Novel Gain-of-Function Mechanism in K+ Channel–Related Long-QT Syndrome:
Altered Gating and Selectivity in the HERG1 N629D Mutant
Abstract—The N629D mutation, adjacent to the GFG signature sequence of the HERG1 A K+ channel, causes long-QT syndrome (LQTS). Expression of N629D in Xenopus oocytes produces a rapidly activating, noninactivating current. N629D is nonselective among monovalent cations; permeation of K+ was similar to that of Na+ or Cs+. During repolarization to potentials between −30 and −70 mV, N629D manifested an inward tail current, which was abolished by replacement of extracellular Na+ (Na+e) with extracellular N-methyl-d-glucamine (NMGe). Because LQTS occurs in heterozygous patients, we coexpressed N629D and wild type (WT) at equimolar concentrations. Heteromultimer formation was demonstrated by analyzing the response to 0 [K+]e. The outward time-dependent current was nearly eliminated for WT at 0 [K+]e, whereas no reduction was observed for homomultimeric N629D or for the equimolar coexpressed current. To assess physiological significance, dofetilide-sensitive currents were recorded during application of simulated action potential clamps. During phase 3 repolarization, WT manifested outward currents, whereas homomultimeric N629D manifested inward depolarizing currents. During coexpression studies, variable phenotypes were observed ranging from a reduction in outward repolarizing current to net inward depolarizing current during phase 3. In summary, N629D replaces the WT outward repolarizing tail current with an inward depolarizing sodium current, which is expected to delay later stages of repolarization and contribute to arrhythmogenesis. Thus, the consequences of N629D resemble the pathophysiology seen in LQT3 Na+ channel mutations and may be considered the first LQTS K+ channel mutation that exhibits gain of function.
Familial long-QT syndrome (LQTS) results from defects in sodium and potassium ion channels that cause prolongation of cardiac repolarization and arrhythmias.1 LQT2 is associated with mutations of the human ether-a-go-go–related gene, HERG1.2 3 4 5 6 7 8 9 10 The HERG1 primary transcript is alternatively processed, giving rise to at least 3 functional mRNAs, HERG1 A, HERG1 A′, and HERG1 B, encoding proteins with distinct physiological properties.11 12 There are several mechanisms by which individual mutations in HERG1 produce LQTS.5 6 7 8 9 Some exert a dominant phenotype through loss of repolarizing current. These include mutations that either cause defects in intracellular transport or result in channels that do not open. Alternatively, V630L forms heterotetramers with wild type (WT) that have reduced open probability due to a negative shift in voltage dependence of inactivation. Abnormally fast deactivation mutations caused by mutations in the N terminus of HERG1 A result in reduction of outward current during phase 3 repolarization and thus LQTS.10 The recently described N629D mutation9 is of particular interest, as it alters the pore selectivity signature sequence from GFGN to GFGD.13 In most K+ channels, including the extensively studied Shaker channel, the signature sequence is GYGD. In Shaker, mutation of GYGD to GYGN results in a nonconducting channel that retains its gating properties.14 Inclusion in the tetramer of 1 or 2 monomers containing D/T, D/N, or D/C mutations yields a conducting channel.15 16 17 Mutation of 2 monomers to D378T in Kv2.1 resulted in a channel with reduced selectivity for K+ over Na+ and strong outward rectification.16 Mutation of Shaker D447 to E in all 4 positions or N in 2 positions causes a large increase in the rate of C-type inactivation.17 18 Accordingly, the objectives of this study were to assess the effects of the N629D HERG mutation on inactivation and ion selectivity.
Materials and Methods
Expression in Xenopus Oocytes
HERG1 A in pSP64 was obtained from Dr M.T. Keating (University of Utah, Salt Lake City, Utah).2 Site-directed mutagenesis was carried out according to Ho et al.19 A dimer was constructed wherein the C terminus of N629D was linked to the N terminus of WT. The linker consisted of SGGSPS.
Currents were expressed in Xenopus oocytes and recorded using 2-microelectrode voltage-clamp techniques.11 Oocytes were perfused with modified frog Ringer’s solution at 21°C to 22°C containing (in mmol/L) NaCl 114, KCl 2.5, MgCl2 1, CaCl2 1.8, and HEPES 10, pH 7.2 adjusted with NaOH. Niflumic acid was included (0.15 mmol/L) to block chloride currents. Glass microelectrodes were filled with 3 mol/L KCl with tip resistances of 0.5 to 2 MΩ. Oocytes were clamped with a Geneclamp 500 amplifier, and voltage-clamp protocols were generated with pClamp software (Axon Instruments). Currents were sampled at a rate of 2 kHz and filtered at 1 kHz using a 4-pole Bessel filter. Oocytes were held at −80 mV between pulses, with an interpulse interval of 30 seconds. Time constants (τs) were measured using Clampfit and τs derived from Chebyshev fits to the deactivating current using the equation y=A0+A1e–t/τ1+A2e–t/τ2. To express N629D homomultimers, 6 or 12 ng of RNA was injected, for HERG1 A WT 6 ng was injected, and for coexpression studies 6 ng of each construct was injected.
Voltage-clamp pulses used in this study are illustrated in the figures. The ramp-clamp protocol is similar to that of Chen et al,10 and the action potential clamp protocol is similar to that of Zhou et al.20 The simulated action potential clamp was modeled to that of monophasic action potentials recorded in human ventricle.21 The simulated action potential clamp duration was 250 ms. An initial pulse from a resting potential of −80 mV was followed by a transient repolarizing wave that returned the potential from +40 to 0 mV over 8 ms, followed by a plateau phase lasting 120 ms, followed by phase 3 repolarization returning from a plateau potential of 0 to −70 mV over 100 ms, followed by a terminal repolarizing phase that returned the voltage from −70 to −80 mV over 20 ms. Dofetilide-sensitive currents elicited by the action potential clamp and ramp clamps are reported.
ANOVA was used to compare data, and the Tukey test was used for multiple comparisons.
N629D Current Is Not Inwardly Rectified and Does Not Inactivate
Figure 1⇓ shows typical currents of WT (injected with 6 ng) in the left column and N629D (12 ng) in the right column. Different amounts of RNA were injected to produce currents of similar magnitude to allow appropriate comparisons. From a holding potential of −80 mV, currents were evoked by a series of depolarizing pulses, P1, for 2500 ms to potentials between −70 and +50 mV followed by a P2 pulse to holding of −60 mV. Tail currents of N629D were negative over the range of voltages tested, which reflects a lack of potassium selectivity (see below). Unlike WT currents, the N629D current activated rapidly at all potentials and exhibited outward rectification.
Mean current-voltage relationships are shown for these examples in Figure 1G⇑ and 1H⇑ for WT (n=16) and N629D (n=14), respectively. Current measured at the end of P1 was related to depolarizing potentials (solid symbols). Inward rectification was evident for WT, whereas outward rectification was seen with N629D. Over the potential range from −20 to −60 mV, N629D time-dependent currents were inward.
Na+ Carries the Inward Current of the N629D Mutant
Figure 1⇑ shows illustrative examples of typical currents elicited before (Figure 1A⇑ and 1B⇑) and after (Figure 1C⇑ and 1D⇑) replacement of extracellular Na+ (Na+e) with extracellular N-methyl-d-glucamine (NMGe) for WT on the left and N629D on the right. The inward portion of the time-dependent current and the inward tail current of N629D were virtually abolished by replacement of Na+e with NMGe. Mean absolute tail current amplitude when pulsing from +20 to −60 mV for WT was 1580±1200 nA before and 1120±480 nA (NS) after replacement of Na+e with NMGe, whereas for N629D the amplitude was 420±260 before and 30±6 nA after (P<0.001) replacement. These data indicate that the inward currents of N629D are carried predominantly by Na+e.
The mean peak of the tail current at the onset of the P2 pulse (in open symbols) was related to the P1 potentials for WT in Figure 1G⇑ and N629D in Figure 1H⇑ (n=16 and 14, respectively). Voltage dependence of the activated tail currents of WT were well fit to a Boltzmann function with V1/2 for activation of −26±7 mV (slope, 9±0.6 mV/e–fold), whereas the V1/2 for N629D measured from inward tail current was −37±7 mV (slope, 8±1 mV/e–fold).
N629D Channel Inward Tail Currents Deactivate More Rapidly Than WT
N629D channel has inward tails using the protocol shown in Figure 1⇑. Representative examples of deactivation currents are shown for WT in Figure 1E⇑ and N629D in Figure 1F⇑. To assess kinetics of deactivation, a double-pulse protocol was used. The first pulse was introduced from a holding potential of −80 mV to a potential of +50 mV for 2 seconds, followed by a second pulse to a variety of test potentials ranging from −20 to −120 mV for 2.5 seconds. The deactivation process was fit to biexponential functions. At −80 mV, the dominant fast component of deactivation τs for WT, 200±25 ms (n=16), were slower than that of N629D, 78±22 ms (n=14), and at −100 mV the τs were 104±20 and 41±2 ms, respectively (P<0.01). Deactivation τs of WT are similar to those reported previously.10
N629D Alters Selectivity to K+
Figure 2⇓ illustrates the relation between reversal potential and [K+]e for WT on the left (Figure 2A⇓) and N629D on the right (Figure 2B⇓). [K+]e was serially increased from 2.5 to 5 and then to 40 mmol/L. With increases in [K+]e, we reciprocally decreased [Na+]e. The plot of reversal potential versus log [K+]e was determined by fitting to the Goldman-Hodgkin-Katz equation. The regression line for this plot for WT has a terminal slope of ≈58 per decade, with a best-fit permeation ratio of [Na+]e/[K+]e of 0.01. During replacement of Na+e with NMG, the curve more closely approached the Nernst slope for a pure potassium-selective channel. In contrast, the regression line for this plot for N629D in normal Ringer’s solution showed relatively little slope, and the best-fit estimate of the permeation ratio PNa/PK was 0.65. However, during replacement of Na+e with NMGe, the slope of the regression line for N629D more closely approximated that of WT with a mean PNMG/PK of 0.03.
To assess relative permeation ratio of Cs+/K+, reversal potentials of WT and N629D were compared in KCl and CsCl2, both at 115 mmol/L. For WT, mean reversal potential was −37±5 in cesium and −9±5 mV in K+ at 115 mmol/L, whereas for N629D, the reversal potential in 115 mmol/L cesium was −10±6 mV and in potassium it was −4±7 mV. These data indicate a differential relative permeation ratio for cesium/potassium in HERG1 A versus N629D. Relative permeation ratio of cesium/potassium was evaluated according to the following formula: The relative cesium/potassium permeation ratio, PCs/PK, was 0.28±0.04 for WT, but it was 0.8±0.05 for N629D (n=3). These data indicate that the N629D channel is relatively nonselective among cations.
Evidence for Formation of Heteromultimers Between WT and N629D
To assess whether functional heteromultimers of WT/N629D are produced, response to 0 [K+]e was evaluated. Figure 3⇓ shows illustrative examples of the effects of removal of all K+e on WT in the left column, N629D in the middle column, and the equimolar coexpressed N629D/WT in the right column. Representative examples at 2.5 mmol/L [K+]e are shown in the top row, at [K+]e at 0 mmol/L in the middle row, and mean responses in the bottom row. Near elimination of outward time-dependent activating current of WT was observed at 0 mmol/L [K+]e, whereas no reduction in this magnitude was observed for homomultimeric N629D. The equimolar WT/N629D-coexpressed time-dependent current had little response to 0 mmol/L [K+]e. Suppose a current was artificially produced that resulted from a superimposition of 50% homomultimeric HERG1 A WT and 50% N629D. The resultant superimposed current would be expected to be reduced by ≈50% at [K+]e of 0 mmol/L. As is shown in this right panel, the time-dependent current during coexpression was reduced by 14% at 0 mmol/L [K+]e. These data indicate that although coexpression of WT/N629D can produce a current with characteristics grossly similar to WT, the response of the functional heteromultimer to [K+]e of 0 mmol/L cannot be explained by a superimposition of homomultimeric currents.
Further evidence for the formation of heteromultimers is provided by the observation that coexpression of the dominant-negative G628S HERG1 A (n=3) with N629D resulted in dominant loss of expression (data not shown). These data taken in concert provide evidence that functional heteromultimers are formed during coexpression of HERG1 A and N629D.
Physiological Significance of the N629D Mutation
To assess the physiological significance of N629D, action potential and ramp clamps were applied. Mean dofetilide-sensitive currents elicited by the ramp protocol (left panel) and the simulated action potential clamp (right panel) are shown for homomultimeric N629D (n=6) and WT (n=6) in Figure 4⇓. Application of these protocols to WT resulted in only outward currents, particularly during phase 3 of the action potential. In contrast, application of identical protocols to N629D resulted in outward currents during the plateau of the action potential followed by an inward current during phase 3 repolarization. For HERG1 A, repolarization ramps result in outward repolarizing currents, whereas for N629D, these ramps result in inward depolarizing currents at voltages more negative than −20 mV (Figure 4D⇓). These data indicate that the loss of potassium selectivity results in inward depolarizing currents during phase 3 of this artificial action potential.
In Figure 5⇓, the upper row shows the spectrum of phenotypes observed after injection of equimolar concentrations of WT and N629D. The left column shows a family of currents that is intermediate between N629D and WT, with no inward tail and a reversal potential of −58 mV. The middle column shows an N629D-like phenotype. An inward tail current is apparent, and the reversal potential of the tail currents is −8 mV. The right column shows a WT-like phenotype, in that it has an outward tail current and the reversal potential is −78 mV. Of 44 oocytes injected, 12 had a phenotype similar to that in the left panel (intermediate phenotype), 11 similar to the middle panel (N629D-like), and 21 similar to the right panel (WT-like). Mean reversal potential of oocytes expressing the intermediate phenotype was −56±9 mV; that of the N629D-like phenotype was −6±4 mV, and that of the WT-like phenotype was −82±6 mV. In contrast, the mean reversal potential of WT was −90±3 mV (P<0.05 by ANOVA).
In contrast to the variability of phenotypes observed when equimolar concentrations are injected, consistent phenotypes are observed when 3-fold excesses of WT or N629D are injected. When WT is injected in 3-fold excess, all measured currents have a phenotype similar to WT, whereas when N629D is injected in 3-fold excess the phenotype is consistently similar to homomultimeric N629D.
To further assess the physiological significance of N629D coexpressed with WT at equimolar concentrations, the same action potential and ramp clamps were applied. Representative examples of dofetilide-sensitive currents elicited by the ramp protocol are shown in the middle row of panels in Figure 5⇑ and those of the simulated action potential clamp are shown in the lower row of panels for the various phenotypes seen during coexpression. Coexpressed current shown in the dotted lines is contrasted with WT shown in the solid lines. Application of these protocols to WT results in outward currents, particularly during phase 3 of the action potential. In contrast, application of identical protocols to coexpressed currents with the intermediate and N629D-like phenotype resulted in inward currents during phase 3 repolarization. In contrast, oocytes with WT-like phenotype manifest reduced repolarizing currents during phase 3 of the action potential.
To assess phenotype of a HERG tetramer with known ratio of WT and N629D subunits, a dimer of N629D connected at its C-terminal end to the N terminus of WT was constructed. Injection of this dimer generated currents that had characteristics intermediate between WT and N629D (Figure 6⇓); moreover, the resultant current showed no variability in phenotype from oocyte to oocyte.
Potential Clinical Relevance
The consequences of the N629D mutation are a loss of C-type inactivation and cation selectivity. Disruption of C-type inactivation results in elimination of the outward tail current that would normally result from recovery of inactivated channels during repolarization. Loss of this outward tail current may be a key determinant of disease phenotype, as it is important in the later phase of repolarization and is thought to be involved in prevention of arrhythmias.22 23 A novel property of N629D is a severe loss of selectivity of K+ relative to Na+, which manifests as an inward Na+ current at membrane potentials below −20 mV. Our action potential clamp data suggest that an inward depolarizing Na+ current will be produced during repolarization between −20 and −70 mV. Such a late inward Na+ current is analogous to mutations found in LQT3 in which inactivation of the cardiac sodium channel is delayed, resulting in a sustained inward Na+ current.24
A limitation of this and all previous studies utilizing mammalian cell lines or Xenopus oocytes to characterize LQT mutations is that they may not represent the exact phenotype that occurs in cardiac myocytes.
Heteromultimers Have Variable Phenotypes
Given that N629D and WT express as homomultimers, it might be expected that heteromultimers would form. We provide evidence that functional heteromultimers are formed by assessing response of homomultimers and coexpression channels to 0 mmol/L [K+]e. The time-dependent current of HERG is markedly reduced at 0 mmol/L [K+]e, whereas that of homomultimeric N629D is unaffected. Coexpression of monomers produces currents that can appear grossly similar to that of WT, including outward tail currents and inward rectification. However, these coexpressed currents were reduced by only 14% when exposed to 0 [K+]e compared with the expected value of ≈50%. Thus, these data cannot be explained by a superimposition of WT and N629D homomultimeric currents.
When N629D is coexpressed at equal concentrations with WT, phenotypes range from N629D-like, intermediate, and WT-like. This unusual phenomenon may have resulted, despite our best efforts, from incomplete mixing of mRNAs. Alternatively, physiochemical differences between oocytes may result in differences in ability to correctly fold and/or incorporate the mutant channel into tetramers. To better understand this phenomenon, we constructed a dimer with a N629D monomer linked to a WT monomer. The resultant current shows no variability in phenotype from oocyte to oocyte, and the phenotype is intermediate between WT and N629D. We speculate that small differences in gene expression or cell physiology may lead to a diversity of phenotypes between individuals or between cells. If our data can be extrapolated from oocytes to heart, intercell diversity in phenotypes could contribute to spatially heterogeneous repolarization that is reported in patients with LQT2.25
In summary, the N629D mutation exerts its effect through removal of C-type inactivation and a reduction in selectivity for K+e over Na+e. This results in a decrease in the outward repolarizing tail current that is replaced with an inward depolarizing sodium current, which is expected to cause a delay in the later stages of repolarization and contribute to arrhythmogenesis.
This study was supported by the Medical Research Council of Canada, the Alberta Heart and Stroke Foundation, and the Andrews Family Professorship in Cardiovascular Science. We thank Drs Robert Clark and Robert French for constructive help and suggestions.
- Received September 13, 1999.
- Accepted December 22, 1999.
- © 2000 American Heart Association, Inc.
Warmke JW, Ganetzky B. A family of potassium channel genes related to EAG in Drosophila and mammals. Proc Natl Acad Sci U S A. 1994;91:3438–3442.
Shi W, Wymore RS, Wang HS, Pan Z, Cohen IS, McKinnon D, Dixon JE. Identification of two nervous system-specific members of the erg potassium channel gene family. J Neurosci. 1997;17:9423–9432.
Sanguinetti MC, Curran ME, Spector PS, Keating MT. Spectrum of HERG K+ channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci U S A. 1996;93:2208–2212.
Nakajima T, Furukawa T, Tanaka T, Katayama Y, Nagai R, Nakamura Y, Hiraoka M . Novel mechanism of HERG current suppression in LQT2: shift in voltage dependence of HERG inactivation. Circ Res. 1998;83:415–422.
Zhou Z, Gong Q, Epstein ML, January CT. HERG channel dysfunction in human long QT syndrome. J Biol Chem. 1998;273:21061–21066.
Chen J, Zou A, Splawski I, Keating MT, Sanguinetti MC. Long QT syndrome associated mutations in the per-arnt-sim (PAS) domain of HERG potassium channels accelerate channel deactivation. J Biol Chem. 1999;274:10113–10118.
Lees-Miller JP, Kondo C, Wang L, Duff HJ, et al. Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circ Res. 1997;81:719–726.
London B, Trudeau MC, Newton KP, Beyer AK, Copeland NG, Gilbert DJ, Jenkins NA, Satler CA, Robertson GA. Two isoforms of the mouse ether-a-go-go-related gene coassemble to form channels with properties similar to the rapidly activating component of the cardiac delayed rectifier K+ current. Circ Res. 1997;81:870–878.
Miller C, Lu Q. Silver as a probe of pore-forming residues in a potassium channel. Science. 1995;268:304–307. Abstract.
Aiyar J, Rizzi JP, Gutman GA, Chandy KG. The signature sequence of voltage-gated potassium channels projects into the external vestibule. J Biol Chem. 1996;271:31013–31016.
Franz MR. Current status of monophasic action potential recording: theories, measurements and interpretations. Cardiovasc Res. 1999;41:25–40.
Compton SJ, Lux RL, Ramsey MR, Strelich KR, Sanguinetti MC, Green LS, Keating MT, Mason JW. Genetically defined therapy of inherited Long QT syndrome: correction of abnormal repolarization by potassium. Circulation. 1997;95:1675–1676.