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Am J Physiol Heart Circ Physiol 274: H477-H487, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 2, H477-H487, February 1998

Mechanism of lidocaine block of late current in long Q-T mutant Na+ channels

R. Dumaine and G. E. Kirsch

Rammelkamp Center for Research and Department of Physiology and Biophysics, Case Western Reserve University, MetroHealth Campus, Cleveland, Ohio 44109

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Inherited long Q-T syndrome is a ventricular arrhythmia associated with delayed repolarization and the risk of sudden death. The chromosome 3-linked form of the disease (LQT3) is associated with mutations in the cardiac Na+ channel (N1325S or R1644H; or deletion of residues 1,505-1,507, Delta KPQ) that increase late inward currents and may cause delayed repolarization. Late currents arise from dispersed reopenings (N1325S and R1644H) or from reopenings combined with prolonged bursts (Delta KPQ). Therefore, we tested whether lidocaine blockade of late current varied among the different LQT3 mutant channels. We found that lidocaine preferentially blocked late over peak current and that the blockade was equally effective in all three channels, expressed in Xenopus oocytes. Lidocaine inhibited both dispersed reopenings and bursting in single channels without affecting mean open times. In the absence of drug, inactivating prepulses inhibited bursting but not dispersed reopenings. We suggest that lidocaine block of late current in LQT3 channels acts via a common mechanism involving stabilization of inactivation. Therefore, blockers that target the inactivated state may be effective therapeutic agents in all three biophysical phenotypes of LQT3.

cardiac arrhythmia; human heart; Romano-Ward syndrome; SCN5A

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IN THE LONG Q-T SYNDROME prolongation of the Q-T interval of the electrocardiogram is associated with polymorphic ventricular tachycardia (torsades de pointes), cardiac arrest, and sudden death (30). The Romano-Ward form of the disease is inherited in an autosomal dominant pattern and is genetically heterogeneous. Linkage analysis has identified four disease-related loci, at least three of which are known to involve genes that encode ion channels. LQT1 (chromosome 11-linked; Ref. 34) and LQT2 (chromosome 7-linked; Refs. 10, 27) are associated with mutations in voltage-gated potassium channel genes (KVLQT1 and HERG, respectively), and LQT3 (chromosome 3-linked; Ref. 35) is associated with mutations in SCN5A, a gene that encodes the cardiac Na+ channel.

The balance between inward, depolarizing and outward, repolarizing currents through ion channels regulates ventricular repolarization. Thus a decrease in outward K+ currents or an increase in inward Ca2+ or Na+ currents has the potential to lengthen the Q-T interval and induce early afterdepolarizations (1, 11, 20). Because membrane resistance during the action potential plateau is very high, small changes in late ionic currents can markedly alter the action potential waveform and the Q-T interval (14, 26). Late Na+ current that persists after fast inactivation has reached a steady state has been shown to prolong the action potential plateau in animal models (3, 9, 16, 21). In heterologous expression systems, human LQT3 mutant Na+ channels exhibit increased levels of late Na+ current (6, 13, 32) that may be directly responsible for the disease.

We previously reported (13) that the LQT3 mutations Delta KPQ (deletion of a lysine, proline, and glutamine at positions 1,505-1,507), R1644H (arginine-to-histidine substitution), and N1325S (asparagine-to-serine substitution) cause an increase in late current by promoting two types of inactivation abnormalities: short reopenings indicative of an accelerated exit from the inactivated state (i.e., reduced stability) and prolonged bursting, indicative of a switch from a normal to a bursting (i.e., noninactivating) mode of gating. Na+-channel blockers, such as class I antiarrhythmics, are potentially useful therapeutic agents. However, drugs that act by promoting inactivation might not be as effective in blocking the noninactivating mode of channel activity as in correcting defects involving reduced stability of the inactivated state. In the present study, we chose lidocaine as a prototypic inactivation-promoting class Ib antiarrhythmic to address this question. Previous studies (2, 13, 33) showed that the late current is more sensitive than the peak current to block by class Ib antiarrhythmics, but the mechanism is unclear, particularly in view of previous observations that noninactivating mutant channels have reduced sensitivity to block by lidocaine (5).

In the present study, we found that the frequency of occurrence of bursting activity was suppressed by inactivating prepulses. Moreover, the effectiveness of lidocaine in blocking late current was enhanced at depolarized holding potentials. Lidocaine, which potentiates the inactivated state, reduced the frequency of occurrence of both bursts and dispersed openings by increasing the closed-time duration between the events but did not change the mean open times. Our results indicate that lidocaine exerts its action on the late current by stabilizing inactivation in LQT3 mutant Na+ channels, thereby reducing the probability of occurrence of the dispersed and bursting activity nonselectively.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Na+-channel clone and mutagenesis. The human heart Na+-channel clone (hH1a, expression plasmid of SCN5A) was the same as previously described (17). The full-length cDNA was cloned into the pGEM3 plasmid vector (Promega, Madison, WI). The final SCN5A expression construct contains cDNA sequence from nucleotide (nt) 123 to nt 6,333 of the published SCN5A cDNA sequence (15).

LQT3 is associated with three defects in the primary structure of the cardiac Na+ channel (35): a substitution of serine for asparagine at position 1,325 (in the intracellular linker between transmembrane segments S4 and S5 of domain III), a substitution of histidine for arginine at position 1,644 (at the intracellular end of transmembrane segment S4 in domain IV), or a three-residue deletion of lysine-proline-glutamine from positions 1,505-1,507. The mutant channels resulting from the expression of these three constructs are denoted in the text by single-letter amino acid abbreviations: N/S, R/H, and Delta KPQ, respectively. The nonmutated (wild type) channel is denoted WT. LQT3 mutations were produced by site-directed, polymerase chain reaction-based mutagenesis using the megaprimer method (28). All three mutant constructs were verified by DNA sequencing.

RNA transcription and oocyte injection. DNA constructs were linearized by digestion with Hind III, and in vitro transcription with T7 RNA polymerase was performed using the mMessage Machine kit (Ambion, Austin, TX). The amount of cRNA product was quantified by incorporation of trace amounts of [32P]UTP in the synthesis mixture, and the integrity of the cRNA was determined using a denaturing agarose gel stained with ethidium bromide. cRNA was resuspended in 0.1 M KCl at a concentration of 250 ng/ml and stored at -80°C. Before use, cRNA was diluted to the desired concentration (generally 1-10 pg/nl). Stage V-VI Xenopus oocytes were defolliculated by collagenase treatment (2 mg/ml for 1.5 h) in a nominally Ca2+-free buffer solution containing (in mM) 82.5 NaCl, 2.5 KCl, 1 MgCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with 100 mg/ml gentamicin, pH 7.6. The oocytes were injected with 46 nl of cRNA solution (in 0.1 M KCl) and incubated at 19°C in culture medium containing (in mM) 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and 2.5 pyruvic acid with 100 mg/ml gentamicin, pH 7.6. Electrophysiological measurements were made 5-10 days after cRNA injection. The amount of cRNA injected was varied according to the purpose of the experiment. For whole cell measurements of peak Na+ currents, the amount of cRNA was adjusted to give peak whole cell currents in the range 5-15 µA to minimize space clamp inhomogeneities and series resistance errors; late current measurements required peak current amplitudes in the range 20-50 µA to maximize the signal.

Electrophysiology and data analysis. Whole cell currents were recorded in oocytes using a two-microelectrode voltage clamp as described previously (12). Beveled microelectrode tips were filled with a solution of 3 M KCl-1% agar and then backfilled with 3 M KCl (29). This method gave sharp-tipped microelectrodes with low electrical resistance (0.2-0.5 MOmega ) required for optimal clamp performance. Whole cell data were analyzed using Clampfit (Axon Instruments, Foster City, CA).

Cell-attached patch recording was performed after dissection of the vitelline envelope. Isotonic KCl bathing solution was used to zero the resting potential; the absence of resting membrane potential was verified by rupturing the membrane patch at the end of each experiment to allow direct intracellular potential measurement. Holding and test potentials applied to the membrane patch during the experiment are reported as conventional intracellular potentials. Data were low-pass filtered at 5 kHz (-3 dB, 4-pole Bessel filter) and then digitized at 20-100 kHz. Linear leakage and capacitative transients were corrected off-line by subtracting the average of records lacking channel activity (null traces).

Data were filtered off-line to a final bandwidth of 3 kHz. Single-channel transitions between closed and open levels were detected using Transit, an interactive event-detection program (31), with the amplitude criterion set at one-half of the maximum amplitude of the unitary current. Computer-detected openings were used to generate idealized records from which histograms of amplitude and closed-time and open-time distribution were constructed. Mean open time and closed time were calculated by fitting the data to the sum of exponential decay functions using a maximum-likelihood estimate. Events of <0.15-ms duration (dead time of the detection system at 3 kHz) were excluded from the fitting to avoid truncation errors introduced by bandwidth limitations (22). Where appropriate, data are expressed as means ± SE. A two-tailed Student's t-test or analysis of variance (ANOVA) was used to evaluate the significance of the difference between means (P < 0.05).

Solutions and drugs. The modified Ringer solution for whole cell recording consisted of (in mM) 120 NaOH, 2 KOH, 122 methanesulfonic acid, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.2 (with NaOH). The patch solution consisted of (in mM) 120 NaCl, 2.5 KCl, 1 CaCl2, 2 MgCl2, and 10 HEPES. Lidocaine (Sigma Chemical, St. Louis, MO) and tetrodotoxin (Calbiochem, San Diego, CA) were diluted in the bath solution from frozen aliquots of concentrated stocks. The depolarizing isotonic KCl bath solution for patch recording consisted of (in mM) 100 KCl, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES, pH 7.3. The pipette solution consisted of (in mM) 100 NaCl, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.2. Bath solution flowed continuously at a rate of 3 ml/min. The experiments were performed at room temperature (21-23°C).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Lidocaine block of whole cell currents in LQT3 mutant channels. In native Na+ channels, lidocaine blocks the inactivated state of the channel more effectively than the resting state (4). Inactivated-state (phasic) block accumulates during sustained depolarization and is relieved by sustained repolarization that allows recovery from inactivation. In contrast, resting-state (tonic) block results in a reduction of peak currents even after recovery from inactivation at strongly negative holding potentials. We compared these two forms of lidocaine block in both WT and LQT3 mutants as shown in Fig. 1. Tonic block (Fig. 1A) was estimated from the lidocaine-induced reduction in peak currents during a short (10 ms) test pulse (-10 or 0 mV) that produced maximum activation of unblocked channels. The oocytes were bathed in drug solution for an equilibration period of 6 min during which the cells were pulsed at 15-s intervals. Figure 1A compares a control record obtained before drug application with that obtained after equilibration of resting Delta KPQ channels with bath-applied lidocaine at 50 µM. This concentration typically reduced peak current by <10%, indicative of the relatively low sensitivity of resting channels to drug block. As shown by the filled symbols in the dose-response relationship (Fig. 1D), similar levels of block were observed in all four types of channels such that tonic block was negligible at drug concentrations <100 µM. Shifting the holding potential to -100 mV, however, resulted in an apparent increase in tonic block that was particularly evident at higher concentrations. As discussed below, the amount of tonic block observed at higher concentrations and more depolarized holding potentials may not provide an accurate estimate of resting channel block caused by drug-enhanced inactivation. Nonetheless, the results suggest that when held in the resting state, all of the channels were highly resistant to drug occupancy.


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  		Fig. 1.   Sensitivity of peak and late currents to lidocaine block. Currents from nonmutated human heart Na+ channel hH1a (WT) and LQT3 mutants Delta KPQ, R1644H (R/H), and N1325S (N/S) were expressed in Xenopus oocytes and measured using 2-microelectrode voltage-clamp technique. A: superimposed records of Delta KPQ currents obtained before and during lidocaine (50 µM) application. Currents were evoked by 10-ms test pulses (inset; only 1st 6 ms are illustrated, capacitative transient is offscale during 1st ms of record) to -10 mV from holding potential (HP) of -120 mV. Peak amplitudes were monitored to assess amount of tonic block during repetitive stimulation at 30-s intervals on external application of lidocaine. black-square, Trace recorded after drug equilibration. In experiment illustrated, peak test pulse currents were reduced 6% in 50 µM lidocaine. Average block from pooled data was 7.1 ± 1.1% (n = 6). B: maximum phasic block obtained under conditions that favored inactivation (see pulse protocol, inset) was measured as reduction of peak test pulse current during a 10-ms pulse to -10 mV after a 960-ms conditioning prepulse and a 10-ms return to -140 mV (to remove residual inactivation) from HP of -100 mV. In experiment illustrated, lidocaine at 50 µM reduced Delta KPQ current by 49%; average value was 54.4 ± 3.3% (n = 6). Traces A and B were filtered at 2,000 Hz, and linear leakage currents were corrected off-line using hyperpolarizing steps. C: late current blockade was measured as reduction of current 300 ms from start of a 500-ms depolarizing pulse to 0 mV from HP of -100 mV. In experiment illustrated, lidocaine (50 µM) reduced Delta KPQ late current by 40%; average value was 35.7 ± 3.4% (n = 6). Nonspecific and capacitative currents were corrected off-line by subtraction of current records obtained after application of 100 µM tetrodotoxin, and traces were filtered at 200 Hz. D and E: dose-response relationships for phasic (D), tonic (D), and late current (E) block. In D, filled and open symbols denote data obtained with HP set at -120 and -100 mV, respectively. Current amplitudes were normalized to control values and plotted against lidocaine concentration. Smooth curves were obtained by fitting pooled data to 1:1 binding isotherms (Hill function); apparent dissociation constant (Kd) = 39, 516, 1,785 and 89 µM, for phasic, tonic (HP -100mV), tonic (HP -120 mV), and late current block, respectively. Data from all 4 channel types was pooled and used to fit tonic (open symbols, D), phasic (closed symbols, D) and late current (E) block data (solid lines). Fits to individual channel data sets gave apparent Kd values for phasic block: 32 ± 1, 25 ± 3, 33 ± 5, and 39 ± 3 µM in WT, R/H, N/S, and Delta KPQ (n = 5-8 cells), respectively. Late current block gave apparent Kd of 113 ± 14, 73 ± 6, and 88 ± 13 µM in R /H, N/S, and Delta KPQ, respectively (n = 6-9 cells).

We measured phasic block using a three-pulse protocol (Fig. 1B). A long conditioning pulse (0-mV amplitude, 960-ms duration) allowed drug equilibration with channels in the inactivated state. Next, a brief prepulse (-140 mV, 10 ms) allowed unblocked channels to recover from inactivation. The amount of block was determined from measurement of peak current during a brief test pulse (0 mV, 10 ms) that maximally activated the channels. Figure 1B shows superimposed Delta KPQ test pulse current records under control conditions and after equilibration with 50 µM lidocaine. An average 55% inhibition of peak current obtained after preconditioning with a long depolarizing pulse indicated a much stronger block of the inactivated than the resting state. Figure 1D shows that this effect was due to a leftward shift of the dose-response relationship such that substantial phasic block (>20%) occurred at a concentration (10 µM) that was ineffective in producing tonic block. Phasic block showed an apparent dissociation constant (Kd) of 32 ± 1, 39 ± 3, 25 ± 3, and 33 ± 5 µM for WT, Delta KPQ, R/H and N/S, respectively. These values were not significantly different by ANOVA test. Our results showed that lidocaine block of channels in the inactivated states was comparable between WT and LQT3 channels and suggest that the affinity for lidocaine was not modified by the mutations.

We next compared the effectiveness of lidocaine in blocking the late current in LQT3 channels (late current in WT channels was too small to resolve lidocaine block). Late current block was measured from inhibition of currents evoked by 500-ms test pulses to 0 mV from a holding potential of -100 mV. Isochronal current amplitudes at 300 ms were compared before and after drug application. Figure 1C illustrates typical results obtained in Delta KPQ channels in 50 µM lidocaine. An average value of 36% block obtained under these conditions was slightly less than the amount of phasic block at the same concentration. Nonspecific currents were corrected by subtraction of current evoked by the same pulse protocol after application of 100 µM tetrodotoxin (a selective Na+ pore blocker). It should be noted, however, that at the 300-ms time point late current block might not have reached steady state, particularly at the lowest concentrations. However, longer test pulses can activate variable levels of endogenous currents in the oocyte that interfere with accurate measurement of the very small, late Na+ currents. As shown in Fig. 1E, late current block was concentration dependent such that an 89 µM dose was required to reach 50% in all three of the LQT3 mutant channels. Thus the concentration dependence of lidocaine block of late currents, although intermediate in effectiveness between tonic and phasic block, more closely resembled inactivated-state block. Given the uncertainty about whether late current blockade was measured in steady state, it seems possible that all of the block was associated with the inactivated state, but we cannot rule out a contribution from drug interaction with the resting or open states. Nonetheless, our results clearly show that when measured under identical conditions, lidocaine did not discriminate between the different LQT3 mutants. Moreover, additional observations described below suggest that late current block occurred primarily from interaction of the drug with the inactivated state.

The identification of tonic and phasic block with resting and inactivated states, respectively, assumes that inactivation is absent at the holding potentials used to measure tonic block. We measured steady-state inactivation (Fig. 2) using a standard voltage protocol of a 25-ms test pulse to -10 mV preceded by a 500-ms conditioning pulse to varying potentials from a holding potential of -100 mV. Peak test pulse currents were normalized to the maximum amplitude recorded under control conditions and plotted against the conditioning potential. The block by lidocaine in both WT and Delta KPQ channels was characterized by a concentration-dependent shift of the steady-state inactivation curves toward more negative potentials and by a distortion of the shape of the curve. Channel maximum availability saturated at -100 mV in the absence of drug, whereas in the presence of high concentrations of drug the curve did not reach plateau in the range -100 to -150 mV. This effect may account for the increased amount of tonic block observed at high lidocaine concentrations (Fig. 1) and suggests that block of late current may also be sensitive to the effects of drug on steady-state inactivation. Therefore, we tested whether depolarizing holding potentials could enhance the effectiveness of late current block. Typical effects of varying the holding potential on late current block in Delta KPQ at 50 µM lidocaine are shown in Fig. 3, A-C. Very negative holding potentials (Fig. 3, A and B) remove steady-state inactivation and appear to have little effect on late current block, but as the holding potential approached the midpoint of the steady-state inactivation curve (Fig. 3C), we observed a significant increase in late current block. A similar pattern is present in pooled data from all three LQT3 channels, but the increased block at a holding potential of -80 mV was only statistically significant for the Delta KPQ mutant. These results indicate that late current block is sensitive to the inactivated state of the channel and that the Delta KPQ mutant channel is at least as sensitive to lidocaine block as the N/S and R/H mutants.


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  		Fig. 2.   Effects of lidocaine on voltage dependence of steady-state inactivation in WT (A) and Delta KPQ (B) channels. We used a conventional steady-state inactivation protocol to measure amount of current available at a fixed test potential of -10 mV (25 ms), following a 500-ms inactivating prepulse to varying potentials. Peak test pulse currents were normalized to the maximum current obtained under drug-free, control conditions. Under these conditions (bullet ), midpoint potentials of -68.3 ± 0.5 mV (n = 6) and -70.7 ± 0.6 mV (n = 6) were observed in WT and Delta KPQ, respectively. Em, membrane potential.


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  		Fig. 3.   HP dependence of late current block. A-C: whole cell recordings of late currents in Delta KPQ-injected oocyte before and after (black-square) application of 50 µM lidocaine. Late currents evoked by a 500-ms test to 0 mV from HP of -120 (A), -100 (B), and -80 (C) mV were reduced by 31, 38, and 72%, respectively. D: summary of late current block by 50 µM in R/H, N/S, and Delta KPQ mutants. Percent block was obtained from ratio of lidocaine-treated cell late current to control. * Statistically significant difference between mean obtained at HP = -120 mV vs. HP = -100 or -80 mV.

Lidocaine blockade in single channels. In native Na+ channels, lidocaine block produces a very stable inactivated, drug-bound state from which recovery is much slower than recovery from inactivation in the absence of drug (19). Therefore, the effectiveness of lidocaine in blocking late currents in N/S and R/H mutants was not surprising, because the gating defect (dispersed reopenings) appears to reduce the stability of the inactivated state at depolarized test potentials. In these channels, lidocaine is likely to increase the stability of the inactivated state and thereby restore normal gating through a reduction in the frequency of the dispersed reopenings. The observation that late currents generated by Delta KPQ had a similar sensitivity to lidocaine was more difficult to rationalize, because roughly one-half of the late current is contributed by burst activity, a gating mode in which the channels fail to inactivate (6). Channels in this mode might be resistant to lidocaine block, because they are in the open state most of the time. If such were the case, resistance to block associated with Delta KPQ burst openings should increase the relative contribution of the burst over dispersed openings to the Delta KPQ late current in the presence of lidocaine. An alternative explanation is that the open state in the Delta KPQ bursting mode might have an unusually high sensitivity to block (compared with normal channels or noninactivating mutants; Ref. 5) such that burst mode activity and dispersed reopenings are equally sensitive. A second alternative is that lidocaine stabilizes the inactivated state, and, as a result, the channels seldom enter the burst mode. We addressed these questions by single-channel analysis of Delta KPQ.

To determine whether lidocaine blocked channel openings during the burst mode, we measured dwell time in the open state for single-channel events during test pulses to 0 mV from a holding potential of -100 mV. Under cell-attached conditions, the midpoint potential of steady-state inactivation was typically shifted from -71 mV observed in whole cell recording to approximately -100 mV (similar observations have been reported for WT channels; Ref. 17). We therefore used 500-ms prepulses to -140 mV to fully remove inactivation. Figure 4 shows representative recordings in controls and after application of 10 µM lidocaine. In both sets of traces we observed burst activity, related to the slow gating mode in Delta KPQ channels, and short dispersed openings characteristic of all three mutant channels. The last pair of traces shows the ensemble average currents as described below. We analyzed open time and amplitude histograms of idealized records to obtain estimates of mean open time and unitary amplitude (Table 1). After application of 10 µM lidocaine, we observed fewer bursts and dispersed openings and more null traces (no detectable activity). However, as shown in Table 1, drug application had no effect on either the long open times associated with bursting or the short open times associated with dispersed reopenings. Moreover, unitary amplitudes were unaffected by drug application. These results indicate that lidocaine does not act by blocking the open state in LQT3 channels.


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  		Fig. 4.   Lidocaine blockade in single Delta KPQ channels. Traces 1-9 show patch-clamp records from a cell-attached patch containing at least 5 channels. Currents were evoked by 0-mV, 150-ms test pulses from HP of -100 mV at a frequency of 0.2 pulses/s. Each test pulse was preceded by a -140-mV, 500-ms prepulse to remove resting inactivation. Dashed line indicates baseline (0 pA), and openings are negative events (downward deflections). Currents were filtered on-line at 3 kHz and digitized at 10 kHz. Traces were analyzed without additional filtering but are illustrated with 2-kHz filtering. Initial 20 ms of each record are blanked; only last 130 ms of each trace (late currents) were analyzed. In control (left) clusters of traces containing long burst openings were separated by more quiescent periods showing short dispersed openings. Bath application of 10 µM lidocaine (right) reduced overall activity and prolonged interval between bursts and between dispersed openings. Bottom panels show total and burst components of ensemble average currents. Ensemble of traces containing bursting activity accounted for 67 and 69% of total in control and treated cells, respectively. Contribution of current from bursts to total ensemble average current was calculated from ratio of averaged current value during last 40 ms (AVG, dashed lines) for each condition. Nearly identical contribution of bursting activity to ensemble average current shows that lidocaine reduced late current in a nonselective fashion between bursting and nonbursting traces. Same result was obtained by analyzing means in 4 additional patches (total n = 5).

                              
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  		Table 1.   Effect of lidocaine on single-channel parameters of Delta KPQ late openings

We next determined whether lidocaine differentially blocked dispersed versus burst activity by estimating the contribution of bursting to the total ensemble average current. Bursts were defined as groups of openings with open times >6 ms, based on our previous results (13), and traces containing at least one group of openings >6 ms were averaged. A closed-time histogram was constructed from this collection of traces and fit to a biexponential distribution. We observed that 90% of the distribution area was described by a time constant of 0.2 ms and the remaining events by a time constant of 11 ms. The shortest time constant was taken as the closed interval between events within a burst, and a burst was defined as a sequence of three or more openings separated by intervals <= 6 ms. A dispersed opening was therefore a single event separated from its neighbors by closed intervals >6 ms. To quantify the relative contributions of the bursts and dispersed openings, we separated the traces containing bursts and calculated an ensemble current from these selected traces. The burst ensemble current was then averaged to the total number of traces and normalized to the ensemble average current from the entire set of recordings. In the experiment illustrated in Fig. 4, the current from burst openings accounted for 67% of the ensemble average current in control and 69% when lidocaine was applied. In three experiments with 10 µM lidocaine, the contribution of bursts to the ensemble average current was 62 ± 9% in control and 63 ± 15% after application of the drug. There was no significant difference between the two conditions compared by Student's t-test or ANOVA. The difference in the contributions between control and 50 µM lidocaine-treated cells were 2 and 4% in two other patches, respectively.

Because lidocaine does not act by blocking the open state in LQT3 channels, we asked whether late current block was associated with the inactivated state, an indication of which would be an increase in closed time between events. As shown in Fig. 5, A and B, the frequency of occurrence of both bursts and dispersed openings was reduced during application of lidocaine. Activity diaries were constructed before (Fig. 5A) and after (Fig. 5B) drug application to quantify the frequency of the bursts. In a very active control Delta KPQ patch (Fig. 5A), 276 traces showed burst activity, indicated by large spikes in the open probability diary, and 47 traces showed no activity (null trace) during the last 120 ms of the test pulse (after peak current has subsided) in a series of 705 steps to 0 mV (150 ms). After application of 50 µM lidocaine (Fig. 5B), we observed 51 bursts in a series of 485 active traces. Lidocaine reduced the occurrence of bursts from 39 to 10% and increased the fraction of null traces from 6 to 19% (Fig. 5C). The number of channels in the patch was estimated from the ratio of maximum peak transient (~50 pA in this patch) to single channel (~1 pA, Table 1) to be roughly 50 channels. In previous experiments (13), the late current accounted for 0.15 and 1.6% of the WT and Delta KPQ peak current, respectively. In a patch containing 50 channels, this translates to a contribution of no more than two channels during the late activity (unitary current ~1 pA). Recordings of late current with overlaps of more than two current amplitude levels were discarded, and single transitions between closed and open states were easily discerned. To quantify the effect of lidocaine on the closed-time distribution between the short openings, we selected traces showing only dispersed activity and constructed a closed-time histogram (Fig. 5, D and E) in the same patch before and after drug application. In the control data of the patch analyzed in Fig. 5, the closed-time distribution was fitted by the sum of two exponentials with fast (tau 1) and slow (tau 2) time constants of 2 ms (61%) and 18 ms (39%), respectively. After lidocaine treatment (Fig. 5E), longer closed-time intervals were more frequent, as indicated by the rightward shift of the peak of the fit, and values of 2 ms (26%) and 29.6 ms (74%) for tau 1 and tau 2, respectively, were obtained. In a total of five patches, we observed shifts of the closed-time distribution toward the longer component and/or an increase in one or both of the time constants. Because the number of channels varied in each patch, we could not pool closed-time data from different patches or ascribe the drug-induced changes to a particular inactivation scheme. However, our qualitative comparison of the closed-time distribution for the dispersed reopenings is consistent with the channels spending more time in closed (drug bound, inactivated) states after lidocaine application.


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  		Fig. 5.   Effect of lidocaine on closed-time intervals between bursts and dispersed openings in Delta KPQ channels. A: diary of late activity during 705 test pulses to 0 mV, 150 ms, at 0.2 pulses/s from HP of -100 mV. Patch recordings were obtained in cell-attached configuration. Only last 130 ms of each trace were analyzed. Percent time spent in open state (open probability product; NPo, %) for each trace was plotted vs. test pulse number. Large spikes indicate traces containing long open times characteristic of bursts. B: activity diary after bath application of 50 µM lidocaine showed diminished frequency of bursting. Patch contained >50 channels (peak current 53 pA). C: number of traces containing either bursts or no activity (nulls) were counted, and sum was normalized to total number of traces in a set to obtain relative frequency of activity. Lidocaine reduced number of bursts and increased number of null traces. D and E: closed-time histograms from control (D) and lidocaine treatment (E) including only traces containing dispersed openings. Distributions were fit to sum of 2 exponentials (thin line) with fast (tau 1) and slow (tau 2) time constants of 2 ms (61%) and 18 ms (39%), respectively (thick lines) (D). After lidocaine treatment (E), longer closed-time intervals were more frequent as indicated by rightward shift of peak of fitted curve (thin line in D). Fitting procedure returned values of 2 ms (26%) and 30 ms (74%) for tau 1 and tau 2. Quantitatively similar results were obtained in another experiment at 50 µM. Application of 10 µM (n = 3) produced qualitatively similar but less dramatic effects on closed-time interval distributions.

Figure 5C shows the effect of lidocaine on the occurrence of bursting and null traces in the unsorted data set from the experiment described above. We observed average reductions of 14 ± 3% (n = 4) in the frequency of the bursts in patches treated with 10 µM and 17 ± 2% (n = 3) in patches treated with 50 µM lidocaine. The average increase in null traces was 3.5 ± 2% (n = 4) and 10.5 ± 0.4% (n = 3) in 10 µM and 50 µM lidocaine, respectively. We therefore observed more frequent and longer closed-time intervals between dispersed openings, a reduction in the frequency of the bursts, and an increase in the number of null traces after application of lidocaine consistent with the hypothesis of lidocaine-induced prolongation of dwell time in the inactivated state.

These results indicated that the drug did not exert an activity-specific blockade. Moreover, the reduction in the activity was not caused by changes in the mean open time during the bursts but rather by a prolongation of the intervals between open events of each type, consistent with the well-known action of lidocaine to slow recovery from the inactivated state (see, e.g., Ref. 4). This explanation presupposes that initiation of both dispersed and burst mode openings is inhibited when the channels are in the drug-bound, inactivated state. In the case of the dispersed reopenings, we postulate that the channels have already entered the inactivated state and that lidocaine inhibits reopening by slowing the recovery reaction. If a similar mechanism applied to burst mode activity, this would predict that the initiation of bursting would be inhibited by inactivation even in the absence of drug. Such an effect also would be consistent with our observation of holding potential dependence of late current blockade (Fig. 3) in Delta KPQ whole cell measurements.

We designed a series of experiments to explore this idea by testing whether inactivating prepulses affect the amount of burst activity recorded from Delta KPQ channels in the absence of lidocaine. Under standard experimental conditions (see, e.g., Fig. 5), diaries of burst activity were obtained by repetitive test pulses preceded by a 500-ms hyperpolarizing prepulse (-140 mV) that removed resting inactivation. With each test pulse the channels start in the resting state and progress to open and inactivated states. Entry into the burst mode could occur from any or all of these states. Figure 6 shows the results of varying the conditioning prepulse voltage such that before the test pulse was applied the channels were either completely resting (with inactivation removed by a prepulse to -140 mV) or partially inactivated (prepulse -70 mV). The peak ensemble average current measured at -10 mV was reduced by 85% after a depolarizing prepulse to -70 mV (Fig. 6, A-C). A diary of the late activity (Fig. 6D) showed that bursts were frequently observed during test pulses preceded by a -140-mV prepulse but strongly suppressed after switching to a prepulse to -70 mV. This experiment also suggests that the frequency of the dispersed reopenings was unaffected by the conditioning prepulse. In Fig. 6E, the cumulative integrals of the current traces are plotted against the trace number. In this plot, clusters of low-open probability product (NPo, where N is no. of channels and Po is mean open probability) traces (Fig. 6D), in which reopenings are the only form of activity, translate into line segments with a constant, shallow slope that occur throughout the experiment, whereas high-NPo traces containing bursts result in brief intervals of steep slope that were less frequent during application of -70 mV prepulses.


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  		Fig. 6.   Effect of inactivating prepulses on Delta KPQ bursting activity under control conditions. A-C: ensemble average current records in a cell-attached patch containing at least 14 Delta KPQ channels (maximum peak current -14.4 pA), in absence of lidocaine, were evoked by test pulses to -10 mV from HP of -100 mV. Test pulses were 144 ms in duration and delivered at a frequency of 1 pulse/s such that interval between end of each pulse and beginning of next pulse was 856 ms. During pulse intervals between traces 1 and 512 (A) and 1,025 and 1,536 (C), a prepulse lasting 100 ms to -140 mV was applied 2 ms before start of each test pulse to remove resting inactivation. A prepulse of -70 mV was applied for traces 513-1,024 (B) to invoke resting inactivation. Transient peak current (0-30 ms) in ensemble averages was reduced by 85% after conditioning prepulse to -70 mV. Scale: horizontal, 5 ms; vertical 5 pA. D: diary of experiment obtained by plotting percentage of time spent in open state (NPo) for each trace vs. test pulse number. In E, data were quantified by plotting cumulatively late current integral (i.e., segment 30-144 ms). Stepwise increases in slope when -140 mV prepulses were applied result from frequent occurrence of clusters of bursts. Depolarizing prepulses reduced number of steps but not background dispersed activity as indicated by constant, nonzero slope. In pooled data, switching from a prepulse of -140 to -70 mV reduced burst activity by 64 ± 11% (n = 7).

The reduction in bursting varied significantly from one patch to another and with the number of channels active under the patch. In two other experiments (not shown), both bursts and peak current were completely abolished by -70-mV prepulses without significant changes in the shallow slopes in the integral plot. The fluctuations in the availability of the channels correlated with the change in bursting activity in these experiments, but overall, inactivating prepulses reduced bursting by 64 ± 11% (n = 6).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We previously reported that mexiletine (a lidocaine analog) was more effective in blocking the late component of Na+ current than the peak current (13), and a similar observation has been made for lidocaine block in Delta KPQ channels expressed in mammalian cells (2). These studies, however, did not address questions about the mechanism of the late current block. Previous reports (6, 13) showed that the Delta KPQ mutation facilitated the entry of the channels into a mode of gating characterized by bursts of long openings, whereas in the N/S and R/H mutations (13) persistent current was generated by short, dispersed reopenings. On the basis of the low affinity of lidocaine for block of the open state (4, 5), we expected to see less effective block in Delta KPQ channels, but our data did not support this hypothesis. The main evidence comes from the equal contribution of the bursting activity to the late current with or without lidocaine. We observed the same reduction of the open probability for both types of activities through a prolongation of the closed times between the events.

It has been proposed that the bursts are caused by the switching of channels into a slow mode of gating (6, 25), in which the entry and the recovery from inactivation are altered, and that the dispersed activity is caused by the destabilization of the absorbing inactivated state, allowing return to the open state. Thus the two types of activity involve different gating transitions of the channel. Our single-channel data, however, showed that depolarizing prepulses that inactivated Delta KPQ channels selectively inhibited the burst openings over the dispersed activity. This selective reduction indicated that channels residing in the resting states are more likely to enter a slow mode of gating than inactivated channels, but channels that become inactivated during sustained depolarization are likely to reopen in a dispersed fashion. This result clearly shows that the initiation of bursting is unlikely to occur in channels that are inactivated, and this observation helps explain why bursts are suppressed by lidocaine, a drug that preferentially binds to and stabilizes channels in the inactivated state. This property of the burst mode in Delta KPQ channels distinguishes it from previously identified modal gating in native cardiac Na+ channels, in which switching from burst mode to dispersed reopenings was found to occur independently of prepulse potentials (7). Therefore, although the ability to switch into a bursting mode appears to be an intrinsic property of Na+ channels, the regulatory mechanism that controls mode switching may be different in normal and mutant channels.

Our results also showed that the late current block was dependent on the holding potential, and part of the block was produced before the test pulse was applied. At the normal resting potential (approximately -80 mV), the LQT3 channels are partly inactivated and the block of the inactivated channels by lidocaine will decrease the probability of slow gating. Therefore, the block of late current by lidocaine reflects binding to the inactivated state and will be increased compared with tonic block. During the test pulse to 0 mV, the Delta KPQ channels fully activate within 2 ms and are maximally inactivated within 20 ms. Lidocaine block, therefore, increased after the channels inactivated, i.e., after the initial 20 ms of the pulse, and an extra block develops after the peak current has resolved. In unclamped cells, during the plateau phase of the action potential, more channels would be inactivated and trapped in an inactivated, blocked state from which reopenings would be less likely to occur (stabilizing effect).

Recently, however, Wang et al. (33) suggested that mexiletine inhibits late activity through an open-state blocking mechanism. They base their conclusions on the observation that the onset of mexiletine block of inactivated channels is too slow to account for late current block and therefore must be caused by open channel block. No single-channel data are presented, and this conclusion conflicts with previous studies of mexiletine's mechanism of action in native Na+ channels. For instance, Ono et al. (24) showed that mexiletine block of activated channels proceeds at a much slower rate than block of inactivated channels and, under physiological conditions, has no effect on single-channel open time (23). Moreover, although a numeric simulation in which open channel blockade can effectively suppress persistent current originating from bursts of long openings (33), the same model cannot account for the observation that the drugs are equally effective against N/S and R/H channels that only exhibit dispersed reopenings. When the drug block rates used by Wang et al. (33) are applied in model channels that exhibit either dispersed reopenings (13) or bursting (6), we find that at 10 µM mexiletine would be predicted to reduce the persistent currents by 27 and 75%, respectively. Moreover, because of the short open times of the dispersed reopenings, the onset of block would be at least three times slower. Our experimental results clearly indicate that open channel block is not essential for suppressing late current in LQT3 mutants. Moreover, from a clinical standpoint, drugs that have a strong open channel blocking potency (i.e., class Ia blockers such as quinidine and disopyramide, unlike class Ib drugs such as lidocaine; Ref. 18) may, in fact, pose a risk of further prolonging the action potential by blocking K+ currents (8) and therefore would be inappropriate treatments for long Q-T syndrome.

    ACKNOWLEDGEMENTS

We thank W.-Q. Dong and C.-D. Zuo for expert oocyte injection and Dr. Q. Wang (Baylor College of Medicine) for providing mutant cDNA constructs.

    FOOTNOTES

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-29473 and by a Grant-In-Aid from the American Heart Association (AHA) and with funds contributed in part by the AHA, Northeast Ohio Affiliate, to G. E. Kirsch. R. Dumaine was supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada and Le Fonds de la Recherché en Santé du Québec.

Present address of R. Dumaine: Masonic Medical Research Laboratory, 2150 Bleeker St., Utica, NY 13501-1787.

Address for reprint requests: G. E. Kirsch, MetroHealth Medical Center, Rammelkamp Center for Research R327, 2500 MetroHealth Dr., Cleveland, OH 44109-1998.

Received 13 May 1997; accepted in final form 6 October 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(2):H477-H487
0363-6135/98 $5.00 Copyright © 1998 the American Physiological Society


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