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Cardiovascular Research 2002 54(1):67-76; doi:10.1016/S0008-6363(02)00240-7
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Copyright © 2002, European Society of Cardiology

Characterization of a novel missense mutation E637K in the pore-S6 loop of HERG in a patient with long QT syndrome

Kenshi Hayashia,*, Masami Shimizua, Hidekazu Inoa, Masato Yamaguchia, Hiroshi Mabuchia, Naoto Hoshib and Haruhiro Higashidab

aMolecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa University 13-1, Takara-machi, Kanazawa, Ishikawa 920-8640, Japan
bBiophysical Genetics, Division of Neuroscience, Graduate School of Medical Science, Kanazawa University 13-1, Takara-machi, Kanazawa, Ishikawa 920-8640, Japan

kenshi{at}im2.m.kanazawa-u.ac.jp

* Corresponding author. Tel.: +81-76-265-2254; fax: +81-76-234-4251

Received 12 July 2001; accepted 21 December 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: In a 32-year-old woman with marked QT prolongation (QTc=0.61 s) and repeated episodes of syncope, we identified a single pertinent base substitution (G to A at 1909) in HERG by genetic analysis. This novel missense mutation is predicted to cause an amino acid substitution of lysine for glutamic acid at position 637 (E637K) in the pore-S6 loop. Therefore, we investigated the role of a glutamic acid at the vicinity of the pore in HERG channels by mutating it to a lysine. Methods: We characterized the electrophysiological properties of the E637K mutation using a Xenopus oocyte heterologous expression system. Results: Injection of the E637K mutant cRNA alone into Xenopus oocytes did not result in any expression of detectable currents. Coexpression of wild-type (WT) and E637K (E637K/WT) elicited only about 30% of the control peak tail current that was expected from expression of WT alone. Kinetic analyses revealed that E637K/WT decelerated the rate of channel activation and enhanced steady-state inactivation. Furthermore, the reversal potentials at low concentrations of K+ showed a positive shift in oocytes injected with E637K/WT compared with WT alone. Conclusions: These results indicated that the E637K mutation causes apparent dominant negative suppression of WT HERG channel function and suggest that E637 at the Pore-S6 is a crucial component of the activation and inactivation gate of HERG channels.

KEYWORDS Arrhythmia (mechanisms); K-channel; Long QT syndrome


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Familial long QT syndrome (LQTS), an inherited cardiac disorder that results in syncopal attacks and sudden death, is characterized by prolongation of the QT interval indicating abnormal cardiac repolarization [1]. Ventricular repolarization involves several different currents controlled by a number of distinct ion channels. Defects in any of these channel genes can result in altered repolarization leading to LQTS. Presently, at least six different genetic loci have been associated with LQTS [2–8].

One of the principal causes of LQTS is the human-ether-a-go-go-related gene (HERG), of which a defect produces the LQT2 form of LQTS. HERG encodes a voltage-gated K+ channel whose properties are similar to the rapidly activating delayed rectifier K+ current (IKr) [9]observed in myocardium. LQTS is known to be comprised of more than 80 HERG mutations [10], most resulting in single amino acid substitutions. There are several mechanisms by which individual mutations in HERG produce LQTS. If the missense mutation results in changing a function vital to the operation of the channel, a dominant-negative effect can occur through loss of repolarizing current [11]. Other missense mutations alter one or more properties of the channel. For example, mutations in the N-terminus accelerate HERG channel deactivation [12]; a mutation in the S4 voltage sensor affects the voltage dependence of activation [13]; mutations in the pore [14] or in the outer mouth of the pore [15] affect HERG channel inactivation; mutations in the pore [16] or in the S5-pore loop [17] affect the ion selectivity function of the HERG channel; and a mutation in the C-terminus modifies activation–deactivation gating properties [18]. These observations indicate that mutations in different regions elicit a variety of functional changes and suggest that each mutation should be examined to clarify the mechanism of HERG current suppression.

We have recently identified in a Japanese LQTS family a novel missense mutation in the pore-S6 loop of HERG which results in an amino acid substitution of lysine for glutamic acid at position 637 (E637K). Since such a mutation has not been reported before, it is of interest to discern whether this mutation is responsible for HERG channel dysfunction and the cause of LQTS in this patient. We examined the channel properties of this mutant by using the heterologous expression system of Xenopus oocytes. We demonstrate that E637K is unable to produce outward currents and acts on wild-type HERG channels in a dominant-negative fashion.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 DNA isolation and mutation analysis
Genetic analysis was performed after obtaining written informed consent. Genomic DNA was purified from subjects* white blood cells after which in vitro amplification was performed by polymerase chain reaction (PCR) [19]. Single-strand conformational polymorphism (SSCP) analysis of the amplified DNA was performed to screen for mutations in KCNQ1, HERG, SCN5A, KCNE1, and KCNE2 as described previously [20] with a slight modification [21]. Normal and aberrant SSCP products were isolated and sequenced by ABI PRISM 310 (PE Applied Biosystems, Foster City, CA, USA). As for the HERG gene, we sequenced all 15 exons in the proband using 19 primer pairs (Table 1) [5,22–24]. To confirm the missense mutation that serves as the basis of this study, restriction enzyme analysis was performed. Using a mutagenic primer, gene amplification by PCR introduced an artificial Tth HB8I site in the PCR product only for the G-to-A allele (E637K). Digestion of the PCR products derived from the mutant allele with Tth HB8I gave rise to 60-bp fragments instead of 79 bp when resolved on polyacrylamide gel.


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  		Table 1 HERG PCR primers

 
2.2 Site-directed mutagenesis and cRNA in vitro transcription
The HERG cDNA in the pGH19 vector was kindly provided by Dr. Gail A. Robertson (University of Wisconsin). The E637K cDNA was constructed by an overlap extension strategy [25]. The E637K cDNA was confirmed by DNA sequencing analyses using 11 primers. Moreover, to eliminate the possibility that the lack of IKr-like current following E637K injection was due to some anomaly arising from the mutagenesis itself, mutagenesis to convert the E637K mutation back to K637E was performed. WT HERG cDNA, E637K cDNA, and K637E cDNA were linearized by digestion with NotI, while cRNAs were prepared with the mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA) using T7 RNA polymerase.

2.3 Oocyte preparation and injection
Defolliculated Xenopus laevis oocytes (stage V–VI) were isolated as described previously [26]. Each oocyte was injected with 50 nl cRNA solution containing either 1.0 ng WT cRNA, 1.0 ng E637K cRNA, or cRNA in combination with both 1.0 ng WT HERG and 1.0 ng E637K. To assess the suppression effect of E637K mutant subunits on HERG channel function, mixtures of WT HERG and E637K cRNAs (2.0 ng total) at various ratios were injected into oocytes. To eliminate the potential for a suppressive effect on HERG current that might be due to an increased amount of RNA being injected into oocytes, WT HERG cRNA and another cRNA (we used a cRNA of metabotropic glutamate receptors, mGluRs) were co-injected into oocytes. The oocytes were incubated at 16 °C in modified Barth's solution (mM: 87.4 NaCl, 1 KCl, 2.4 NaHCO3, 10 Hepes, 0.82 MgSO4, 0.66 NaNO3, 0.74 CaCl2, at pH 7.5 adjusted with NaOH), supplemented with penicillin (100 µg/ml) and streptomycin (100 µg/ml). The oocytes were studied 2–4 days after injection.

2.4 Electrophysiological experiments
Membrane currents were studied using the two-microelectrode voltage clamp technique with an amplifier AXOCLAMP-2A (Axon Instruments, Foster City, CA, USA) at a room temperature of 23–25 °C as previously described [27]. During recording, oocytes were continuously perfused with ND 96 solution (mM: 96 NaCl, 2 KCl, 5 MgCl2, 0.3 CaCl2, 5 Hepes, pH 7.6, adjusted with NaOH). To examine for permeability of K+ relative to Na+ on expressed currents, [K+]o was varied as 0.2, 0.5, 2, 5, 10, or 20 mM by replacing with equimolar Na+.

Data acquisition and analysis were performed by a Digi DATA 1200 A/D converter (Axon Instruments) and pCLAMP (version 5.5.1, Axon Instruments).

2.5 Voltage clamp protocols and data analysis
All pulse protocols are described in the figure legends. Data analysis was carried out using Clampfit (version 6.1, Axon Instruments). The voltage dependence of HERG current activation was determined for each oocyte by fitting peak values of tail current (Itail) versus test potential to a Boltzmann function in the following form: Itail=Itail-max/{1+exp[(V1/2–Vt)/k]}, where Itail-max is peak Itail, Vt is the test potential, V1/2 is the voltage at which Itail is half of Itail-max, and k is the slope factor. The binomial distribution was applied to relative tail current amplitudes at –60 mV following a test pulse to –10 mV for different coinjection mixtures as described previously [28]. The ideal predicted curve plotted was derived from the binomial function:

Formula
where I is the relative tail current, P is the fraction of wild-type subunits, (1–P) is the fraction of mutant subunits, and Zx is the fraction of wild-type current passed by channels with x mutant subunits. If the mutant is completely dominant over the wild type, Zx is zero for x=1.

Steady-state inactivation was analyzed as described previously [29]. Briefly, the corrected steady-state inactivation curves were fitted with a Boltzmann function in the following form:

Formula
where I is the amplitude of inactivating current corrected for deactivation, Imax is the maximum of I, Imin is the minimum of I, Vt is the prepulse of test potential, V1/2 is the voltage at which I is half of Imax, and k is the slope factor.

The K+ selectivity of HERG was determined by measuring the reversal potential of currents in oocytes bathed in solution containing different concentrations of KCl (0.2–20 mM). Tail currents were measured at a variable test potential after current activation by a pulse to 0 mV. The voltage at which the tail current reversed from an inward to an outward current was defined as the reversal potential, Erev. The plot of Erev versus log[K+] was determined by fitting to the Goldman–Hodgkin–Katz equation.

2.6 Statistical analysis
All values are expressed as mean±S.E.M. Differences within these values were evaluated by ANOVA and unpaired Student's t-test when appropriate. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Clinical diagnosis
Fig. 1 shows the 12-lead ECG of the proband (Fig. 2A, II-6), who exhibited a markedly prolonged QTc (610 ms) with notched T waves. She is a 32-year-old woman who had experienced syncopal episodes at rest. She had been diagnosed as having epilepsy and was treated with neuroleptic drugs. ECG monitoring during her electroencephalogram showed a polymorphic ventricular tachycardia (torsade de pointes) which was accompanied by heart palpitations and chest discomfort. Her audiometrics were normal as were the serum K+, Mg2+, and Ca2+ concentrations. The father (I-1) and the daughter (III-4) of the proband had prolonged QTc intervals of 520 and 512 ms, respectively, but neither had a history of syncope (Fig. 2A).


Figure 1
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  		Fig. 1 Twelve-lead ECG of the proband. ECG shows a markedly prolonged QTc (0.61s) with notched T waves.

 

Figure 2
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  		Fig. 2 Mutation analysis. (A) The pedigree and QTc intervals. I, II, and III indicate generations. The asterisk in the pedigree indicates the proband. Numbers show QTc intervals. Half-closed square and half-closed circles indicate heterozygous male and female patients with the HERG E637K mutation revealed in sequence analysis in (B). Result of PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Digestion of the PCR products with Tth HB8I generates polymorphic restriction fragments of 79 and/or 60 bp. (B) DNA sequence analysis in the proband. A single nucleotide transition from G to A at nucleotide position 1909 of HERG occurs in three affected patients. This mutation leads to a missense mutation of the 637th glutamate to lysine. (C) Primary sequence alignment of the pore-S6 loop of the HERG E637K mutant, seven potassium channels and one cation channel (and their GenBank accession numbers); eag family of channels HERG (U04270), Seizure (U36925), Drosophila Eag (M61157), and Elk (U04246); plant inward rectifiers KAT1 (M86990) and AKTI (X62907); cyclic nucleotide gated cation channel cAMP (X55519); and Drosophila Shaker family of voltage-gated channel Shaker (M17211). (D) Two schemes indicate the location of LQT-associated mutation (Lys 637) in mutant HERG and the corresponding site (Glu 637) in WT HERG, according to a model of partial single KcsA K+ channel [36].

 
3.2 Molecular genetic analyses
Screening for mutations in the HERG gene with SSCP analysis identified an abnormal SSCP conformer in the DNA samples from the three family members with prolonged QTc intervals. These SSCP anomalies were not observed in the DNA samples from more than 100 control subjects. Sequence analysis of the abnormal SSCP conformer revealed a mutation leading to a single base substitution (G to A) (Fig. 2B). All of the individuals with the E637K mutation showed both 79- and 60-bp fragments by PCR RFLP analysis, indicating heterozygosity for the mutation (Fig. 2A). This mutation is predicted to result in an amino acid substitution of a conserved glutamic acid with lysine at codon 637 (E637K), located in the pore-S6 loop in the vicinity of the pore of the HERG subunit (Fig. 2C,D). Sequence analysis of all 15 exons of the HERG gene did not show any other mutation. Screening for mutations in the KCNQ1, SCN5A, KCNE1, and KCNE2 with SSCP did not also show any abnormal SSCP conformers. In order to obtain information regarding how and which dysfunction may be elicited by this mutation, we constructed E637K mutant cDNA. We confirmed that the E637K cDNA revealed no mutation existed except for lysine at codon 637 by DNA sequence analysis. Armed with these preparations, we expressed wild-type and mutant HERG cRNAs in oocytes and analyzed the resultant currents in the following experiments.

3.3 Electrophysiological characterization of E637K mutation in HERG
3.3.1 Influence of the E637K mutation on channel function
Oocytes injected with 1.0 ng WT HERG cRNA alone expressed a slowly activating outward current reaching >0.5 µA in amplitude by step depolarizations from –20 to +20 mV (Fig. 3). This current was associated with an inwardly rectifying property (Figs. 3C and 4AGo), as described previously [30]. By contrast, oocytes injected with 1.0–9.0 ng of E637K cRNA did not express functional channels at any time during the 1–5-day period following cRNA injection (Fig. 3A). The generated E637K HERG currents were not different from those in H2O-injected oocytes (Fig. 3B). The current induced by injection of 1.0 ng K637E cRNA, of course, behaved like WT HERG (data not shown). In this way, it was confirmed that no anomaly arose from the mutagenesis procedure itself in the E637K HERG constructs or experimental errors. And we could eliminate the possibility that the lack of IKr-like current following E637K microinjection was due to these anomaly.


Figure 3
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  		Fig. 3 Voltage-clamp recordings of Xenopus oocytes expressing wild-type (WT) and E637K HERG. Representative whole cell current traces of oocytes injected with 1.0 ng E637K cRNA (E637K, A), H2O (control, B), 1.0 ng WT cRNA (WT, C), and coinjected with 1.0 ng WT cRNA plus 1.0 ng E637K cRNA (E637K/WT, D). Depolarizing pulses were applied to potentials ranging between –80 and +50 mV for 3 s, followed by a hyperpolarizing pulse to –60 mV for 6 s. Holding potential of –60 mV. The voltage protocol is illustrated in the inset in panel (A).

 

Figure 4
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  		Fig. 4 (A) Current–voltage (I–V) relationship. Peak currents in oocytes expressing WT (closed square), E637K/WT (open square), E637K (open circle), control (closed circle), and 1.0 ng WT cRNA plus 1.0 ng metabotropic glutamate receptors, mGluRs (mGluRs/WT, open triangle). Currents were plotted as a function of depolarizing voltages in 5 to 10 experiments. Bars indicate standard error of the mean (S.E.M.). (B) I–V relationship for amplitudes of tail currents recorded during depolarizing pulses for WT (closed square), E637K/WT (open square), and mGluRs/WT (open triangle). (C) Normalized I–V relationships for mean amplitudes of tail currents for WT (closed square) and E637K/WT (open square) in 11 experiments. (D) Normalized tail currents recorded at –60 mV after +20 mV depolarizing pulse in oocytes injected with varying ratios of WT cRNA and E637K (solid line with open square) or mGluRs cRNA (solid line with open triangle). Experiments were numbered from 13 to 21. Solid line describes the binomial function of a completely dominant mutant in a tetrameric assembly.

 
When E637K and WT HERGs were coexpressed by injecting into oocytes the equal amount of 1.0 ng WT and 1.0 ng E637K (i.e., 2.0 ng cRNA altogether, E637K/WT), evoked currents were detectable. The peak current was obtained at a depolarizing step to +20 mV as WT currents, but E637K/WT currents were significantly smaller than those for WT alone (Fig. 3D). The mean amplitude (±S.E.M.) of the tail currents measured at –60 mV after a depolarizing test pulse to +20 mV was 1701±175 nA (n=11) for WT 1.0, and 588±41 nA (n=11) for E637K/WT (Fig. 4B, P<0.01). To exclude the possibility that the additional cRNA could nonspecifically attenuate WT HERG expression, we injected into oocytes 1 ng of WT cRNA and 1 ng of mGluRs cRNA (i.e., 2.0 ng cRNA altogether, mGluRs/WT). The mGluR cRNA was chosen to use, because mGluR is expressed in membrane but may not interact directly with HERG. The peak currents and tail currents for mGluRs/WT were similar to those for WT (Fig. 4A,B). Amplitudes of tail currents were plotted as a function of the test potential and were fitted to a Boltzmann function (Fig. 4C). The voltage at which the current was half-activated (V1/2) was –23.3±1.2 mV (n=11) for WT and –14.5±1.8 mV (n=11) for E637K/WT. The V1/2 for E637K/WT was shifted significantly to positive potentials compared with that for WT alone (P<0.01). The slope factor for E637K/WT (6.9±0.18 mV, n=11) was also reduced compared with that for WT alone (8.3±0.32 mV, n=11) (P<0.01).

Fig. 4D shows a more detailed analysis of the effects of the mutant E637K on WT HERG current. Oocytes were injected with a constant amount (2 ng) of WT HERG cRNA with varying ratios of E637K. In oocytes injected with mixtures of WT and E637K cRNAs at various ratios, as the ratios of E637K cRNA were increased, the relative current amplitudes at –60 mV after depolarizing test pulses to +10 mV were significantly reduced. The suppressive effect was slightly weaker than that expected for ideal dominant negative mutants (Z1=0.37, Z2=0.18) (open square in Fig. 4D). In contrast, in oocytes injected with mixtures of WT and mGluRs cRNAs at various ratios, the relative current amplitudes gradually decreased (open triangle in Fig. 4D). These results suggest that an E637K mutant causes dominant-negative suppression of WT HERG; however, some fraction of E637K/WT forms heterotetramers to function as active channels. We next examined the kinetics of HERG channels and reversal potentials between coinjected oocytes and oocytes injected with WT HERG alone.

3.3.2 Deactivation of E637K mutation channels
Deactivation currents during test pulses could be fitted to a double exponential function. At test potentials between –100 and –50 mV, both fast and slow time constants of deactivation for E637K were similar to that for WT alone (Fig. 5A,B). Our results differ from those of a previous report [16] in which a mutation in the channel pore N629D accelerated the deactivation time course.


Figure 5
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  		Fig. 5 (A,B) Deactivation time constants of expressed HERG currents in oocytes injected with WT (closed square) and E637K/WT (open square). To examine the deactivation time course, a current was activated with 3 s pulses to +20 mV, followed by a return to test potentials ranging from –50 to –100 mV (10-mV steps). The deactivation process was fit to biexponential functions. Fast (A) and slow (B) components of deactivation time constants as a function of test potential. The values are the means of 10 measurements. (C) Normalized steady-state inactivation curves of expressed currents in oocytes injected with WT (closed square) and E637K/WT (open square). To construct inactivation curves, a voltage protocol (inset) was employed: a 900-ms depolarizing pulse to inactivate HERG channels followed by varying repolarizing pulses to potentials between –130 and +50 mV for a short period of 60 ms, followed by a test pulse to +20 mV. Current amplitude at test potential was normalized and plotted against prepulse potentials. Symbols are experimental data (n=6, ±S.E.M.) and curves represent best fits to a Boltzmann function. (D) Comparison of K+ selectivity through expressed currents in oocytes injected with WT (closed square) and E637K/WT (open square). Plot of the reversal potential versus log[K+]o was determined by fitting to the Goldman–Hodgkin–Katz equation. The relative permeability of Na+ to K+ determined from this fit was 0.010 for WT and 0.018 for E637K/WT. The values are the means of five measurements.

 
3.3.3 Steady-state inactivation of E637K mutation channels
The steady-state inactivation for E637K/WT was shifted significantly to a negative potential (V1/2 value was –101.9±3.9 mV (n=6)) compared with that for WT alone (V1/2 value was –82.5±3.0 mV (n=7)) (Fig. 5C, P<0.01). Thus, at the same depolarized potential, channel availability was diminished for E637K/WT resulting in enhanced inward rectification. Our results are in accord with previous reports [14,29] on HERG with nearby mutants. For instance, a mutation in the channel pore V630L caused a negative shift in the voltage dependence of steady-state inactivation [14], while mutations in the outer mouth of the channel pore S631C produced a substantial increase in the rate of inactivation [29].

3.3.4 Ion permeability of expressed currents
The reversal potentials at low concentrations of K+ (0.2, 0.5, and 2 mmol/l) showed a positive shift in oocytes injected with E637K/WT (–105.9±2.4 mV for 0.2 mmol/l, n=7; –103.7±1.3 mV for 0.5 mmol/l, n=11; –87.4±1.1 mV for 2 mmol/l, n=10) compared with WT alone (–118.9±1.3 mV for 0.2 mmol/l, n=6; –110.2±2.3 mV for 0.5 mmol/l, n=9; –93.2±1.7 mV for 2 mmol/l, n=13) (Fig. 5D, P<0.05). The relative permeability of Na+ to K+ for WT determined from this fit was 0.010. For E637K/WT, the best fit estimate of the permeability ratio, PNa/PK, was 0.018.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we identified in one Japanese LQTS family a novel single base substitution (from G to A) at the nucleotide position 1909 of HERG. This mutation may lead to the substitution of a positively charged lysine for the highly conserved, negatively charged glutamic acid at position 637 located at the outer mouth, the pore-S6 loop, of HERG channels (Fig. 2C,D). Interestingly, the primary consequence of the E637K mutant was an inability to produce HERG outward currents when corresponding cRNA was injected to express E637K HERG in Xenopus oocytes (Figs. 3A and 4AGo). Since the identical injection of wild-type HERG produced inwardly rectified currents [31] as shown in Fig. 3, the absence of current in E637K HERG may seem to be related to its fundamentally defective properties.

On the contrary, co-expression of E637K with WT in oocytes, which is expected to form heteromultimers rather than two distinct homomultimers, resulted in outward currents whose amplitudes were far less than one-third of the control currents that were expected from expression of WT alone (Fig. 4A,B). However, most of the current properties tested were similar between WT and WT/E637K currents. For instance, the peak of current–voltage (I–V) relationships for peak currents recorded during depolarizing pulses (Fig. 4A) and the deactivation time course (Fig. 5A,B) were similar between the two experimental conditions. In contrast, the two currents were not identical with respect to the following properties: the voltage dependence of activation was shifted in a positive direction (Fig. 4C), the voltage dependence of steady-state inactivation was shifted in a negative direction (Fig. 5C), and the reversal potentials at low concentrations of K+ showed a positive shift (Fig. 5D). These results suggest that E637K mutant subunits co-assemble with those of wild-type channels, and that such heteromultimers function as potassium channels but suppress normal channels. Thus, it can be concluded that the E637K mutation is pathogenic in producing LQTS in these patients, because no genetic abnormality accountable for this was detected in other portions of the K+ channels (KCNQ1, KCNE1, KCNE2) or the Na+ channels (SCN5A). As shown by PCR-RFLP, our patients are heterozygous for this mutation. Thus, native HERG channels consist of wild-type and E637K mutant channels. Although we have no exact information about this patient at the HERG protein level, it is highly possible that two types of HERG proteins are expressed and that channels in these patients function similarly to those of channels expressed in our experimental oocytes.

The dominant-negative effects of E637K found in co-expressed currents can be easily generated when we assume that one or two mutant subunit(s) form a composite as members of the four-subunit potassium channel. As mentioned before, injection of this mutant alone formed nonfunctional channels. The reasons for why and how such dysfunction is produced by the E637K mutation are not clear. However, at least two explanations can be considered. According to some reported mechanisms [32,33], one possibility involves defective protein trafficking of mutant HERG proteins to the plasma membrane, while another involves a primary dysfunction in gating, even when the channel protein is correctly positioned in the plasma membrane. Our co-expression experiments indicate that the gating properties are less effective, but such dysfunction is not enough to explain all the observed dominant-negative effects. Therefore, the first possibility suggested above should not be disregarded. Experiments to test protein processing are currently underway using cell biological techniques, such as fusion with green fluorescence protein, and immunocytochemical techniques to detect membrane insertion properties.

The data demonstrating a negative shift in the voltage dependence of HERG inactivation by the E637K mutation suggests that the residue E637, which is located in the vicinity of the channel pore, may be related to conformational changes during inactivation. It has been reported that one mode of suppression of outward currents in HERG channels is caused by fast C-type inactivation which involves a conformational change near the outer mouth of the pore [34]. In a previous report [14], a mutation in the channel pore or in the outer mouth of the channel pore altered the voltage dependence of inactivation of the HERG channel. As examples, substitution of V630L caused a negative shift in the voltage dependence of steady-state inactivation, substitution of S631C accelerated the rate of inactivation, and substitution of S631A reduced the fast inactivation of HERG channels [15].

In this study, the permeability of K+ relative to Na+ in the expressed currents with E637K/WT was altered 1.8-fold that of WT alone. Several reports have shown that mutations in the pore region, the S6, the S4–S5 loop, and the S5-pore loop altered ion selectivity. For example, substitution of N629D [16] and substitution of H587 to proline or lysine [17] disrupted C-type inactivation and reduced the selectivity for K+ over Na+. Mutations of E395Q and L385A in the S4–S5 linker of Shaker K+ channels increased the permeability ratio of Rb+ relative to K+ [35]. In the structure of the KcsA K+ channel [36], the residue corresponding to E637 (G88) is located at the external end of S6 and faces the pore loops (Fig. 2D). We speculate that the mutation of E637 to lysine may change the geometry of the pore resulting in the alteration in ionic selectivity.

In conclusion, we have identified a novel missense mutation in the pore-S6 loop of the HERG gene of a Japanese family diagnosed with LQTS. The results of electrophysiological studies indicate that the E637K mutation in the pore-S6 loop of the HERG K+ channel alters the kinetic properties of activation and inactivation as well as the channel's K+ selectivity. This results in a decrease in the outward repolarizing tail current which should cause a delay in the repolarization phase of action potentials along cardiomyocytes and thus contribute to arrhythmogenesis.

Time for primary review 24 days.


   Acknowledgements
 
We thank Gail A. Robertson (University of Wisconsin) for supplying the HERG cDNA.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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C. E. Clarke, A. P. Hill, J. Zhao, M. Kondo, R. N. Subbiah, T. J. Campbell, and J. I. Vandenberg
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