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(Circulation Research. 1996;78:916-924.)
© 1996 American Heart Association, Inc.

Articles

Multiple Mechanisms of Na⁺ Channel– Linked Long-QT Syndrome

Robert Dumaine, Qing Wang, Mark T. Keating, Hali A. Hartmann, Peter J. Schwartz, Arthur M. Brown, Glenn E. Kirsch

From the Rammelkamp Center for Research (R.D., A.M.B., G.E.K.), MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio; the Howard Hughes Medical Institute (Q.W., M.T.K.), University of Utah Health Sciences Center, Salt Lake City; the Department of Molecular Physiology and Biophysics (H.A.H.), Baylor College of Medicine, Houston, Tex; and the Department of Cardiology (P.J.S.), University of Pavia (Italy).

Correspondence to Dr G.E. Kirsch, MetroHealth Medical Center, Rammelkamp Bldg R327, 2500 MetroHealth Dr, Cleveland, OH 44106. E-mail gkirsch@mhnet.mhmc.org.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Inheritable long-QT syndrome (LQTS) is a disease in which delayed ventricular repolarization leads to cardiac arrhythmias and the possibility of sudden death. In the chromosome 3–linked disease, one mutation of the cardiac Na⁺ channel gene results in a deletion of residues 1505 to 1507 (KPQ), and two mutations result in substitutions (N1325S and R1644H). We compared all three mutant-channel phenotypes by heterologous expression in Xenopus oocytes. Each produced a late phase of inactivation-resistant, mexiletine- and tetrodotoxin-sensitive whole-cell currents, but the underlying mechanisms were different at the single-channel level. N1325S and R1644H showed dispersed reopenings after the initial transient, whereas KPQ showed both dispersed reopenings and long-lasting bursts. Thus, two distinct biophysical defects underlie the in vitro phenotype of persistent current in Na⁺ channel–linked LQTS, and the additive effects of both are responsible for making the KPQ phenotype the most severe.

Key Words: human heart • cardiac arrhythmia • Romano-Ward syndrome • site-directed mutagenesis • Na⁺ channels

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inheritable LQTS is characterized by delayed ventricular repolarization (QT interval of the electrocardiogram) associated with arrhythmia (particularly torsades de pointes), syncope, and cardiac arrest.¹ Since monophasic action potential recordings in LQTS patients show an abnormally slow repolarization phase,^{2 3 4 5} a defect in the ion channels that control myocardial excitability was suspected. K⁺ channels and Na⁺ channels became candidates, based on experimental models in which blockade of K⁺ current^{6 7 8 9} or potentiation of late Na⁺ current^{10 11} lengthens the QT interval and enhances early afterdepolarizations. Although rapidly inactivating Na⁺ currents are primarily responsible for the initial upstroke of the action potential, a persistent late component is known to contribute to inward currents that maintain the plateau phase.^{12 13 14 15} Genetic linkage mapping of the autosomal-dominant form of LQTS (Romano-Ward syndrome)^{16 17} has identified abnormalities in four different chromosomes.^{18 19 20} The defective gene is not yet known in the chromosome 4–linked form of LQTS,²⁰ but chromosome 7– and possibly chromosome 11–linked forms are associated with mutations in cardiac K⁺ channel genes.^{21 22 23} The chromosome 3–linked form, however, is associated with mutations^{24 25} in the cardiac Na⁺ channel gene (SCN5A).²⁶ Three SCN5A mutations (Fig 1) have been identified in DNA from affected members of LQTS families²⁵ : KPQ (a deletion of residues 1505 to 1507), R/H (an argininehistidine substitution at position 1644), and N/S (an asparagineserine substitution at position 1325). A biophysical phenotype for KPQ has been reported previously,²⁸ and an inactivation defect has been proposed. However, the biophysical phenotypes of the point mutations are unknown. We tested the hypothesis that all three mutations involve abnormalities of Na⁺ channel inactivation²⁵ by comparing their electrophysiological phenotypes in a Xenopus oocyte expression system. All three mutations potentiate a late phase of TTX-sensitive inward current. Two types of single-channel activity were responsible: brief openings dispersed throughout the trace and infrequent long-lasting bursts of openings. Both mechanisms contributed to the phenotype of KPQ and make it the most severe, whereas the milder N/S and R/H phenotypes showed increased numbers of dispersed openings only.

View larger version (14K): [in this window] [in a new window]   Figure 1. Location of LQTS mutations in the human cardiac Na+ channel subunit. IFM indicates the cluster of hydrophobic residues previously shown to be critical for inactivation gating.27

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Na⁺ Channel Clone and Mutagenesis
Wild-type human heart Na⁺ channel clone (hH1a, expression plasmid of SCN5A) was the same as previously described.^{29 30} The full-length cDNA was cloned into the pGEM3 plasmid vector (Promega). Three LQTS mutations (Fig 1) were introduced into the WT construct: a substitution of serine for asparagine at position 1325 (in the intracellular linker between transmembrane segments S4 and S5 of domain III), a substitution of histidine for arginine at position 1644 (at the intracellular end of transmembrane segment S4 in domain IV), and a three-residue deletion of lysine-proline-glutamine from positions 1505 to 1507 (in the intracellular linker between domains III and IV). Mutations were produced by site-directed polymerase chain reaction–based mutagenesis³¹ and verified by DNA sequencing. The mutant channels resulting from expression of these three constructs are abbreviated N/S, R/H, and KPQ, respectively.

RNA Transcription and Oocyte Injection
DNA constructs were linearized by digestion with HindIII for runoff transcription. In vitro transcription with T7 RNA polymerase was performed using the mMessage Machine kit (Ambion). The amount of cRNA synthesized (20 to 100 µg) was quantified by the incorporation of trace amounts of [³²P]UTP in the synthesis mixture. The final cRNA product was resuspended in 0.1 mol/L KCl at 250 ng/µL and stored at -80°C. The integrity of the final product and the absence of degraded RNA was determined by denaturing agarose gel stained with ethidium bromide. The cRNA was diluted to the desired concentrations (generally 1 to 10 pg/nL) immediately before oocyte injection. Stage V-VI Xenopus oocytes were defolliculated by collagenase treatment (2 mg/mL for 1.5 hours) in a Ca²⁺-free buffer solution (mmol/L): NaCl 82.5, KCl 2.5, MgCl₂ 1, and HEPES 5 (+100 µg/mL gentamicin), pH 7.6. The defolliculated oocytes were injected with 46 nL of cRNA solution (in 0.1 mol/L KCl) and incubated at 19°C in culture medium (mmol/L): NaCl 100, KCl 2, CaCl₂ 1.8, MgCl₂ 1, HEPES 5, and pyruvic acid 2.5 (+100 µg/mL gentamicin), pH 7.6. Electrophysiological measurements were made 5 to 10 days after cRNA injection.

Electrophysiology and Data Analysis
Whole-cell currents were recorded in oocytes by using conventional two-intracellular microelectrode voltage-clamp methods.³² Beveled microelectrode tips were filled with a solution of 3 mol/L KCl+1% agar and then backfilled with 3 mol/L KCl to give low tip resistance (0.2 to 0.5 M).³³ Linear leakage and capacitative transient currents were subtracted on-line using a P/4 subtraction routine unless otherwise noted. Cell-attached patch recording was performed after manual removal 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, four-pole Bessel filter) and then digitized at 20 to 100 kHz. Linear leakage and capacitative currents were subtracted on-line using a P/4 subtraction routine.

Whole-cell data were analyzed using Clampfit (Axon Instruments). Exponential decay functions were fit to current waveforms using a nonlinear least-squares procedure. Single-channel data were analyzed using Transit (Dr A.M.J. VanDongen, Duke University, Durham NC) as described previously.³⁰ Appropriate data are expressed as mean±SEM. A two-tailed Student's t test was used to evaluate the significance of the difference between the mean (P<.05) obtained from each mutant channel compared with that obtained from each WT channel on a one-to-one basis.

Solutions and Drugs
A modified Ringer's solution for whole-cell and patch recording consisted of (mmol/L) NaCl 120, KCl 2.5, CaCl₂ 1, MgCl₂ 2, and HEPES 10, pH 7.2 (with NaOH). TTX (Calbiochem) and mexiletine (Boehringer Mannheim) were diluted in the bath solution from frozen aliquots of concentrated stocks. Depolarizing isotonic KCl bath solution for patch recording consisted of (mmol/L) KCl 100, EGTA 10, and HEPES 10, pH 7.3. The pipette solution consisted of (mmol/L) KCl 120, CaCl₂ 1, MgCl₂ 2, and HEPES 10, pH 7.2. The bathing solution flowed continuously at a rate of 3 mL/min. All electrophysiological measurements were made at room temperature (21°C to 23°C).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Late Currents in LQTS Mutant Na⁺ Channels
We tested the ability of the mutant channels to produce persistent late currents by using test pulses of 150- to 500-ms duration. TTX subtraction was used to show that a late component of whole-cell current was conducted by Na⁺ channels (Fig 2A). Test potentials to -10 mV, 500-ms duration, were applied to voltage-clamped oocytes bathed in normal Ringer's solution, and the ionic currents were recorded without leakage subtraction (Fig 2A, recording 1). The measurement was repeated in Ringer's solution+100 µmol/L TTX (Fig 2A, recording 2), a selective Na⁺ channel blocker. Digital subtraction of the two current waveforms (Fig 2A, recording 3) gives the time course of the TTX-sensitive component. In WT channels at a time point 300 ms from the start of the test pulse, inward currents inactivated almost completely; residual current was only 0.15% of the peak transient current (note that peak currents are off scale at the amplification used to illustrate the late currents). By contrast, each of the three mutant channels (Fig 2B, recordings 1 through 3) showed significant increases in TTX-sensitive inward current at 300 ms compared with WT channels. As shown in Fig 2C, the relative magnitude of the late current in the mutant channels was as follows: KPQ>N/S>R/H. At times shorter than 25 ms, the major time constant of inactivation at a test potential of -10 mV was unchanged in the mutant compared with WT channel (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 2. Late inward currents in WT and mutant channels. A, Recordings of the WT currents during the control condition (Ctrl) (recording 1) and after extracellular application of 100 µmol/L TTX (recording 2). Currents were elicited by 500-ms pulses to -30, -10, and +10 mV from a holding potential of -100 mV and digitally filtered (500 Hz). Note that at the high amplification necessary to illustrate the late current, the early peak currents are off scale. The late components (recording 3) of inward current were obtained by digitally subtracting the currents recorded in TTX from Ctrl recordings. The solid line preceding each set of traces indicates the zero-current baseline. Recordings from oocytes in which holding current changed by >10 nA after application of TTX were rejected. The dotted line indicates the 300-ms isochron used to compare the amplitude of the late current from each phenotype. B, Late currents from the three mutant channels, R/H (recording 1), N/S (recording 2), and KPQ (recording 3), recorded under the same conditions as in panel A. Traces were selected so that the maximal peak currents (off scale) from oocytes expressing the mutant and WT channels had comparable amplitudes (25 to 30 µA). C, The relative conductance at a test potential of -10 mV and a duration of 300 ms normalized to the maximum peak conductance. The number of cells tested was as follows: WT, 5; R/H, 4; N/S, 6; and KPQ, 8. *P<.05, **P<.01, and ***P<.001 compared with WT.

Blockade of late Na⁺ currents by class Ib antiarrhythmics, such as lidocaine, is thought to be responsible for shortening of the action potential in ventricular myocytes.³⁴ Therefore, we tested whether the late currents in mutant channels could be blocked by application of mexiletine, a lidocaine analogue available for oral use. Fig 3A shows a typical effect at high dosage (200 µmol/L) in the N/S mutant channel, where blockade of the late current (eg, at the 300-ms time point, vertical line) was 75% complete, compared with only 20% block of the peak current (off scale). As shown in Fig 3B, the effect was dose dependent, with a significant effect observed at concentrations as low as 10 µmol/L. Fitting the data to a 1:1 binding isotherm gave an apparent K_d of 60 µmol/L. Similar results were obtained for R/H and KPQ mutants, where 50 µmol/L mexiletine block of the late current was 48±7% (n=3) and 60±10% (n=3) complete, respectively. At this concentration, peak currents were blocked by only 9±1% (n=3) and 14±2% (n=3), respectively, in R/H and KPQ channels. Drug block of late currents in WT channels could not be determined accurately because of the low signal-to-noise ratio, but based on blockade of peak current, sensitivity of the WT late current similar to that obtained in the mutant channels would be predicted.

View larger version (13K): [in this window] [in a new window]   Figure 3. Sensitivity of the late current to mexiletine. A, Currents elicited by 500-ms pulses to -10 mV showed a 69% block of the N/S late current (control-TTX), at 300 ms (dotted line), after extracellular application of 200 µmol/L mexiletine (Mex). B, Dose-response curve for the block of the late Na+ current by Mex. The remaining TTX-sensitive current after subtraction of the Mex-sensitive component was normalized to the maximal TTX-sensitive current. Data from three experiments were averaged, and their mean values were plotted against drug concentration. The data, fit to the Hill equation for a 1:1 binding kinetics, yielded an apparent Kd of 59.9 µmol/L (Hill coefficient=0.93).

Mutation-Induced Gating Defects
We next asked whether the underlying biophysical defect was the same for all three mutations at the single-channel level (Fig 4). All of the data were obtained using -10-mV test pulses of 150-ms duration from a holding potential of -100 mV. Each test pulse was preceded by a 500-ms prepulse to -140 mV to allow full repriming. As in the whole-cell experiments, the amplitude of the test pulse was chosen to measure persistent current that might contribute to the plateau phase of the action potential. The shorter duration of the test pulse was a necessary compromise to allow resolution of brief openings late in the trace. The most severe defect was associated with the KPQ mutation (Fig 2C), and as shown in Fig 4B, two distinct forms of channel activity contributed to the late currents: brief dispersed openings (Fig 4B, traces 1 through 6) that are present in nearly every trace and long-lasting bursts (Fig 4B, trace 2) that occur infrequently. The ensemble average of 128 traces (Fig 4B, trace 7) shows that the combined effect of both types of activity produced a small persistent current that continued long after the initial transient current (off scale; peak, 6 pA) had subsided. By contrast, late channel activity of any type was extremely rare in WT channels (Fig 4A, trace 5), and in the experiment illustrated, the ensemble average of 96 traces (Fig 4A, trace 7) showed no persistent current.

View larger version (41K): [in this window] [in a new window]   Figure 4. Single-channel activity in WT and mutant channels. Six consecutive current traces were evoked by a test potential of -10 mV from a holding potential of -100 mV at a frequency of 0.5 pulse per second. Each test pulse lasted 140 ms and was preceded by a 500-ms conditioning pulse to -140 mV to remove resting inactivation. Channel openings are downward deflections in the current traces. The start of the test pulse is coincident with the residual uncorrected capacitative current. Trace 7 is the ensemble average of 96 WT traces (A, 13.7-pA peak current) and of 128 traces each in KPQ (B, 6.0-pA peak current), R/H (C, 9.6-pA peak current), and N/S (D, 3.3-pA peak current), shown at high gain to demonstrate the difference in persistent current; initial peak currents are off scale. Bandwidth is 2 kHz. Similar results were obtained in replicate experiments from nine WT, five KPQ, five R/H, and seven N/S patches.

Compared with KPQ, the N/S and R/H mutations were less effective in generating late current (Fig 2C) in whole-cell recordings, and as shown by single-channel recording (Fig 4C and 4D), the point mutants showed only the dispersed form of late channel activity. Nonetheless, as indicated in the ensemble-averaged traces (Fig 4, bottom traces), the frequency of occurrence of dispersed openings was sufficient to generate persistent current.

We purposely chose multichannel patches to increase the probability of observing infrequent late events in both WT and mutant channels for this series of experiments (Fig 4). Therefore, when comparing activity levels in different patches, we normalized for differences in the number of channels in the patch by calculating the ratio of late current to peak current, under the assumption that the kinetic components that determine peak conductance were unchanged. Analysis of ensemble-averaged currents at -10 mV yielded inactivation time constants of 0.32±0.08 ms (WT, n=9 patches), 0.41±0.07 ms (N/S, n=7), 0.3±0.08 ms (R/H, n=5), and 0.4±0.10 ms (KPQ, n=5) and time-to-peak values of 0.38±0.03 ms (WT, n=9 patches), 0.30±0.03 ms (N/S, n=7), 0.34±0.02 ms (R/H, n=5), and 0.36±0.04 ms (KPQ, n=5). Since the mean values from each of the mutant channels were not significantly different from WT channels, our assumption is valid. Comparison of the average peak currents shows that even though from 1.4 to 4.2 times as many channels were available in the WT patch (Fig 4A), they produced negligible late current compared with the mutant channels (Fig 4B through 4D). Nonetheless, in WT patches rare instances of both dispersed openings and bursts were observed, but their frequency was much less than in the mutant patches. Fig 5A shows the progress of two experiments in which patches were stimulated 600 times in succession (at 2-s intervals). The percent of time spent in the open state was calculated for the latter portion of each recorded trace (excluding the initial transient) in the 30- to 140-ms range and plotted versus pulse number (Fig 5C and 5D) to give a diary of late activity. In WT channels, even though many channels were present (peak current, 21.9 pA), only three instances of long bursts were observed (Fig 5A, traces 56, 98, and 214), and these appeared at widely spaced intervals as large spikes in the P_open diary (Fig 5C). The vast majority of traces were either devoid of activity or showed only one or two brief dispersed openings. In contrast, the KPQ patch, even though it contained fewer channels (maximum peak current, 15.0 pA), showed much more late activity, including several instances of consecutive traces that displayed burst activity (eg, Fig 5B, traces 211 and 212). The vast majority of traces showed multiple dispersed openings, which account for the higher level of baseline activity in the P_open diary (Fig 5D). It should be noted, however, that the long bursts observed in both WT and KPQ patches were very similar in form; the bursts lasted tens of milliseconds and consisted of repetitive long openings separated by brief closures.

View larger version (35K): [in this window] [in a new window]   Figure 5. Patterns of late activity in WT and KPQ channels. Examples of late-burst activity in WT (A) and KPQ (B) channels obtained during a train of 600 stimuli (the pulse number is indicated to the left of the trace) under conditions identical to those described in the previous figure. Only the last 110 ms of the 140-ms recordings is shown. Diaries of late activity were obtained (C and D) by plotting the percentage of time spent in the open state (Popen) for each trace versus test pulse number during repetitive stimulation. Similar patterns of activity were obtained in additional experiments from eight WT and four KPQ patches.

The dispersed openings in both WT and mutant channels are likely to result from return to the same open state that is entered during the initial phase of channel activation. Analysis of the distribution of open time intervals (Fig 6) revealed that in WT channels the histogram of open times for traces showing only dispersed openings was described by a single exponential with a mean open time of 0.3 ms (Fig 6B), a value identical to the mean open time of the initial openings (Fig 6A). Burst openings, in contrast, had much longer mean open times (Fig 6C). Although the observations were drawn from many patches because of their low probability, the mean value was roughly 10 times that of either the late dispersed openings or the initial openings of the WT channels and was comparable to channels in which the inactivation gate was disabled by mutating the IFM cluster to QQQ.^{27 30} Traces containing only dispersed openings were often observed in the KPQ mutant. Interval analysis (Fig 6D and 6E) revealed that the characteristic brief open times seen in the WT channel were preserved in the mutant channel. Furthermore, in traces containing long bursts, the open-time histogram (Fig 6F) was dominated by long openings similar to those seen in the WT channel but quite distinct from the brief openings that characterized either the initial openings or the late dispersed activity. Therefore, in both channels, openings during bursts appear to represent sojourns in a different open state from that visited briefly during the initial phase of activation or revisited later to generate dispersed reopenings.

View larger version (28K): [in this window] [in a new window]   Figure 6. Histogram analysis of open times in WT (A through C) and KPQ (D through F) patches. Open-time duration was measured from idealized recordings of the initial 15 ms of the test pulse (A and D) or the final 110 ms of traces that displayed either dispersed activity (B and E) or prolonged bursts (C and F). The histograms are plotted with logarithmic binning in which the mean open time corresponds to the peak of the smooth curve obtained by fitting the histogram to single-exponential decay functions with the indicated time constants (). Complications arising from overlapping openings in the analysis of initial open times were avoided by adjusting the conditioning prepulse to reduce the number of available channels through inactivation. The probability of observing late activity was sufficiently low, such that overlap was not a problem. Graphs were obtained in single experiments from WT (A) or KPQ (D through F) patches. Graphs B and C contain pooled data from eight WT patches. Overall, WT channels showed initial, dispersed, and burst open times, respectively, of 0.34±0.05 ms (from five patches), 0.32 ms (pooled data from eight patches), and 4.51 ms (pooled data from eight patches). In KPQ channels, initial, dispersed, and burst open times, respectively, were 0.32±0.03 ms (four patches), 0.33±0.02 ms (five patches), and 3.9±0.35 ms (five patches).

We summarized all the single-channel data by calculating the average amount of persistent current in the 30- to 140-ms range after the start of the test pulse, which was normalized to the average peak current and expressed as a percentage (Fig 7A). The resulting comparison among the four types of channel shows that (as in the whole-cell measurements) each of the three mutant channels produced significantly larger amounts of persistent current than the WT channel. KPQ was the most severe defect, whereas R/H and N/S each produced roughly one half as much persistent current. Furthermore, ensemble averages constructed from KPQ data in which traces containing burst activity were excluded (Fig 7A, rightmost bar) reduced the magnitude of the persistent current to the level of the point mutants. Thus, the additive effects of both types of activity were responsible for the severity of the KPQ phenotype.

View larger version (17K): [in this window] [in a new window]   Figure 7. Summary of single-channel data. The relative amount of persistent current (A) was obtained by measuring the average current in the ensemble of idealized recordings from each patch over the 30- to 140-ms range after the start of the pulse. Each trace was corrected for nonzero baseline in a two-step procedure: leakage currents were offset by digital subtraction of recordings with no openings, and then before idealization each recording was adjusted to a zero baseline by an automatic baseline correction method that removes slow drift during the test pulse. The average persistent current was normalized by dividing by the peak current in the 0- to 25-ms range after the start of the pulse and was expressed as a percentage. The occurrence of traces containing bursts (B) and dispersed openings was obtained by identifying bursts according to the following procedure: A closed-time histogram was constructed from a collection of traces containing obvious bursts (eg, Fig 3B, trace 2) and fit to a biexponential distribution. It was observed that 90% of the area was described by a time constant of 0.2 ms; the remaining events, by a time constant of 11 ms. The shortest time constant was taken as the closed interval between events within the burst, and a burst was defined as a sequence of three or more openings separated by intervals no longer than 0.6 ms. A burst index (B) was calculated from the ratio of traces containing bursts to the total number of traces. The ratio was corrected for differences in the number of channels in the patch (unitary amplitude, 1.0 pA) by normalizing to the maximum peak current. Panels A and B show pooled data from nine WT, five R/H, seven N/S, and five KPQ patches. Traces containing dispersed openings were identified by the criterion that a dispersed opening was a single open event separated from its neighbors by a closed interval longer than 0.6 ms. The dispersion index (C) was calculated as the percent of traces in which dispersed openings were observed in patches selected to have similar numbers of channels, as described in the text. *P<.05 vs WT.

In each channel type, the two mechanisms of persistent current were qualitatively the same but occurred with much different frequencies. We quantified burst activity by calculating a burst index (Fig 7B) in which the frequency of occurrence of traces with long bursts was expressed as a percentage of the total number of traces, which was normalized to the maximum peak current observed in each patch. The burst index approximates the probability of observing slow-mode activity in a single channel, since the average unitary current amplitude was 1.0 pA and simultaneous bursting of two or more channels per trace, seen only twice, was counted as two bursts. To the extent that peak ensemble-averaged current underestimates the total number of channels in a patch (because the maximum P_open of a single channel is <1.0), the burst index overestimates the absolute frequency of slow-mode bursting. As a comparative measure, however, the calculation shows that burst-mode activity was rarely observed in WT and in the point mutant channels but was nearly 10 times more frequent in KPQ. The relative frequency of occurrence of dispersed openings (dispersion index, Fig 7C) was obtained simply by dividing the number of traces in which late dispersed openings were observed by the total number of traces and was expressed as a percentage. Since the openings of different channels in a single trace could not be distinguished, we could not correct the probability for the different numbers of channels in each patch. Therefore, much more late activity would be observed in patches containing many channels than those containing fewer channels, even if the single-channel properties were uniform. Instead, for the mutants, we selected patches that had similar peak currents and compared these with WT patches in which the peak currents exceeded the average in the mutant patches by at least 40%. The average peak currents were 16.6±1.4 pA (WT, n=7), 11.0±3.5 (R/H, n=5), 9.8±1.5 (N/S, n=6), and 11.5±1.9 (KPQ, n=5). Thus, the dispersion index overestimates the relative frequency in WT patches, since more channels were available, as indicated by the significantly larger average peak current compared with the mutant-channel patches, which were not significantly different from one another. Nonetheless, as shown in Fig 7C, each of the three mutant channels showed significant increases in the frequency of occurrence of dispersed openings compared with WT channels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results provide strong support for the hypothesis that an intrinsic defect underlies the LQT phenomenon through characterization of the molecular phenotypes of all three LQTS-linked defects in the primary sequence of the human cardiac Na⁺ channel. All the mutations result in the potentiation of a late persistent component of inward Na⁺ current, which would have the effect of delaying myocardial repolarization. The main evidence is that when expressed in a heterologous system, the mutant channels show elevated levels of residual inward current that persist during long test pulses at a time when normal Na⁺ currents have decayed to nearly zero. This late current was isolated from unrelated background currents by its sensitivity to the specific Na⁺ channel blockers, TTX and mexiletine. There are mixed reports of the value of mexiletine and other class Ib agents in LQTS patients of unknown genotype.^{35 36 37 38} Recently, however, mexiletine has been shown to markedly shorten QTc (from 0.54 to 0.45 s) in chromosome 3–linked, but not in chromosome 7–linked, LQTS.³⁹ Therefore, our results offer a molecular rationale for assessing the value of mexiletine in the treatment of chromosome 3–linked, but not chromosome 7–linked, patients.

Late Na⁺ currents have been noted previously in native Na⁺ channels, and two mechanisms have been proposed: a slow phase of inactivation¹³ or an increase in window current generated by the overlap between steady state activation and inactivation curves over a restricted voltage range.¹² The latter mechanism can be ruled out as a major cause of late current in our experiments, because the observations were made at test potentials well outside the range of overlap. In the former mechanism, late currents result from channels in which the inactivation mechanism is nonabsorbing; ie, the latching mechanism that prevents inactivated channels from reopening is defective.

Defective latching mechanisms have been induced by mutation of the IFM cluster in the III-IV linker region^{27 30} and are thought to occur spontaneously when native channels were observed to switch into a slow mode of gating characterized by long-lasting bursts. Infrequent long-lasting bursts have been reported both in native Na⁺ channels recorded in ventricular myocytes^{15 40} and in cloned Na⁺ channels expressed heterologously.⁴¹ Modal bursting has been observed previously in KPQ and has been proposed as the basis for LQTS,²⁸ but the mutation-induced bursts were reported to have properties quite different from slow-mode activity in native channels. Burst openings in KPQ were reported to have the same mean open time as the initial openings in WT channels. By contrast, previous observations in rat cardiac Na⁺ channels have shown that slow-mode gating is characterized by much longer than normal mean open time.^{15 40} Thus, the interpretation²⁸ that KPQ causes more frequent mode switching is difficult to accommodate unless the burst mode in WT hH1 channels is significantly different from the slow-mode bursting observed in native nonhuman cardiac channels.

Our results show that slow-mode activity occurs in WT human channels as well as KPQ and that slow-mode openings are as long as those previously reported for inactivation-deficient mutants.³⁰ Furthermore, we have identified a second mechanism of late current that takes the form of brief dispersed openings, with mean open times identical in WT and mutant channels. The differences between our results and those of Bennett et al²⁸ may be due to differences in recording conditions and/or analysis. Our results were obtained from cell-attached patches to avoid the possibility of kinetic changes that might occur under cell-free recording conditions.⁴² Under these conditions, we obtained clear evidence for two types of late openings with distinctly different open times (0.3 ms for dispersed and initial openings and 5 ms for burst openings), whereas Bennett et al fit their open-time distributions to a monoexponential function with a time constant of 0.6 ms. Nonetheless, traces containing either dispersed openings or bursts of long-lasting openings are evident in their sample recordings. Therefore, our interpretation of the mutation-induced defects is different. We propose that the dispersed openings, which show no apparent modelike clustering, are related to the destabilization of inactivation that allows revisitation of the initial open state, whereas the burst openings belong to a sporadically visited slow mode of gating in which both the entry to and recovery from inactivation are altered,³⁰ producing an abnormally prolonged open state and long bursts.

Fig 8 shows a simple nonunique kinetic scheme, which illustrates the point that dispersed openings, such as those observed in all three mutants, may be related to a change in the kinetics of inactivation. Inactivation is thought to proceed via the movement of an inactivation particle located in the III-IV linker into a receptor site to block the cytoplasmic end of the pore. We have shown previously³⁰ that fast inactivation of hH1a expressed in oocytes has both fast and slow phases that account for roughly 10% and 90%, respectively, of the decay of currents evoked by test potentials in the range of -40 to +80 mV. Since inactivation shows a similar pattern in native human cardiac Na⁺ channels,⁴³ we have modeled inactivation as a two-step process. The first step, which is fast and reversible (O₄I₅), may represent the initial binding of the inactivation particle with its receptor. The reversibility allows the channel to reopen briefly,^{44 45} until a second step occurs in which the particle-receptor complex becomes stabilized, thus causing the channel to enter an almost completely absorbing inactivated state (I₅ I₆). WT channels rarely escape back to the open state (Fig 8D), but for LQT mutant channels (Fig 8E), an increase in the rate constant h, which controls the I₆I₅ transition, would produce dispersed openings as channels recycle back through the open state and reenter I₅. In this scheme, since there is only one open state, the mean open times of early and late openings would be identical, as was observed. Structurally, an increase in the rate constant h could represent a destabilization of the second, absorbing inactivated state that normally requires the concerted action of both the inactivation particle and its receptor. Such a destabilization could be produced by a structural change in either the receptor site (N/S and R/H mutations) or in the inactivation particle (KPQ).

View larger version (18K): [in this window] [in a new window]   Figure 8. A kinetic model of gating in WT and mutant channels operating in the fast gating mode. States C1 through C3 are nonconducting states visited during activation, and I5 and I6 are nonconducting inactivated states; state O4 is the conducting open state (A). A pathway between the proximal closed state, C3, and proximal inactivated state, I5, which bypasses O4, was provided to give a realistic value for the peak probability of opening (Po) (B and C). Simulations of the probability of opening Po vs time of WT and LQT channels are superimposed and plotted on short and long time bases in panels B and C, respectively. Panels D and E show simulated single-channel recordings at a bandwidth comparable to that of the actual experiments. All of the rate constants are the same, except rate constant h is 0.001 ms-1 in WT (D) and 0.1 ms-1 in LQT (E). The invariant rate constants were as follows (ms-1): , 8.1; ß, 0.2; c, 1.5; d, 0.01; e, 2.2; f, 0.3; and g, 0.35. The simulation shows late dispersed openings that produce a persistent nonzero Po throughout the 70-ms simulation (C) without changing the initial decay phase (B).

The unique ability of KPQ to enhance entry into a slow mode of gating that closely resembles inactivation-deficient mutants may be related to its location in the III-IV linker. We speculate that in the slow mode the channels fail to inactivate because of a transient conformational change in the III-IV linker that prevents inactivation particle-receptor binding (O₄I₅) in a manner similar to the permanent change produced by the IFMQQQ mutation^{27 30} and that the KPQ mutation produces a structural modification that makes this conformational change occur more often. On the other hand, N/S and R/H mutations, which are not located in the III-IV linker and do not affect the O₄I₅ step, also do not affect the frequency of transitions into the slow mode of gating. Therefore, we suggest that the two mechanisms reflect destabilization of different inactivated states. Furthermore, since dispersed activity is elevated equally in all three mutants, whereas burst activity is enhanced only in the KPQ deletion, the additive effects of both types of activity must be responsible for the severity of the gating defect in the deletion mutant. Therefore, our results suggest that in the absence of unique adaptations, patients exhibiting this form of the disease would experience the greatest delay in ventricular repolarization.

	Selected Abbreviations and Acronyms

 

LQT = long QT LQTS = LQT syndrome Popen = open probability TTX = tetrodotoxin WT = wild-type

	Acknowledgments

 
This study was supported by National Institutes of Health grants NS-29473 (Dr Kirsch), NS-23877 and HL-37044 (Dr Brown), and HL-48074 and RR-00064 (Dr Keating). Dr Dumaine was supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada and Le Fonds de la Recherche en Santé du Québec. We thank W.-Q. Dong and C.-D. Zuo for expert oocyte injection.

Received September 26, 1995; accepted January 29, 1996.

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