This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bennett, P. B. Articles by Kallen, R. G. Search for Related Content PubMed PubMed Citation Articles by Bennett, P. B. Articles by Kallen, R. G.

(Circulation Research. 1995;77:584-592.)
© 1995 American Heart Association, Inc.

Articles

On the Molecular Nature of the Lidocaine Receptor of Cardiac Na⁺ Channels

Modification of Block by Alterations in the -Subunit III-IV Interdomain

Paul B. Bennett, Carmen Valenzuela, Li-Qiong Chen, Roland G. Kallen

From the Departments of Pharmacology and Medicine (P.B.B., C.V.), Vanderbilt University Medical School, Nashville, Tenn, and the Department of Biochemistry and Biophysics (L.-Q.C., R.G.K.), University of Pennsylvania School of Medicine, Philadelphia.

Correspondence to Paul B. Bennett, PhD, Department of Pharmacology, 560 MRBII, Vanderbilt University Medical School, Nashville, TN 37232.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract The mechanism of inhibition of Na⁺ channels by lidocaine has been suggested to involve low-affinity binding to rested states and high-affinity binding to the inactivated state of the channel, implying either multiple receptor sites or allosteric modulation of receptor affinity. Alternatively, the lidocaine receptor may be guarded by the channel gates. To test these distinct hypotheses, inhibition of Na⁺ channels by lidocaine was studied by voltage-clamp methods in both native and heterologous expression systems. Native Na⁺ channels were studied in guinea pig ventricular myocytes, and recombinant human heart Na⁺ channels were expressed in Xenopus laevis oocytes. Fast inactivation was eliminated by mutating three amino acids (isoleucine, phenylalanine, and methionine) in the III-IV interdomain to glutamines or by enzymatic digestion with -chymotrypsin. In channels with intact fast inactivation, lidocaine block developed with a time constant of 589±42 ms (n=7) at membrane potentials between -50 and +20 mV, as measured by use of twin pulse protocols. The IC₅₀ was 36±1.8 µmol/L. Control channels inactivated within 20 ms, and slow inactivation developed much later (time constant of slow inactivation, 6.2±0.36 s). The major component of block developed long after activated and open channels were no longer available for drug binding. Control channels recovered fully from inactivation in <50 ms at -120 mV (time constant, 11±0.5 ms; n=50). In 30 µmol/L lidocaine, 49±3% of the current recovered with a second larger time constant of 398±46 ms (n=5). After removal of fast inactivation, either enzymatically or by mutagenesis, the channels were no longer blocked by 50 µmol/L lidocaine (n=8). Much higher concentrations of lidocaine produced a tonic block (IC₅₀, 0.4±0.07 mmol/L; n=13) and time dependence suggestive of open-channel block. The results indicate the importance of inactivated state block by therapeutic concentrations of lidocaine and suggest that the molecular site of action is the structural region of the channel that is responsible for inactivation.

Key Words: Na⁺ channels • Na⁺ currents • local anesthetics • antiarrhythmic drugs

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lidocaine is an effective and widely used local anesthetic and antiarrhythmic agent that acts by inhibiting Na⁺ flux through voltage-gated Na⁺ channels. The block of Na⁺ channels by lidocaine has been extensively studied in nerve (squid axon, frog node of Ranvier, etc) and heart (Purkinje fibers and rat, rabbit, and guinea pig cardiocytes) by using both indirect (V_max) and direct measures of Na⁺ current and both whole-cell and single-channel recordings.^{1 2 3 4 5 6 7 8 9 10 11 12}

Hille^{1 4} has proposed that the binding site for both neutral and charged drugs that are used as local anesthetics and antiarrhythmic agents lies between the channel gates and the selectivity filter. Starmer and coworkers^{13 14 15 16} have attempted, for the sake of parsimony, to simplify and formalize some of these concepts mathematically. Thus, in one such model, the binding site is guarded by the channel gates; a drug can bind to its receptor (and inhibit current) only after the gate is open and the receptor is unguarded. Alternatively, it has been proposed that lidocaine binds with highest affinity to the inactivated state of the channel.^{4 5 6} In each of these models, the concept of drug binding and channel gating being interdependent is prominent.^{2 5 17 18 19 20 21} Interpretation of these studies is likewise confounded by the interrelation between channel gating and drug block of the channels and the inability to independently resolve these reactions. Efforts have been made to simplify gating reactions by slowing inactivation with toxins.^{22 23 24} In principle, this approach should permit investigation of blocking reactions without the complication of inactivation gating. This method suffers from the often large changes in channel gating that occur in toxin-bound channels and from the interactions between drug and toxin binding.²³ An alternative to using toxins is to modify structurally the channel protein by enzymatically directed cleavage or site-directed mutagenesis of an inactivation domain. Armstrong et al²⁵ first showed that Na⁺ channel inactivation could be removed in squid axons after internal proteolysis. Patlak and Horn²⁶ demonstrated removal of fast inactivation by N-bromoacetamide in excised patches. The purpose of the present study was to investigate lidocaine block of cardiac Na⁺ channels after removal of fast inactivation using two approaches: (1) Intracellular domains were covalently modified by proteolytic cleavage to remove fast inactivation.²⁷ (2) Site-directed mutagenesis of specific amino acids in the recombinant cardiac Na⁺ channel was carried out. Such an approach allows us to determine whether the inactivated state is necessary for lidocaine-induced channel inhibition or whether lidocaine binds when given continuous access to the open channel by removing a component of gating.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Isolation
Ventricular myocytes were obtained from 200- to 250-g guinea pigs by standard collagenase dispersion as described previously.²⁷

Xenopus oocytes were obtained from frogs purchased from Xenopus 1 (Ann Arbor, Mich). Defolliculated oocytes were prepared for RNA injection and electrical recording by exposure to collagenase (1 mg/mL, type II, Worthington) for 1 to 2 hours at 20°C. After injection of 30 to 50 nL (5 to 20 ng) of 5' capped cRNA, the oocytes were kept for 2 to 8 days at 20°C, during which time they were tested for expression of Na⁺ current by two-electrode voltage clamp as described previously.²⁸

Molecular Biology
Mutations in human cardiac Na⁺ channel cDNA were created by synthetic oligonucleotide site-directed mutagenesis by high-efficiency methods using pSelect-1 and protocols from Promega. All mutations were confirmed by dideoxynucleotide sequencing. cRNA was synthesized by using T7 RNA polymerase and truncated templates as described earlier.²⁹ Synthetic oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer.

Solutions
Patch Clamp
The cells were superfused with a solution of the following composition (mmol/L): NaCl 145, CaCl₂ 1.8, MgCl₂ 1.0, KCl 4.0, glucose 10, and HEPES 10, titrated to pH 7.35 with NaOH. The patch electrodes were filled with a solution containing (mmol/L) NaCl 145, MgCl₂ 2.0, CaCl₂ 0.1, and HEPES 10, titrated to pH 7.35 with NaOH. After gigaohm seal formation, the bath solution was changed to a solution containing (mmol/L) NaF 10, CsF 110, CsCl 20, MgCl₂ 2.0, EGTA 2, and HEPES 10, titrated to pH 7.35 with CsOH. -Chymotrypsin (type VII, treated with N-p-tosyl-L-lysine chloromethyl ketone to avoid trypsin activity, Sigma Chemical Co) was dissolved in the following (mmol/L): CsCl 80, CsF 80, EGTA 10, and HEPES 10, titrated to pH 7.35 with CsOH.

Two-Electrode Clamp: Oocytes
The bath solution was the standard ND-96 solution for oocytes and contained (mmol/L) NaCl 96, KCl 2, CaCl₂ 1.8, MgCl₂ 1, HEPES 5, and pyruvic acid 2.5, and pH was adjusted to 7.50 at 22°C with NaOH. Although there are numerous references in the literature to endogenous channels in Xenopus oocytes, we minimized endogenous channel activity by selecting batches of oocytes that showed no ionic currents in the absence of injected RNA. Oocytes with endogenous currents >0.1 µA were discarded.

Patch-Clamp Recordings
Experiments were performed at room temperature (20°C) by the inside-out patch-clamp method.³⁰ Recordings were made with an Axopatch-1C patch-clamp amplifier (Axon Instruments). Patch pipettes were constructed from capillary tubes (Radnoti Glass Technology) and heat-polished after being coated near the tip with a hydrophobic polymer (Sylgard, Dow Corning). Electrode resistances were 5 to 10 MA. Additional details of patch-clamp methods can be found in Valenzuela and Bennett.²⁷

Two-Electrode Voltage Clamp
Oocytes were voltage-clamped by using standard two-microelectrode voltage-clamp techniques. Electrodes were pulled from Radnoti starbore glass and filled with 3 mol/L KCl. Electrode resistances were 1 to 3 MA for voltage-recording electrodes and 0.15 to 0.3 MA for current-passing electrodes. Membrane potentials were controlled by a high-compliance voltage-clamp amplifier (Clampator, Dagan). Only currents <10 µA were analyzed to minimize series resistance problems associated with larger currents. A grounded metal shield was inserted between the two electrodes to minimize electrode coupling and to speed the clamp rise time.

Voltage commands were generated by a 12-bit D/A converter driven by customized PCLAMP software (Axon Instruments). Currents were filtered at 5 kHz (-3 dB, four-pole Bessel filter) and sampled at 50 kHz by a 12-bit A/D converter. Data were saved for subsequent analysis on the hard disk and were archived on an optical disk drive. The holding potential was typically -120 mV. In drug-free conditions, recovery from inactivation was <200 ms, and the cycle time for any protocol was 5 s (with the exception of specific pulse trains). Between -120 and -80 mV, only passive linear leak was observed. Linear least-squares fits to these data were used for leak correction if necessary. All measurements were made with custom-analysis programs designed to read and analyze PCLAMP binary data files.

Data Analysis
Inactivation curves were fitted with a Boltzmann equation:

where I is current, I_max is maximal current, V is voltage, V_1/2 is half-maximal voltage, and s is the slope factor. The time courses of the falling phase of the macroscopic Na⁺ current ("apparent inactivation") as well as onset and recovery from block were fitted with exponential functions: y(t)= A+B_i · exp(-t/), where is a time constant. Curve fitting was based on a nonlinear least-squares algorithm. The results were displayed in linear and semilogarithmic formats together with a plot of the residuals. Goodness of fit was judged by visual inspection. Results are presented as mean±SEM. Statistical significance was taken as P<.05. For single-channel data, capacitative and linear leakage currents were subtracted by averaging the traces without activity and subtracting the averaged "null" from each trace. Data were analyzed with custom software written in BASIC (Microsoft, QUICKBASIC) by use of an event-detection scheme based on a half-amplitude criterion.^{31 32} Single-channel recordings were first analyzed by generation of amplitude histograms, which allowed identification of current levels associated with channel openings. Amplitude histograms were fitted with gaussian curves by using a simplex algorithm. The mean±SD of the all-points current amplitude histograms were then used in further analysis of open and closed durations. Additional details on the analysis have been published.²⁷

Simulations and Modeling
Models were written in FORTRAN and BASIC and run on a 33-MHz 80486 computer. State models were solved by numerical integration using a Runge-Kutta algorithm. The basic Na⁺ channel state diagram and rate constants were modified from the method of Horn and Vandenberg.³³ Slow inactivation was incorporated by assuming a 0.25-s^-1 transition rate constant into the slow inactivated state.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Attempts to resolve changes in channel open times caused by lidocaine are complicated by the very brief nature of these events, which is due to normal fast inactivation of the channel. If block can only occur while the channel is open, then closing into the inactivated state is expected to preempt drug binding. In this case, lidocaine will be permitted to bind to its receptor only during a very brief temporal aperture while the channel is open.^{1 4 13 14 15 16} Removal of fast inactivation should then permit free access of lidocaine to its receptor site in the period of time that the channel is open. The effects of a supratherapeutic concentration of lidocaine on Na⁺ channels after removal of fast inactivation by brief proteolysis with -chymotrypsin are shown in Fig 1. If lidocaine acts as an open-channel blocker, we expect to see an abbreviation of the channel open times if block occurs on a time scale similar to channel gating or to observe a reduction of single-channel current if block is very fast relative to the recording bandwidth. The effects of lidocaine on channel opening and closing during a voltage jump are not obvious from the raw data (Fig 1). Exposure of the intracellular side of the excised patch to lidocaine caused a small decrease in open times suggestive of open-channel block.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effects of supratherapeutic concentrations (10 µg/mL=43 µmol/L) of lidocaine on single Na+ channels in a patch excised from a guinea pig ventricular myocyte after proteolytic removal of fast inactivation. Top, Representative raw current traces of Na+ channel activity in the control condition (left) and after lidocaine administration (right). Middle, Open-time frequency distributions in the control condition (left) and after lidocaine administration (right). A1 indicates probability density; , time constant. Bottom, Closed-time frequency distributions in the control condition (left) and after lidocaine administration (right).

A small reduction of open times in the presence of lidocaine can be seen in Fig 1 along with an increase in the number of closed (blocked) events. The appearance of a second component in the plot of closed durations is apparent. Although these effects of lidocaine on open and closed durations were detectable and statistically significant (P<.05, n=5), the effect on ensemble-averaged current was small, and the change in open times cannot account for the decrease in macroscopic current that is seen with this concentration of lidocaine in intact Na⁺ channels. This suggests that open-channel block even at this relatively high concentration is not very important.⁶ Likewise, a small effect on the latency to first opening could be seen (data not shown), but this effect was hardly measurable from the macroscopic current. Although these data confirm previous reports of an effect of lidocaine on a preopen state, they also demonstrate that this mechanism plays at best only a modest role in the inhibition of Na⁺ channel by lidocaine at therapeutic concentrations (10 µmol/L).³⁴

In contrast to the block that is apparent during a single voltage-clamp step, a large amount of block can be induced when channels are first placed into the inactivated state by a prepulse (Fig 2, left). In these experiments, control channels were placed into the inactivated state with a 500-ms voltage-clamp step to +20 mV. A subsequent test pulse was delivered 100 ms later (to allow recovery of unblocked/inactivated channels) to assess the amount of block. When fast inactivation is intact (Fig 2, left), placing the channels in the inactivated state leads to a large amount of block. When fast inactivation is removed, the same pulse protocol causes little or no block (Fig 2, right).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of lidocaine on ensemble-averaged Na+ current in a membrane patch excised from a guinea pig ventricular myocyte. The holding potential was -120 mV, Na+ channels were driven into the inactivated state by a 500-ms prepulse to +20 mV, and block was assessed during a subsequent test pulse (data shown) delivered 100 ms after the prepulse (left). After proteolytic removal of fast inactivation by -chymotrypsin, lidocaine block was lost (right).

A potential problem with experiments using -chymotrypsin to remove fast inactivation is that the enzyme may not only cleave the structures responsible for fast inactivation but it may also modify the lidocaine receptor. To avoid this potential problem and to begin to identify the region of the channel that binds lidocaine, we have used recombinant wild-type and mutated human heart Na⁺ channels (hH1).³⁵ When recombinant channels were expressed in oocytes, they displayed gating characteristics similar to those of native cardiac Na⁺ channels. Channels began to open near -60 mV, and the peak inward current occurred near -20 mV. At membrane potentials more positive than -30 mV, steady state inactivation developed within 20 ms. The channels had a steady state inactivation midpoint (-79±0.4 mV) and slope factor (5.1±0.1 mV) (n=5) that were similar to those obtained in an earlier study, suggesting that the measurement of Na⁺ current was adequate for the purposes of the present study.^{35 36}

When lidocaine block was induced by a single 500-ms prepulse to +20 mV, a large fraction of channels recovered much more slowly than normal (Fig 3 and the Table). The fast recovery process was not altered by 30 µmol/L lidocaine. As shown in Fig 3 (bottom), the small time constants in the control condition and after the administration of lidocaine are superimposed. After lidocaine administration, a second larger time constant became apparent, and 50% of the Na⁺ current recovered from block, with time constants ranging between 0.4 and 2 s, depending on membrane potential (Fig 3). These recovery-rate changes indicate a voltage or state dependence for this process. Such an apparent voltage dependence could derive from an intrinsic voltage dependence of the unbinding reaction, which could occur if the charged form of lidocaine binds within the electrical field. Alternatively, it could result because the receptor affinity is determined by the channel state, the occupancy of which is voltage dependent.

View larger version (21K): [in this window] [in a new window]   Figure 3. Recovery from lidocaine block (30 µmol/L) of human heart Na+1 channels. Block was induced by a single 500-ms prepulse to +20 mV. Top, Recovery from inactivation and block was assessed by a test pulse given at different time intervals (abscissa) after the prepulse. The voltage-clamp pulse protocol is shown as an inset. Peak Na+ current during the test pulses was normalized by the current recorded after a 10-s interval at -120 mV. Control data during recovery at -90, -100, or -120 mV are shown. The recovery curves were fitted with a one-exponential (control, ) or two-exponential (lidocaine, ) equation (see Table). Unblocked (inactivated) channels recover fully in <50 ms at -120 mV. In 30 µmol/L lidocaine, 50% of the current recovered slowly, reflecting recovery of channels from block. This unblocking was slower at -90 mV and fastest at -120 mV. Bottom, Time constant of recovery (recovery ) from inactivation and/or block (unblock) was assessed as a function of membrane potential. Control channels could be fitted with a single time-constant process (). In lidocaine, a second time constant was required (). Recovery was identical in the control condition and in the presence of lidocaine. The second, large time constant reflects the lidocaine unblocking process. This slow recovery rate changes with membrane potential, indicating a voltage or state dependence of unblock.

View this table: [in this window] [in a new window]   Table 1. Recovery of Na+ Channels at Different Membrane Potentials

Recently, it has been demonstrated that a mutation in the III-IV interdomain of the rat brain II Na⁺ channel leads to loss of fast inactivation.³⁷ We have mutated the same three amino acids (IFM, indicating isoleucine [I], phenylalanine [F], and methionine [M]) in the III-IV interdomain of the human cardiac Na⁺ channel to glutamine (QQQ). Since this mutation also leads to complete loss of fast inactivation in the cardiac isoform without other measurable changes in channel function, we can independently test the competing hypotheses that (1) lidocaine block requires fast inactivation or (2) the lidocaine receptor is guarded by the channel gates. If hypothesis 2 is correct, then we expect block to occur as soon as the receptor becomes unguarded upon channel activation in the IFM/QQQ mutant, since inactivation is no longer present. Fig 4 shows use-dependent block in wild-type and mutant hH1 channels. During the 5-Hz pulse trains, the wild-type channel was markedly blocked by 25 µmol/L lidocaine. Lower concentrations also produce a large amount of block under these conditions in wild-type channels.³⁵ The fast inactivation-deficient IFM/QQQ channel shows no use-dependent lidocaine block at 0.1 mmol/L (>4 times the maximum therapeutic level) even at 10 Hz (Fig 4). Clearly, the loss of inactivation correlates with the lack of block.

View larger version (14K): [in this window] [in a new window]   Figure 4. Lidocaine block during pulse trains for wild-type and mutant Na+ channels. Raw current tracings were recorded during trains of pulses delivered at 5 Hz for the wild-type channel or at 10 Hz for the QQQ (where Q is glutamine) mutant Na+ channel. The pulse duration was 40 ms, and the holding potential was -120 mV. The interval between pulses at -120 mV was 160 ms (200-ms cycle length) for the wild-type and 90 ms (100-ms cycle length) for the QQQ mutant. The vertical calibration bars are 0.5 µA for the two wild-type tracings and 1.0 µA for the QQQ mutant. The wild-type tracings are from the same oocyte. Shown are tracings during a 16-pulse train; for clarity, certain sweeps are omitted. Sweeps 1 through 6 and sweep 16 are shown for the wild-type channel. Only the sweeps 1 and 16 are shown for both control and lidocaine in the mutant channel.

In channels with intact fast inactivation, the onset of block by 25 µmol/L lidocaine at three membrane potentials at which the Na⁺ channels are open (-50, -30, or +20 mV) developed with a time constant of 550 ms, independent of membrane potential (Fig 5). The average time constant for onset of block from seven oocytes was 589±42 ms. In 13 experiments using different concentrations of lidocaine, the average IC₅₀ for block of Na⁺ current using this protocol was 36±1.8 µmol/L. For prepulses > 1 s, the control current decreases because of slow inactivation. This slow inactivation developed with an apparent time constant of 5.5±0.2 s (n=6). Thus, onset of lidocaine block was much slower than onset of fast inactivation (=1 to 20 ms) and much faster than the onset of slow inactivation (=5 s). Lidocaine block develops sometime after channel activation but well before slow inactivation takes place. Measuring the onset of block with twin pulse protocols, as was done in the experiments illustrated in Fig 5, we observed a complete loss of prepulse-induced block even in 50 µmol/L lidocaine in the inactivation-removed Na⁺ channel mutant (Fig 5). This demonstrates that the fast inactivation process is essential for high-affinity lidocaine block of the channel.

View larger version (21K): [in this window] [in a new window]   Figure 5. Top, Time course and voltage dependence of the onset of block of wild-type human heart Na+ channels by 25 µmol/L lidocaine are shown. Twin-pulse protocols were used to induce block at three different membrane potentials: -50, -30, or +20 mV in channels with intact inactivation. For given fixed prepulse membrane potential, the prepulse durations were varied, and their durations are plotted on the logarithm abscissa. After a complete run at one prepulse potential, the level of the prepulse was changed. The interval between the prepulse and test pulse was 300 ms. The same degree of block occurred with the same time course independent of membrane potential (-50 to +20 mV). The time-dependent reduction of current had one component in the control condition (time constant =6 s) and two components in the presence of lidocaine (=550 ms and 6 s). The slow process reflects slow inactivation and is not altered by lidocaine. Lidocaine induced the faster process with the 550-ms time constant. Onset of lidocaine block was much slower than onset of fast inactivation (=1 to 20 ms) and much faster than the onset of slow inactivation (=6 s). Bottom, The same pulse protocols were applied in the inactivation-deficient mutant Na+ channel (III-IV interdomain [IFM to QQQ, where I is isoleucine, F is phenylalanine, M is methionine, and Q is glutamine]). A protocol identical to that in the top panel was used, except the lidocaine concentration was increased to 50 µmol/L. Only data from the prepulses to +20 mV are shown. No block was observed even with a twofold higher lidocaine concentration.

Gingrich et al¹¹ have recently reported that the permanently charged lidocaine derivative QX-314 blocks open cardiac Na⁺ channels with a K_i of 4.4 mmol/L at a site deep within the pore (73% into the electrical field from the intracellular side). We observed only modest open-channel block in the therapeutic range of concentrations we have used. To determine whether we could observe the component of block described by Gingrich et al, we used much higher concentrations of lidocaine with the inactivation-deficient mutant Na⁺ channel (QQQ). Results of such an experiment are shown in Fig 6. The time- and voltage-dependent block in a high concentration of lidocaine is illustrated in Fig 6B. This concentration of lidocaine (0.4 mmol/L) induced a rapid decay of the macroscopic Na⁺ current. The rate of this decay phase was dependent on the lidocaine concentration (Fig 6C). This behavior is precisely what one can predict from a first-order blocking reaction in the open channel.^{1 38} In such a scheme, the on-rate of block should increase with drug concentration as shown in Fig 6C. The average IC₅₀ for tonic reduction of peak current was 0.4±0.07 mmol/L (n=13) (not shown). The steady state reduction of current had an apparent IC₅₀ of 0.14±0.02 mmol/L (not shown). These data suggest that activated or open-channel block does occur in the human cardiac Na⁺ channel but at concentrations much higher than those used in the treatment of arrhythmias.

View larger version (24K): [in this window] [in a new window]   Figure 6. Block of inactivation removed (QQQ mutant, where Q is glutamine) Na+ channels by high concentrations of lidocaine. In all experiments, the holding potential was -120 mV. A, Voltage dependence of QQQ mutant in the absence of lidocaine. Na+ currents recorded during voltage steps to different membrane potentials are shown. The step potential is indicated next to each record. B, Effects of 0.4 mmol/L lidocaine. Conditions were identical to those in panel A. The voltage dependence of block (ratio of the current in the presence of lidocaine to the control current) was fitted with a Boltzmann function (1+e-zF(V-V1/2)/RT)-1 where F, R, and T have their usual thermodynamic meanings, z has a value of +1 (charged lidocaine), and is the fractional electrical distance of the blocking site. The best fit for was 0.63 (not shown). The decay phase of the current in the presence of lidocaine was fitted by using a nonlinear least-squares algorithm with an exponential function to obtain the time constant of block (Block). C, The effect of two different concentrations of lidocaine on the rate of decay of the normalized Na+ current (INa) shown on a logarithmic ordinant for convenience. These results can be described by using a simple one-siteblocking model: CCO[L]konkoffOD, where Cindicates closed states, O is the open state, [L] is the lidocaine concentration, kon and koff are drug association and dissociation rate constants, respectively, and OD is the lidocaine-blocked open state. After the channel is open, the time constant () of open-channel block is as follows: ([L] · kon+koff)-1. Therefore, a plot of the reciprocal time constant (-1) versus lidocaine concentration should be a line with a slope equal to kon and a y intercept equal to koff. D, Plot of -1 vs lidocaine. The rate of development of block was measured during steps to -20 mV. The best fit values for kon and koff were 0.33 (mmol/L)-1 · ms-1 and 0.2 ms-1, respectively. These values can be used to calculate an apparent Kd (koff /kon) for open-channel block equal to 0.6 mmol/L.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The goal of the present study was to elucidate the molecular mechanisms of action of lidocaine on cardiac Na⁺ channels. We wished to determine more directly than could be done previously the channel states with which the lidocaine interacts and whether the affinity for drug binding to these states is relevant at therapeutic concentrations of lidocaine. We can discuss our results in the context of a number of subhypotheses, depicted as partial-state diagrams in Fig 7, by focusing on the dominant property of each model.

View larger version (14K): [in this window] [in a new window]   Figure 7. Proposed models of lidocaine block. Left, Partial-state model previously suggested to explain lidocaine block of Na+ channels. Models are as follows: 1, activated-state (preopen) drug-channel interaction model; 2, open-channel block model; 3, inactivated-state drug-channel interaction model; 4, slow inactivated-state drug-channel interaction model; and 5, guarded-receptor model (inactivation removed). Right, Lifetimes of various channels states (binding sites) plotted as solid lines on a logarithmic abscissa. Time courses for various channel states were generated from a modified Horn and Vandenberg33 Na+ channel model as shown. The rate constants (per second) for each transition are indicated above the arrows. The slow inactivated state is not shown in the model. The time course of lidocaine block at +20 mV (BLOCK) is plotted as a dotted line (1.0, no block; 0, 100% block). The open-state (lifetime, 1 to 10 ms) and activated-state (lifetime, 0.1 to 2 ms) lifetimes are too short to allow significant drug binding with low lidocaine concentrations. The receptor availability is too brief to account for the time course of block. Only the inactivated (3) or guarded (5) models have receptor site availabilities consistent with the data. Removal of fast inactivation allows discrimination of these two models. The open-channel blocking site appears to be guarded from the charged form of lidocaine, but this component of block is only apparent at high concentrations (see Fig 6). Inactivation is essential for high-affinity block to occur. R indicates rested states; A, activated states (closed or open); O, the open state; I, inactivated states; and SI, slow inactivated states. AD, DD, ID, and SID represent the drug-associated forms of these states.

Previous work on lidocaine block of Na⁺ channels has led to several different proposed mechanisms diagrammed in Fig 7: model 1, binding of lidocaine to a preopen (activated) state³⁴ ; model 2, open-channel block; model 3, binding to the fast inactivated state^{2 4 6 10 20} ; model 4, modulation of slow inactivation; or model 5, binding to a fixed affinity receptor that is guarded by the channel gates.^{13 14 15 16} The hypothesis that the local anesthetic (antiarrhythmic) receptor of the Na⁺ channel has at least three major states differing in their binding affinities is called the modulated receptor hypothesis. The idea has its roots in Armstrong's earlier analysis of the interactions of tetraethylammonium with K⁺ channels³⁸ and is formally equivalent to the concepts of conformational-dependent binding affinities of allosteric enzymes.^{1 39} In the modulated receptor hypothesis, different channel conformations vary quantitatively in affinity, and a complete version includes features of models 1 to 3. Model 5 depicts a fixed affinity receptor, but the channel must have open gates for the drug to bind. In all of the models, the concept of drug binding and channel gating being interdependent is prominent.

The Na⁺ channel -subunit consists of four homologous domains or repeats coupled by intracellular or extracellular amino acid loops.^{40 41} There is now substantial evidence that the intracellular amino acids linking domains III and IV are involved in channel inactivation, and it has been speculated that this region is the "inactivation gate."^{37 42} Specific antibodies directed toward this domain interfere with inactivation. Three amino acid residues (IFM) in this III-IV interdomain are essential for inactivation.³⁷ We adopted a strategy of removing fast inactivation both by limited proteolysis of the protein and by site-directed mutagenesis in order to test the hypothesis that fast inactivation is essential for lidocaine-induced channel inhibition. This strategy also allowed us to explore the importance of open-channel block (in the absence of fast inactivation) and the possibility that a channel "gate" guards the lidocaine receptor. Our data, although confirming that drug binding occurs in both open and preopen states of the channel, indicate that these are not the primary mechanisms for block at low concentrations of lidocaine used therapeutically. Rather, our data suggest that the drug interacts with a structural region of the channel that is responsible for inactivation. After activation in a channel that does not inactivate, the open state is almost continuously available for drug binding. Our results show that although open/activated channel block does occur, the IC₅₀ is much greater than that for the inactivated state of the channel. Our conclusion is that at low concentrations, lidocaine binding primarily requires the inactivated conformation of the channel. Our data show that a very large degree of block can be induced at concentrations in the therapeutic range (2 to 6 µg/mL=8.5 to 26 µmol/L). The time course of lidocaine block (see Figs 5 and 7) was slower than the establishment of fast inactivation (<20 ms) and faster than slow inactivation (>4 s). This precludes the dominant effect of lidocaine being on preopen activated or open Na⁺ channels. Thus, the major component of lidocaine block develops long after activation (opening) and well before slow inactivation develops. The fact that block develops more slowly than fast inactivation does not preclude slow binding to the inactivated state. The inactivated state becomes available for binding in <20 ms; thus, the slow onset of block does not depend on the kinetics of availability of this state. A possible explanation for the loss of prepulse-induced block in inactivation-deficient mutant channels is that block occurs but unblock at negative potentials is accelerated. This is clearly not the case, at least within the limits of resolution in our experiments. We used a twin-pulse recovery protocol (as in Fig 3) with the IFM/QQQ mutant Na⁺ channel to determine whether the apparent lack of block resulted from a rapid recovery. In five experiments, this analysis using twin-pulse recovery protocols failed to reveal any such increase in the unblocking rate at negative membrane potentials. Since block fails to develop in the IFM/QQQ mutant channel, there was no recovery from block (data not shown).

During the course of our work Ragsdale et al⁴³ reported that mutations in the S6 segment of domain IV modified block by the local anesthetic etidocaine. Specifically, mutation of phenylalanine 1764 to an alanine caused a loss of block. The interpretation of their data is complicated by the presence of fast inactivation and by the fact that etidocaine has a chiral atom potentially giving rise to four active species of drug, including the charged and uncharged forms of each stereoisomer. Clearly, however, their mutations in the S6 region altered channel block of etidocaine. One explanation of their data is that the open-channel blocking site(s) lies in the pore (S6). Since this site is unchanged in our mutant channel, one might conclude that the site responsible for inactivated state block is distinct from the open-channel block site. Alternatively, block of the wild type channel may involve initial interactions with the S6 site, followed by a stabilization of the inactivated state when it occurs. This could possibly involve a coordination between one end of the drug that is bound to the S6 site and the other end that is associated with the inactivation gate when it closes. This second interaction would not occur in a channel lacking fast inactivation. Our data show that the inactivated-state blocking site is not the electrical field, whereas the open-channel blocking site is deep within the field (60% to 70%).^{1 11} It will be important to test experimentally whether or not the S6 site (rat brain II 1764) is in the electrical field.

On the basis of our results, we can eliminate models 1, 2, 4, and 5 as the mechanism responsible for the major component lidocaine block at therapeutic concentrations in human cardiac channels. The main conclusions of the present study are as follows: (1) The onset of block at low concentrations was not voltage dependent. (2) The onset rate of block did not equal the recovery rate, suggesting modulation of affinity. (3) Block develops after activated and open states are no longer available (models 1 and 2). (4) Block develops before the slow inactivated state is available (model 4). (5) The main mechanism of lidocaine–Na⁺ channel interactions at therapeutic concentrations appears to be a stabilization of the inactivated state, and this component of block is lost if inactivation is eliminated.

The observed time course of action of lidocaine is consistent with the behavior of the mammalian cardiac cycle and permits substantial block to develop during the several hundred–millisecond cardiac action potential. In contrast, the relatively brief neuronal action potentials (approximately a millisecond) would not be expected to allow significant lidocaine block by this mechanism. In neuronal cells, open- or activated-channel block at higher concentrations may be much more important. This likely forms the basis for the relative tissue selectivity of lidocaine action at low concentrations.

	Acknowledgments

 
This study was supported by grants HL-51197, HL-46681 (Dr Bennett), and AR41762 (Dr Kallen) from the National Institutes of Health and a Grant-in-Aid from the American Heart Association and the Research Foundation of the University of Pennsylvania. Dr Bennett is an Established Investigator of the American Heart Association. The authors thank Dr R. Horn for his constructive review of the manuscript, C. Short for oocyte injections, and T. Sheffer and M. Austin for editorial assistance.

Received May 24, 1994; accepted May 5, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Hille B. Ionic Channels of Excitable Membranes. 2nd ed. Sunderland, Mass: Sinauer Associates Inc; 1992.

2. Hondeghem LM, Katzung BG. Antiarrhythmic agents: the modulated receptor mechanism of action of sodium and calcium channel blocking drugs. Annu Rev Pharmacol Toxicol. 1984;24:387-423. [Medline] [Order article via Infotrieve]

3. Lee KS, Hume JR, Giles W, Brown AM. Sodium current depression by lidocaine and quinidine in isolated ventricular cells. Nature. 1981;291:325-327. [Medline] [Order article via Infotrieve]

4. Hille B. Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction. J Gen Physiol. 1977;69:497-515. [Abstract/Free Full Text]

5. Hondeghem LM, Katzung BG. Time- and voltage-dependent interactions of antiarrhythmic drugs with cardiac sodium channels. Biochem Biophys Acta. 1977;472:373-398. [Medline] [Order article via Infotrieve]

6. Bean BP, Cohen CJ, Tsien RW. Lidocaine block of cardiac sodium channels. J Gen Physiol. 1983;81:613-642. [Abstract/Free Full Text]

7. Fozzard HA, January CT, Makielski JC. New studies of the excitatory sodium currents in heart muscle. Circ Res. 1985;56:475-485. [Abstract/Free Full Text]

8. Bennett PB. Mechanisms of antiarrhythmic drug action: block of sodium channels in voltage clamped cardiac cell membranes. J Appl Cardiol. 1987;2:463-488.

9. Grant AO, Dietz MA, Gilliam FR III, Starmer CF. Blockade of cardiac sodium channels by lidocaine: single-channel analysis. Circ Res. 1989;65:1247-1262. [Abstract/Free Full Text]

10. Clarkson CW, Follmer CH, Ten Eick RE, Hondeghem LM, Yeh JZ. Evidence for two components of sodium channel block by lidocaine in isolated cardiac myocytes. Circ Res. 1988;63:869-878. [Abstract/Free Full Text]

11. Gingrich KJ, Beardsley D, Yue DT. Ultra-deep blockade of Na⁺ channels by a quaternary ammonium ion: catalysis by a transition-intermediate state? J Physiol (Lond). 1993;471:319-341. [Abstract/Free Full Text]

12. Grant AO. Evolving concepts of cardiac sodium channel function. J Cardiovasc Electrophysiol. 1990;1:53-67.

13. Starmer CF, Grant AO, Strauss HC. Mechanisms of use-dependent block of sodium channels in excitable membranes by local anesthetics. Biophys J. 1984;46:15-27. [Medline] [Order article via Infotrieve]

14. Starmer CF, Grant AO. Phasic ion channel blockade: a kinetic model and parameter estimation procedure. Mol Pharmacol. 1985;28:348-356. [Abstract]

15. Starmer CF, Courtney KR. Modeling ion channel blockade at guarded binding sites: application to tertiary drugs. Am J Physiol. 1986;251:H848-H856.

16. Starmer CF, Packer DL, Grant AO. Ligand-binding to transiently accessible sites: mechanisms for varying apparent binding rates. J Theor Biol. 1987;124:335-341. [Medline] [Order article via Infotrieve]

17. Hondeghem LM. Antiarrhythmic agents: modulated receptor applications. Circulation. 1987;75:514-520. [Free Full Text]

18. Hondeghem L, Anno T, Bennett PB, Johns J, Murray K, Snyders DJ. Modulated receptor: voltage- and time-dependence of sodium channel block. In: Kojima M, Hondeghem LM, eds. Current Topics in Antiarrhythmic Agents: Mode of Action and Clinical Usage. Tokyo, Japan: Excerpta Medica; 1989:43-54.

19. Hondeghem LM, Bennett PB. Models of antiarrhythmic drug action. In: Hondeghem L, ed. Molecular and Cellular Mechanisms of Antiarrhythmic Agents. Mount Kisco, NY: Futura Publishing Co; 1989:201-239.

20. Khodorov BI. Role of inactivation on local anesthetic action. Ann N Y Acad Sci. 1991;625:224-248. [Medline] [Order article via Infotrieve]

21. Strichartz GR. The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine. J Gen Physiol. 1973;62:37-57. [Abstract/Free Full Text]

22. Moczydlowski E. Blocking pharmacology of batrachotoxin-activated sodium channels. In: Miller C, ed. Ion Channel Reconstitution. New York, NY: Plenum Publishing Corp; 1986:405-428.

23. Postma SW, Catterall WA. Inhibition of binding of [³H]batrachotoxin in A 20- benzoate to sodium channel by local anesthetics. Mol Pharmacol. 1984;25:219-227. [Abstract]

24. Wang GK, Brodwick MS, Eaton DC, Strichartz GR. Inhibition of sodium current by local anesthetics in chloramine-T-treated squid axons: the role of activation. J Gen Physiol. 1987;89:645-667. [Abstract/Free Full Text]

25. Armstrong CM, Bezanilla F, Rojas E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J Gen Physiol. 1973;62:375-391. [Abstract/Free Full Text]

26. Patlak J, Horn R. The effect of N-bromoacetamide on single sodium channel currents in excised membrane patches. J Gen Physiol. 1982;79:333-351. [Abstract/Free Full Text]

27. Valenzuela C, Bennett PB. Gating of cardiac Na⁺ channels in excised membrane patches after modification by -chymotrypsin. Biophys J. 1994;67:161-171. [Medline] [Order article via Infotrieve]

28. Po S, Roberds S, Snyders DJ, Tamkun MM, Bennett PB. Heteromultimeric assembly of human potassium channels: molecular basis of a transient outward current? Circ Res. 1993;72:1326-1336. [Abstract/Free Full Text]

29. Chen LQ, Chahine M, Kallen RG, Barchi RL, Horn R. Chimeric study of sodium channels from rat skeletal and cardiac muscle. FEBS Lett. 1992;309:253-257. [Medline] [Order article via Infotrieve]

30. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100. [Medline] [Order article via Infotrieve]

31. Colquhoun D, Hawkes AG. The principles of the stochastic interpretation of ion-channel mechanisms. In: Sakmann B, Neher E, eds. Single-Channel Recording. New York, NY: Plenum Publishing Corp; 1983:135-175.

32. Colquhoun D, Sigworth FJ. Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E, eds. Single-Channel Recording. New York, NY: Plenum Publishing Corp; 1983:chap 11.

33. Horn R, Vandenberg CA. Statistical properties of single sodium channels. J Gen Physiol. 1984;84:505-534. [Abstract/Free Full Text]

34. McDonald TV, Courtney KR, Clusin WT. Use-dependent block of single sodium channels by lidocaine in guinea pig ventricular myocytes. Biophys J. 1989;55:1261-1266. [Medline] [Order article via Infotrieve]

35. Gellens ME, George AL, Chen L, Chahine M, Horn R, Barchi RL, Kallen RG. Primary structure and functional expression of the human cardiac voltage-dependent sodium channel. Proc Natl Acad Sci U S A. 1992;89:554-558. [Abstract/Free Full Text]

36. Chahine M, Bennett, PB, Horn R, George AL. Functional characterization of the human skeletal muscle Na⁺ channel. Biophys J. 1993;64:A4. Abstract.

37. West JW, Patton DE, Scheuer T, Wang Y, Goldin AL, Catterall WA. A cluster of hydrophobic amino acid residues required for fast Na⁺-channel inactivation. Proc Natl Acad Sci U S A. 1992;89:10910-10914.[Abstract/Free Full Text]

38. Armstrong CM. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J Gen Physiol. 1971;58:413-437. [Abstract/Free Full Text]

39. Monod J, Changeux JP, Jacob F. Allosteric proteins and cellular control systems. J Mol Biol. 1963;6:306-329. [Medline] [Order article via Infotrieve]

40. Catterall WA. Structure and function of voltage-sensitive ion channels. Science. 1988;242:50-61. [Abstract/Free Full Text]

41. Stühmer W, Conti F, Suzuki H, Wang X, Noda M, Yahagi N, Kubo H, Numa S. Structural parts involved in activation and inactivation of the sodium channel. Nature. 1989;339:597-603. [Medline] [Order article via Infotrieve]

42. Patton DE, West JW, Catterall WA, Goldin AL. Amino acid residues required for fast Na⁺-channel inactivation: charge neutralizations and deletions in the III-IV linker. Proc Natl Acad Sci U S A. 1992;89:10905-10909. [Abstract/Free Full Text]

43. Ragsdale DS, McPhee JC, Scheuer T, Catterall WA. Molecular determinants of state-dependent block of Na⁺ channels by local anesthetics. Science. 1994;265:1724-1728.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. A. Hanck, E. Nikitina, M. M. McNulty, H. A. Fozzard, G. M. Lipkind, and M. F. Sheets Using Lidocaine and Benzocaine to Link Sodium Channel Molecular Conformations to State-Dependent Antiarrhythmic Drug Affinity Circ. Res., August 28, 2009; 105(5): 492 - 499. [Abstract] [Full Text] [PDF]

				M. F. Sheets and D. A. Hanck Outward stabilization of the S4 segments in domains III and IV enhances lidocaine block of sodium channels J. Physiol., July 1, 2007; 582(1): 317 - 334. [Abstract] [Full Text] [PDF]

				C. E. Clancy, Z. I. Zhu, and Y. Rudy Pharmacogenetics and anti-arrhythmic drug therapy: a theoretical investigation Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H66 - H75. [Abstract] [Full Text] [PDF]

				J. A. M. Smith, S. M. Amagasu, J. Hembrador, S. Axt, R. Chang, T. Church, C. Gee, J. R. Jacobsen, T. Jenkins, E. Kaufman, et al. Evidence for a Multivalent Interaction of Symmetrical, N-Linked, Lidocaine Dimers with Voltage-Gated Na+ Channels Mol. Pharmacol., March 1, 2006; 69(3): 921 - 931. [Abstract] [Full Text] [PDF]

				W. Ulbricht Sodium Channel Inactivation: Molecular Determinants and Modulation Physiol Rev, October 1, 2005; 85(4): 1271 - 1301. [Abstract] [Full Text] [PDF]

				K. Fukuda, T. Nakajima, P. C Viswanathan, and J. R Balser Compound-specific Na+ channel pore conformational changes induced by local anaesthetics J. Physiol., April 1, 2005; 564(1): 21 - 31. [Abstract] [Full Text] [PDF]

				S. Y. Tsang, R. G. Tsushima, G. F. Tomaselli, R. A. Li, and P. H. Backx A Multifunctional Aromatic Residue in the External Pore Vestibule of Na+ Channels Contributes to the Local Anesthetic Receptor Mol. Pharmacol., February 1, 2005; 67(2): 424 - 434. [Abstract] [Full Text] [PDF]

				M. M. McNulty and D. A. Hanck State-Dependent Mibefradil Block of Na+ Channels Mol. Pharmacol., December 1, 2004; 66(6): 1652 - 1661. [Abstract] [Full Text] [PDF]

				S.-Y. Wang, J. Mitchell, E. Moczydlowski, and G. K. Wang Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells: Evidence for Drug Binding Along the Activation Pathway J. Gen. Physiol., November 29, 2004; 124(6): 691 - 701. [Abstract] [Full Text] [PDF]

				M. E. O'Leary, M. Digregorio, and M. Chahine Closing and Inactivation Potentiate the Cocaethylene Inhibition of Cardiac Sodium Channels by Distinct Mechanisms Mol. Pharmacol., December 1, 2003; 64(6): 1575 - 1585. [Abstract] [Full Text] [PDF]

				Y.-C. Yang and C.-C. Kuo Inhibition of Na+ Current by Imipramine and Related Compounds: Different Binding Kinetics as an Inactivation Stabilizer and as an Open Channel Blocker Mol. Pharmacol., November 1, 2002; 62(5): 1228 - 1237. [Abstract] [Full Text] [PDF]

				M E O'Leary and M Chahine Cocaine binds to a common site on open and inactivated human heart (Nav1.5) sodium channels J. Physiol., June 15, 2002; 541(3): 701 - 716. [Abstract] [Full Text] [PDF]

				J T Kimbrough and K J Gingrich Quaternary ammonium block of mutant Na+ channels lacking inactivation: features of a transition-intermediate mechanism J. Physiol., November 15, 2000; 529(1): 93 - 106. [Abstract] [Full Text] [PDF]

				B.-H. Ong, G. F. Tomaselli, and J. R. Balser A Structural Rearrangement in the Sodium Channel Pore Linked to Slow Inactivation and Use Dependence J. Gen. Physiol., November 1, 2000; 116(5): 653 - 662. [Abstract] [Full Text] [PDF]

				M. F. Sheets, J. W. Kyle, and D. A. Hanck The Role of the Putative Inactivation Lid in Sodium Channel Gating Current Immobilization J. Gen. Physiol., May 1, 2000; 115(5): 609 - 620. [Abstract] [Full Text] [PDF]

				Z. Chen, B.-H. Ong, N. G Kambouris, E. Marban, G. F Tomaselli, and J. R Balser Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels J. Physiol., April 1, 2000; 524(1): 37 - 49. [Abstract] [Full Text] [PDF]

				K. Ono, T. Kaku, N. Makita, A. Kitabatake, and M. Arita Selective Block of Late Currents in the Delta KPQ Na+ Channel Mutant by Pilsicainide and Lidocaine with Distinct Mechanisms Mol. Pharmacol., February 1, 2000; 57(2): 392 - 400. [Abstract] [Full Text]

				F. Lehmann-Horn and K. Jurkat-Rott Voltage-Gated Ion Channels and Hereditary Disease Physiol Rev, October 1, 1999; 79(4): 1317 - 1372. [Abstract] [Full Text] [PDF]

				J. R. Balser Structure and function of the cardiac sodium channels Cardiovasc Res, May 1, 1999; 42(2): 327 - 328. [Abstract] [Full Text] [PDF]

				V. Vedantham and S. C. Cannon The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na+ Channels J. Gen. Physiol., January 1, 1999; 113(1): 7 - 16. [Abstract] [Full Text] [PDF]

				T. Scheuer Commentary: A Revised View of Local Anesthetic Action: What Channel State Is Really Stabilized? J. Gen. Physiol., January 1, 1999; 113(1): 3 - 6. [Full Text] [PDF]

				H.-L. Li, A. Galue, L. Meadows, and D. S. Ragsdale A Molecular Basis for the Different Local Anesthetic Affinities of Resting Versus Open and Inactivated States of the Sodium Channel Mol. Pharmacol., January 1, 1999; 55(1): 134 - 141. [Abstract] [Full Text]

				N. G Kambouris, L. A Hastings, S. Stepanovic, E. Marban, G. F Tomaselli, and J. R Balser Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat {micro}1 skeletal muscle sodium channel J. Physiol., November 1, 1998; 512(3): 693 - 705. [Abstract] [Full Text] [PDF]

				J. Pu, J. R. Balser, and P. A. Boyden Lidocaine Action on Na+ Currents in Ventricular Myocytes From the Epicardial Border Zone of the Infarcted Heart Circ. Res., August 24, 1998; 83(4): 431 - 440. [Abstract] [Full Text] [PDF]

				G. K. Wang, C. Quan, and S.-Y. Wang Local Anesthetic Block of Batrachotoxin-Resistant Muscle Na+ Channels Mol. Pharmacol., August 1, 1998; 54(2): 389 - 396. [Abstract] [Full Text]

				R. L. Sah, R. G. Tsushima, and P. H. Backx Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation Am J Physiol Cell Physiol, August 1, 1998; 275(2): C389 - C400. [Abstract] [Full Text] [PDF]

				Y.-F. Xiao, S. N. Wright, G. K. Wang, J. P. Morgan, and A. Leaf Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the alpha -subunit of the human cardiac Na+ channel PNAS, March 3, 1998; 95(5): 2680 - 2685. [Abstract] [Full Text] [PDF]

				R. Dumaine and G. E. Kirsch Mechanism of lidocaine block of late current in long Q-T mutant Na+ channels Am J Physiol Heart Circ Physiol, February 1, 1998; 274(2): H477 - H487. [Abstract] [Full Text] [PDF]

				S. Kellenberger, J. W. West, T. Scheuer, and W. A. Catterall Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels J. Gen. Physiol., May 1, 1997; 109(5): 589 - 605. [Abstract] [Full Text] [PDF]

				S. Kellenberger, T. Scheuer, and W. A. Catterall Movement of the Na+ Channel Inactivation Gate during Inactivation J. Biol. Chem., November 29, 1996; 271(48): 30971 - 30979. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bennett, P. B. Articles by Kallen, R. G. Search for Related Content PubMed PubMed Citation Articles by Bennett, P. B. Articles by Kallen, R. G.