon (1998). The pipette solution con-tained (mM): 100 NaCl, 10 HEPES, 2 CaCl lidocaine reduces the apparent fraction of channels The bath contained 100 KCl, 10 HEPES, 5 EGTA, 1 MgCl that fail to inactivate and attenuates the increase in 7.6. Lidocaine powder (hydrochloride salt; Sigma Chemical Co.) peak current associated with MTS-ET modification.
was added to the bath solution in appropriate amounts to obtain These effects of lidocaine on F1304C-ET are similar to a final concentration of 0.5, 1, 2, 4, or 8 mM lidocaine. Stock so- its effects on fast inactivation–defective Naϩ channels lutions (2 mM) of MTS-ET (Toronto Research Chemicals Inc.)were prepared from the solid in 1 ml of distilled, deionized H studied in other contexts (Balser et al., 1996; Bennett and placed on ice at the beginning of each recording day. Appro- et al., 1995; Wang et al., 1987). When lidocaine is sub- priate amounts were diluted into 10 ml of bath solution (to a fi- sequently washed out, the current traces were indistin- nal concentration of 8 ␮M) after suitable patches were obtained guishable from the case when no lidocaine was present and immediately before use. MTS-ET solutions were never used during the modification (data not shown), demonstrat- for Ͼ10 min after dilution from the stock. Our method for deter-mining the fidelity of solution exchange is described in Vedan- ing that lidocaine does not prevent the modification re- tham and Cannon (1998), with one change: patches in which the Lidocaine and Naϩ Channel Inactivation Modification of F1304C with MTS-ET in the presence Measurement of the rate of MTS-ET modification of of lidocaine. (A) Naϩ currents recorded from inside-out patches F1304C in the presence of lidocaine. The experimental protocol excised from Xenopus oocytes injected with ␮1 F1304C and human shown in A consists of a series of 20-ms exposures of excised inside- out patches to 8 ␮M MTS-ET with test pulses between each expo- 1 subunit are shown before and after 5 s of exposure to 8 ␮M MTS-ET. Each trace is an average of 10 individual depolarizations sure. 1.0 mM lidocaine was present at all times. In B, selected from Ϫ120 to Ϫ20 mV. F1304C-ET has an increased peak current traces from the modification experiment described in A are super- (62%) and a dramatic failure of inactivation as compared with un- imposed. To determine the degree of modification after each modified F1304C. B shows experiments performed as in A with 1.0 trace, the value of the macroscopic current between 3 and 3.5 ms mM lidocaine added to the bath. Under these conditions, MTS-ET after depolarization was averaged. Rates were determined by fit- causes a modest increase in peak current (22%). Lidocaine re- ting the degree of modification of each trace as a function of cu- duces the steady state current of unmodified F1304C and causes mulative exposure time with a monoexponential containing a partial current decay of the noninactivating F1304C-ET current.
maximum value after complete modification, a nonzero initialvalue before modification, and a time constant as free parameters.
C shows these averages, normalized to the difference between themaximum value and the initial value, plotted against cumulative Measurement of the Modification Rate of in the Presence exposure time, with the normalized curve fit superimposed.
For accurate measurement of the rate of modification posure. The rate of modification was determined by av- of F1304C by MTS-ET, we used a rapid perfusion sys- eraging the value of the macroscopic current between tem (Vedantham and Cannon, 1998) to apply con- 3.0 and 3.5 ms after depolarization (Fig. 2 B), plotting trolled, brief exposures of MTS-ET to the intracellular the averages from successive traces against cumulative face of inside-out patches. Fig. 2 A shows an example of exposure time, and fitting the resulting curve with a such an experimental protocol: a series of 20-ms expo- monoexponential (Fig. 2 C). For the experiment sures to 8 ␮M MTS-ET at Ϫ120 mV, with a test pulse shown in Fig. 2, the time constant of this curve was 0.16 s, used to assay the macroscopic current between each ex- giving a rate of 0.781 ␮molϪ1 sϪ1.
Concentration Dependence of Site 1304 Accessibility in lidocaine concentrations below the Kd, the majority of tonic block is caused by lidocaine binding to noninacti-vated Naϩ channels. However, as drug concentration is We estimated tonic block in F1304C by measuring the increased, the fraction of inactivated channels appears peak current elicited from excised inside-out patches to increase as well, although we cannot rule out the by infrequent depolarization from Ϫ120 to Ϫ20 mV possibility that nonspecific effects cause the reduction under control conditions, in the presence of a fixed of reaction rate at such high drug concentrations.
concentration of lidocaine applied to the intracellularface (0.1, 0.5, 1.0, 2.0, 4.0, or 8.0 mM), and then back The Voltage Dependence of Site 1304 Accessibility Is Shifted in in control solution. The fraction of tonic block was ob- tained by dividing the peak current measured in thepresence of lidocaine by the average of the peak cur- We confirmed that lidocaine causes a hyperpolarizing rent measured before exposure to lidocaine and after shift in the steady state availability curve for F1304C us- washout. These data (Fig. 3, ᭹) were fit to a binding ing 1.0 mM lidocaine. In five experiments, the steady curve with a hill coefficient of 1.0, yielding a K state availability curve was measured with 200-ms We compared the relative accessibility of site 1304 to prepulses, first in control solution, and then in 1.0 mM the degree of tonic block at specific lidocaine concen- lidocaine. Each curve was fit independently to a Boltz- trations by dividing the modification rate in the pres- mann (Fig. 4). We observed a hyperpolarizing shift in ence of 0.5, 1.0, 2.0, or 4.0 mM lidocaine by the rate the half-maximal voltage (10.8 Ϯ 2.6 mV), and a reduc- measured with no lidocaine present (Fig. 3, ᭺). We tion in the maximum value of 22 Ϯ 6%.
found that the accessibility of site 1304 was less sensitive Although such a shift strongly suggests a stabilizing to lidocaine than the peak current (K ϭ effect of lidocaine on fast-inactivated channels, it is in pared with 1.9), indicating that, at Ϫ120 mV and Effect of lidocaine on the steady state availability curve of F1304C. hϱ curves were recorded with 200-ms prepulses, fol-lowed by a test pulse to Ϫ20 mV. For each patch, the hϱ curve wasmeasured in the absence and presence of 1.0 mM lidocaine (n ϭ5) in rapid succession. In other experiments without lidocaine, we Effect of tonic block on accessibility of site 1304. Tonic did not observe significant left shifts in gating after patch excision block of F1304C (᭹) was evaluated by dividing the peak current on the time scale of these experiments. Data from each curve was obtained from infrequent depolarizations to Ϫ20 from Ϫ120 mV fit with a Boltzmann with maximal value (Imax), half-maximal volt- in the presence of 0.1, 0.5, 1.0, 2.0, 4.0, or 8.0 mM lidocaine by the age (V1/2), slope (k), and nonzero plateau (c) as free parameters.
peak current in the absence of drug (n ϭ 4 for each point). These The graph shows data normalized to the maximum value for data were fit to a binding curve with a hill coefficient of 1.0, giving curves measured in the absence of lidocaine, and then averaged a Kd of 1.9 mM. Also shown in the graph is the modification rate of across trials. Without lidocaine, V1/2 ϭ Ϫ78.9 Ϯ 1.9 mV, k ϭ 6.2 Ϯ F1304C with 8 ␮M MTS-ET at Ϫ120 mV in the presence of 0.5 (n ϭ 0.7 mV, and c ϭ 0.11 Ϯ 0.02. In 1.0 mM lidocaine, V1/2 ϭ Ϫ89.7 Ϯ 4), 1.0 (n ϭ 10), 2.0 (n ϭ 8), or 4.0 (n ϭ 10) mM lidocaine divided 1.8 mV, k ϭ 7.1 Ϯ 0.4 mV, c ϭ 0.08 Ϯ 0.02, and Imax in 1.0 mM by the rate measured without lidocaine present (n ϭ 4) (᭺). The lidocaine was 78 Ϯ 6% of Imax in the absence of lidocaine. This rep- rates were measured using the protocol shown in Fig. 2 A. A fit to resents a hyperpolarizing shift in the apparent hϱ curve by 10.8 Ϯ the modification rate data gives a Ka of 6.6 mM.
Lidocaine and Naϩ Channel Inactivation principle possible that the decrease in available chan- data were fit with Boltzmanns containing maximum nels due to lidocaine does not involve closure of the rates (Rmax), minimum rates (Rmin), slope (k), and half- fast-inactivation gate, but rather to the intrinsic proper- maximal voltage (V1/2) as free parameters. With no ties of drug-block. Indeed, a hyperpolarizing shift in lidocaine present, Rmax ϭ 0.71 ␮molϪ1 sϪ1, Rmin ϭ 0.12 the apparent hϱ curve does not preclude the possibility ␮molϪ1 sϪ1, k ϭ 3.3 mV, and V1/2 ϭ Ϫ79.7 mV; while in of competition between lidocaine binding and closure 1.0 mM lidocaine, Rmax ϭ 0.68 ␮molϪ1 sϪ1, Rmin ϭ 0.08 of the fast-inactivation gate (for example, if lidocaine ␮molϪ1 sϪ1, k ϭ 6.6 mV, and V1/2 ϭ Ϫ89.9 mV.
and the fast-inactivation gate compete for a single bind- The voltage at half-maximal modification is shifted ing site). In the case of a stabilizing interaction, the by 10.2 mV in the hyperpolarizing direction, similar to voltage dependence of site 1304 accessibility should the 10.8-mV shift seen in the steady state availability shift in the same direction as the steady state availability curves (Fig. 2 A). Thus, depolarization increases the curve, while in the case of competition it would not.
fraction of blocked channels that are inactivated: at We therefore measured the modification rate at a va- Ϫ120 mV, very few of the blocked channels are inacti- riety of conditioning voltages using the protocol shown vated, while at Ϫ90 mV, nearly all of the blocked chan- in Fig. 5 A in the absence of lidocaine (Fig 5 B, ᭹), and nels (ف40–50% of the total) are inactivated. Both the in the presence of 1.0 mM lidocaine (Fig. 5 B, ᭺). The direction of the voltage shift and the fact that Rmin isunchanged demonstrate that lidocaine does not com-pete with the fast-inactivation gate. Rather, it tends to favor closure of the fast gate in a voltage-dependentmanner. Also, the Rmax was not significantly differentfor control and 1.0 mM lidocaine. To confirm this con-vergence of Rmax values, further experiments were per-formed at conditioning voltages of Ϫ160 and Ϫ200mV. We found no significant difference between con-trol and 1.0 mM lidocaine conditions (data not shown).
Lidocaine Does Not Impede Recovery of Site 1304 Accessibility Use-dependent block results from the slowing of the re-covery of Naϩ channel availability, and explains the ability of lidocaine to prevent rapid, high-frequency dis-charges in excitable tissues (Bean et al., 1983). We mea-sured this effect in F1304C inside-out macropatches us-ing a two-pulse recovery protocol with a 20-ms condi-tioning pulse to 0 mV, a variable recovery period, and atest pulse to 0 mV. The peak current from the test pulsedivided by the peak current from the conditioningpulse is a measure of the amount of repriming thattakes place during the recovery period. Experimentswere performed in control solution (Fig. 6, ᭹), and in1 mM lidocaine (Fig. 6, ᭺). Consistent with previousstudies, lidocaine dramatically slowed recovery of Naϩchannel availability—by ف100-fold under our experi- Effect of lidocaine on the voltage dependence of site 1304 accessibility. The rate of MTS-ET modification of F1304C was If use-dependent block involves accumulation of fast- measured at a variety of voltages, as shown in A. Excised, inside-out inactivated channels, then the accessibility of site 1304 patches underwent a series of 300-ms depolarizations: 200 ms to should be reduced for hundreds of milliseconds after a achieve steady state fast inactivation, followed by 50 ms for expo- brief depolarization, in accordance with the reduction sure to 8.0 ␮M MTS-ET, and a final 50-ms period after exposure toinsure complete washout of MTS-ET before repolarization. After in Naϩ channel availability. However, if recovery from each 300-ms depolarization, the patch was maintained at Ϫ120 mV fast inactivation is unaffected by lidocaine, then the ac- for enough time to insure complete recovery (2–8 s, depending on cessibility of site 1304 after a short depolarization the conditioning voltage) before assaying macroscopic current should not change in the presence of lidocaine, even with a test pulse to Ϫ20 mV. Modification rates are shown in B though Naϩ channel availability is reduced.
when the experiments were conducted in the absence (᭹) andpresence ( The protocol we used to resolve this issue (Fig. 7 A) ᭺) of 1.0 mM lidocaine. (On average, n ϭ 6, n ϭ at least consisted of a series of 20-ms conditioning pulses to 0 expected if site 1304 accessibility mirrored the availabil-ity of Naϩ current (ف0.13 ␮molϪ1 sϪ1; see Fig. 7 legendfor calculation). These data demonstrate that recoveryfrom fast inactivation is not significantly affected by lido-caine, and thus that use-dependent block does not in-volve the accumulation of fast-inactivated Naϩ channels.
Lidocaine slows the repriming of Naϩ channels after brief depolarizations. A two-pulse recovery protocol was used to as-sess the rate of repriming of F1304C channels after a 20-ms depo-larization to 0 mV. The peak current measured during the testpulse was divided by the peak current measured during the condi- tioning pulse, and plotted as a function of the recovery period. Inthe absence of lidocaine, the current recovered almost completelywithin 10 ms, while in the presence of 1.0 mM lidocaine, the timeconstant of recovery is roughly 100-fold slower. The shaded areaindicates the duration of MTS-ET exposure relative to Naϩ chan-nel repriming for the protocol shown in Fig. 7 A.
Lidocaine does not slow the return of site 1304 accessi- mV, each followed by a brief, experimentally measured bility after brief depolarizations. In the protocol diagrammed in A, 7.5-ms delay at Ϫ120 mV, and then a 20-ms exposure to 20-ms exposures to 8 ␮M MTS-ET were timed to occur 7.5 ms after 8 ␮M MTS-ET, also at Ϫ120 mV. Relative to the time 20-ms depolarizations to 0 mV. The macroscopic current was as- course of Naϩ channel repriming, the duration of MTS- sayed between each exposure, and modification rates were mea- ET exposure corresponds to the shaded area of Fig. 6 sured as shown in Fig. 2. The 7.5 ms reflects the experimentallymeasured delay between the voltage command to the piezoelectric B. After each depolarization and MTS-ET exposure, stack (which occurred exactly at the end of the conditioning sufficient time was allowed for complete recovery be- pulse) and the commencement of solution exchange (for details, fore assaying the macroscopic current. Experiments see Vedantham and Cannon, 1998). B shows the average of several were performed in control solution and in 1.0 mM modification experiments conducted with or without 1.0 mM lidocaine. Averages of modification time courses, anal- lidocaine. The solid lines are exponentials whose time constantsare the mean values of the time constants obtained from fits to ogous to the individual time course in Fig. 2 C, are data from individual experiments. The dashed line reflects the shown in Fig. 7 B (᭹, control; ᭺, 1.0 mM lidocaine).
curve that would be expected if the accessibility of site 1304 paral- The reaction rates were not significantly different for leled the degree of Naϩ channel availability in 1.0 mM lidocaine the two conditions: 0.63 Ϯ 0.07 ␮molϪ1 sϪ1 for control, during the exposure. The fraction of current recovered in the and 0.60 Ϯ 0.07 ␮molϪ1 sϪ1 for 1.0 mM lidocaine, and presence of 1.0 mM lidocaine after 8-, 10-, and 20-ms recoverytimes was averaged (corresponding to the shaded area in Fig. 6), are within 10–14% of Rmax, the maximum rate of modi- giving 0.2962. An accessibility of 0.30 predicts a rate of 2.1 sϪ1 at 8 fication estimated from the data of Fig. 5. The dashed ␮M MTS-ET (predicted rate ϭ 0.30 ϫ (Rmax Ϫ Rmin) ϩ Rmin), or line shows the modification time course that would be Lidocaine and Naϩ Channel Inactivation Our next set of experiments on the voltage depen- dence of site 1304 accessibility in the presence of Accessibility of Site 1304 During Lidocaine Block lidocaine showed a 10.2-mV hyperpolarizing shift of The major findings of this study are that (a) lidocaine the half-maximal modification rate, similar to the 10.8- does not compete with the fast-inactivation gate, (b) mV hyperpolarizing shift of the V1/2 of the hϱ curve.
lidocaine potentiates the degree to which depolariza- However, Rmax and Rmin were not significantly changed, tion favors closure of the fast-inactivation gate, and (c) even though the maximum value of the hϱ curve was re- lidocaine does not measurably affect the rate of recov- ery of the fast-inactivation gate. These observations Most state-dependent models predict that block at were made possible by our ability to follow the position very hyperpolarized voltages reflects binding of drug to of the fast-inactivation gate with a conformational noninactivated channels. Rmax, the limiting modifica- marker, the reactivity of site 1304 with MTS-ET, charac- tion rate at such hyperpolarized voltages, reflects the terized in detail in a previous study (Vedantham and position of the fully accessible fast-inactivation gate and should not, according to a state-dependent model, be In the first set of experiments, we determined the reduced in the presence of 1.0 mM lidocaine, even if position of the fast-inactivation gate as a function of 22% of the channels are blocked. As the channels are lidocaine concentration during tonic block, the inhibi- depolarized, however, a state-dependent mechanism fa- tion of peak sodium current that occurs with infre- vors binding to channels further along in the activation quent depolarization. Our results indicate that at Ϫ120 pathway and predicts that the fraction of blocked chan- mV, for lidocaine concentrations below the K nels that are fast-inactivated will increase. This explains block, the majority of blocked channels are not fast- the observed left shift of the voltage dependence of the that lidocaine favors closure of the fast-inactivation gree of gate closure in F1304C, is not significantly gate, although the certainty of this conclusion is under- changed in the presence of lidocaine, a finding that is mined by the possibility of nonspecific effects interfer- also predicted by a state-dependent mechanism favor- ing with the reaction between MTS-ET and site 1304 at such high drug concentrations. (Our data on the volt- The final set of experiments was directed at the effect age dependence of the reaction rate show that nonspe- of lidocaine on the recovery of site 1304 accessibility af- cific reduction of the reaction rate is not occurring at ter brief depolarizing pulses. We first confirmed that 1.0 mM lidocaine: the reaction rates in 1.0 mM 1.0 mM lidocaine dramatically slows the recovery of lidocaine and control conditions are equal at very hy- F1304C availability at Ϫ120 mV after a 20-ms depolar- ization to 0 mV. In the absence of lidocaine, the time Assuming that the modification rate faithfully reports constant of recovery is on the order of 1–2 ms, while in the position of the fast-inactivation gate even above the K 1.0 mM lidocaine, it is ف100–200 ms. This effect pro- for tonic block, our observations on concentration de- duces use-dependent block, a frequency-dependent, pendence are consistent with state-dependent binding of cumulative inhibition of sodium current with repetitive lidocaine to channel conformations that are populated depolarizations. Between 7.5 and 37.5 ms, only ف20– significantly only at depolarized potentials in the absence 30% of channels recover in the presence of lidocaine, of drug. As the lidocaine concentration is increased, whereas Ͼ90% recover with no lidocaine present. By the population of channels that are in these “depolar- contrast, the modification rate was not changed at all in ized” conformations will increase by mass action, even the presence of 1.0 mM lidocaine, demonstrating that at Ϫ120 mV. Because depolarized states favor closure lidocaine does not significantly alter the kinetics of re- of the fast-inactivation gate, increasing lidocaine con- centration should also favor closure of the fast gate.
A Possible Mechanism of Lidocaine Action That the modification rate is reduced by 40–50% in the presence of 4.0 mM lidocaine at Ϫ120 mV predicts At first glance, the results of these experiments seem to a dramatically altered hϱ curve: at Ϫ120 mV, a signifi- be in conflict: on the one hand, lidocaine shifts the hϱ cant fraction of channels must be unavailable. We curve in a way that favors fast inactivation, suggesting a found, consistent with our data, that in 4.0 mM stabilizing interaction between lidocaine block and fast- lidocaine, availability at Ϫ120 mV is somewhere on the inactivated channels. On the other hand, lidocaine has steep portion of the hϱ curve, although we could not no measurable effect on the off rate of the fast-inactiva- accurately estimate the relative availability at Ϫ120 mV tion particle, suggesting that it does not preferentially because patches do not survive the strong hyperpolar- izations (less than Ϫ140 mV) that would be required to One model that reconciles our results is shown in determine the maximum availability (data not shown).
Fig. 8. Following Kuo and Bean (1994), we employ a pendent, and the rightmost equilibria favor inactivated,rather than noninactivated, channels. According to thismodel, depolarization moves the distribution of chan-nels to the right and down, while hyperpolarizationtends to shift the distribution to the left and up.
Fig. 8 B presents a qualitative model for how lidocaine affects the states depicted in Fig. 8 A. We as-sume that each state can bind lidocaine, since our datasuggest that both inactivated and noninactivated chan-nels may experience block. We incorporate state de-pendence by postulating that lidocaine binds more fa-vorably to channels that are further along in the activa-tion pathway (towards the right), regardless of whetherthey are noninactivated or inactivated. In other words,lidocaine is sensitive to position along the horizontal,voltage-dependent axis of the state diagram, but notthe vertical, voltage-independent axis. In this model,lidocaine does not directly affect the equilibrium con- stants between inactivated and noninactivated channels(the equilibrium distributions for Cn ↔ In and CnL ↔InL are equal). Consequently, lidocaine binding doesnot affect the rate of recovery from fast inactivation byvery much, in agreement with our findings on the re-covery of accessibility of site 1304. However, the volt-age-dependent equilibria in the activation pathway are altered in lidocaine-bound channels, shifting the over-all distribution of channels to the right in Fig. 8 B.
A model for lidocaine action. In A, a section of the ac- The model also explains why lidocaine causes a hy- tivation pathway for sodium channels is shown, in which each non- n) is connected to an inactivated state (In). The length of the vertical arrows between inactivated and noninacti- tion of lidocaine at any given voltage will tend to shift vated states indicate the degree to which the equilibrium favors in- the distribution of channels towards the right in the activated channels: the longer the arrow, the greater the fraction state diagram of Fig. 8 B. Since the vertical equilibria of inactivated channels. Thus, depolarization causes rightward will favor fast-inactivated states as the distribution of movement and increases the fraction of inactivated channels. In B, channels moves sufficiently rightward along the activa- a set of states is added to the model that incorporate lidocainebinding. The arrows that move between unbound (C tion pathway, the addition of lidocaine will indirectly lidocaine-bound states (CnL or InL) indicate the degree to which promote fast inactivation. This phenomenon also ex- the equilibrium favors lidocaine binding: the longer the arrow, the plains our tonic block measurements: the greater the greater the fraction of lidocaine-bound channels. Thus, as for the lidocaine concentration, the greater the rightward shift case of inactivation, depolarization favors lidocaine block as well as along the activation pathway, and hence the greater the inactivation. The model implies that addition of lidocaine causes arightward shift in the distribution of channels owing to coupling between activation and lidocaine binding, while the vertical equi- The model also predicts a reciprocal effect of fast in- libria experience no such coupling (explaining why recovery from activation on lidocaine action: the presence of the fast- fast inactivation is not altered in lidocaine-bound channels). The inactivation gate promotes block, because (like lido- rightward movement of the distribution will tend to increase the caine) the fast-inactivation particle binds more tightly fraction of channels that are inactivated, thereby causing a shift inthe h to the rightmost states on the activation pathway. This ϱ curve. The slowing of repriming is a result of the slow disso- ciation of lidocaine from the CnL states.
would partly explain why channels with disrupted fastinactivation show a reduction in sensitivity to lidocaine model for sodium channel gating consisting of several effects (Cahalan, 1978; Yeh, 1978; Bennett et al., 1995; closed, noninactivated states, each in equilibrium with Balser et al., 1996). We need not attribute this reduc- a fast-inactivated state (Fig. 8 A). For convenience, only tion in sensitivity to an essential role played by inactiva- a few such equilibria are depicted. Horizontal equilib- tion in the mechanism of lidocaine action.
ria represent the voltage-dependent transitions along Use-dependent block, in our model, is a conse- the activation pathway, with depolarization favoring a quence of a slow off rate of drug from the drug-bound, rightward shift in the distribution of populated states.
non–fast-inactivated states. Recall that at depolarized The vertical transitions, by contrast, are voltage inde- potentials, our data show that both lidocaine and the Lidocaine and Naϩ Channel Inactivation fast-inactivation particle are bound (i.e., the back, Wang, 1986; Wang et al., 1987). As noted above, this is lower row in Fig 8 B is populated), and that on repolar- consistent with the predictions of our model: the inacti- ization the fast-inactivation particle dissociates rapidly, vation gate potentiates the effects of local anesthetics, populating the back, upper row of Fig. 8 B. The transi- but is not necessary to generate those effects. There is tions from the back, upper row to the front, upper row, also evidence that at least some local anesthetic mole- along with full leftward movement along the activation cules can be trapped by closure of the activation gate, pathway, is rate limiting and slow (100-fold slower than suggesting a possible mechanism for use-dependent recovery from fast inactivation), and generates use- block that does not involve the fast-inactivation gate dependent block when further depolarization occurs (Strichartz, 1973; Yeh and Tanguy, 1985).
Gating-current studies have revealed that lidocaine A remaining question concerns the kinetics of left- can produce a hyperpolarizing shift in the Q/V curve ward movement along the activation pathway upon re- (Hanck et al., 1994; Josephson and Chi, 1994) along polarization. Because inactivation is not intrinsically with a reduction in the total amount of on-gating cur- voltage dependent, but derives its voltage dependence rent. A possible interpretation of this finding is that from activation, some leftward movement along the ac- some of the voltage sensors of drug-bound channels tivation pathway must precede recovery from inactiva- move outward at less depolarized potentials than nor- tion. In other words, some inward charge movement mal. This would entail, at any given voltage, a drug- must occur if recovery from inactivation is to occur.
induced rightward shift in the distribution of channels Unfortunately, whether and to what extent lidocaine along the activation pathway diagrammed in Fig. 8, as impedes inward charge movement upon repolarization has not been examined carefully. Our results predict Finally, site-directed mutagenesis has placed the re- that some component of the gating charge must re- ceptor for lidocaine roughly in the middle of the S6 main relatively free to move even in lidocaine-bound transmembrane segment (Ragsdale et al., 1994). Ex- channels, and that inward movement of this fraction trapolation to Naϩ channels of a recent substituted cys- must be sufficient for complete recovery of the fast- teine accessibility study in segment S6 of Shaker Kϩ inactivation gate. Further experiments will be required channels (Liu et al., 1997) suggests that the position of to elucidate the details of the coupling between inacti- the activation gate is likely to be very close to the local vation and gating charge movement in the presence of anesthetic binding site. Thus, it would not be surpris- lidocaine, and thereby to determine how far the distri- ing if the primary action of lidocaine is to interact with bution of channels must move to the left on repolariza- activation gating, perhaps by stabilizing the channel in tion for full recovery from inactivation to occur.
We wish to emphasize that our results are not suffi- cient to determine uniquely our particular model of Relation to Previous Work on Lidocaine lidocaine action. Although our results do suggest a very Our model is a version of the modulated receptor hy- limited role for fast inactivation in generating use- pothesis (Hille, 1977; Hondeghem and Katzung, 1977), dependent block, it is still possible that the affinity of in which the affinity of a single receptor site for lidocaine for its receptor is increased by closure of the lidocaine is altered by the conformational state of the fast-inactivation gate in the intact channel (i.e., with a channel. Our model differs from Hille’s original pre- phenylalanine at site 1304). Another possibility is that sentation and from that of Bean et al. (1983) by not the Naϩ channel slow inactivation mechanism plays a treating the inactivated state as the high-affinity state.
role in lidocaine action. Our finding that recovery from Instead, we propose that transitions along the activa- fast inactivation precedes recovery of the ionic current tion pathway (outward movement of S4 segments and/ in the presence of lidocaine parallels an earlier finding or opening of the activation gate) affect the affinity of that recovery from fast inactivation precedes recovery lidocaine for its receptor, following the proposals of from slow inactivation (Vedantham and Cannon, 1998), Wang et al. (1987), Strichartz and Wang (1986), and and raises the possibility that the two slowly recovering Yeh and Tanguy, 1985. Several lines of evidence sup- states are related in some way. For example, lidocaine might accelerate the rate of entry into slow-inactivated First, numerous studies have shown a reduction in the potency of local anesthetics in fast-inactivation de- Also, the mechanism of lidocaine action might vary fective sodium channels (Cahalan, 1978; Yeh, 1978; among sodium channel isoforms. Our experiments Bennett et al., 1995; Balser et al., 1996). However, de- were conducted in skeletal muscle sodium channels, spite the loss of potency, local anesthetics do retain which have a lower apparent lidocaine affinity that car- their ability to generate tonic and use-dependent block diac channels (Hille, 1978; Nuss et al., 1995). However, in these channels (Shepley et al., 1983; Strichartz and most of this difference is attributable to relative shifts in voltage-dependent gating between the two isoforms, These uncertainties aside, our data do enable us to rather than to differences in the putative binding site place important new constraints on the possible forms (Wright et al., 1997), suggesting that our results with that models for lidocaine action can take. Any such skeletal muscle channels will probably hold for cardiac model must involve cooperativity between lidocaine channels as well. We should also emphasize that our re- binding and fast inactivation, and must incorporate a sults may not hold for all local anesthetics, which ex- state that is slowly recovering, but not fast inactivated, hibit considerable variation at the chemical level as well as in their effects on sodium channels (Hille, 1977).
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Lidocaine and Naϩ Channel Inactivation

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