Cardiovascular Research

Cardiovascular Research 2001 51(4):670-680; doi:10.1016/S0008-6363(01)00350-9
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

A spectrum of functional effects for disease causing mutations in the Jervell and Lange-Nielsen syndrome

Lingqian Huang^a, Maria Bitner-Glindzicz^b, Lisbeth Tranebjærg^c and Andrew Tinker^a,*

^aCentre for Clinical Pharmacology, Department of Medicine, University College London, The Rayne Institute, 5 University Street, London WC1E 6JJ, UK
^bClinical and Molecular Genetics Unit, Institute of Child Health, 30 Guildford Street, London WC1N 1EH, and Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London WC1N 3JH, UK
^cDepartment of Medical Genetics, University Hospital Tromsø, Tromsø, Norway

^* Corresponding author. Tel.: +44-20-7679-6174; fax: +44-20-7679-6212 a.tinker{at}ucl.ac.uk

Received 24 January 2001; accepted 11 May 2001

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Jervell and Lange-Nielsen syndrome (JLNS) is a recessively inherited long QT syndrome (LQTS) characterised by profound sensorineural deafness and predisposition to syncope and sudden cardiac death. Mutation analysis has established the presence of mutations in affected individuals in the genes KCNQ1 and KCNE1: the potassium channel complex responsible for the cardiac I_Ks current involved in repolarisation of the ventricular action potential. Our objective was to determine the functional effects of disease causing mutations in JLNS. Methods: In this study we have investigated the electrophysiological effects of eight distinct JLNS mutations after expression of cRNA in Xenopus laevis oocytes. Results: KCNE1 mutant T59P/L60P showed no dominant negative effect and was a pure loss of function mutation. KCNQ1 mutant E261D showed a strong dominant-negative effect. KCNQ1 mutant R243H produced a moderate dominant-negative effect, right shifted the steady-state activation curve and led to an increased deactivation rate. The behaviour of KCNQ1 mutants 572–576del, 1008delC, R518X, Q530X, R594Q depended on the relative quantities of mutant and wild-type proteins (with a weak dominant-negative effect present at 1:3 but not 1:1 injection ratios). These data indicate the presence of an additional assembly domain before S2–S3 and the importance of the S4–S5 region in channel function and gating. Conclusions: Our data suggest a spectrum of behaviour for disease causing mutations from simple loss of function through to prominent dominant negative behaviour.

KEYWORDS Arrythmia (mechanisms); Long QT syndrome; K-channel; Repolarization; Sudden death

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This article is referred to in the Editorial by A.A.M. Wilde and D. Escande (pages 627–629) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Long QT syndrome (LQTS) is characterised by prolongation of the QTc interval (QT interval corrected for heart rate) on the electrocardiogram. It predisposes to syncope, seizures and sudden death due to the development of a characteristic ventricular tachycardia known as torsades de pointes and subsequently fatal ventricular fibrillation. Long QT syndromes can be acquired in origin most commonly due to drugs or more rarely occur as a part of an inherited syndrome. The autosomal recessive Jervell and Lange-Nielsen syndrome (JLNS) was first described in 1957 and is characterised by profound sensorineural deafness in association with a prolonged QT interval [1]. The more common Romano Ward syndrome (RWS) is inherited in autosomal dominant fashion and patients have only cardiac symptomatology with normal hearing [2,3].

Advances in molecular genetics have established mutations in genes encoding the cardiac sodium channel and two potassium channels and their ancillary proteins [4–9]. JLNS and RWS are allelic at the molecular level as homozygous mutations in the potassium channel gene KCNQ1 cause JLNS [9–12] and heterozygous mutations in KCNQ1 cause RWS [11,13–19]. Mutations in the gene encoding a regulatory protein KCNE1 are also known to cause both JLNS and RWS [7,9,20,21]. KCNQ1 together with KCNE1 encodes the slowly activating delayed rectifier potassium current (I_Ks) channel; a current responsible for repolarisation of the cardiac action potential [22,23].

Clinical and genetic data suggest that the functional effects of mutations ascertained through RWS families may differ from those ascertained through JLNS families. Clinically, carriers of JLNS mutations are only occasionally symptomatic, although they may have prolonged QT intervals [24]. Consequently the mild phenotypic effects of these mutations may allow them to breed to homozygosity in the population. Of the mutations causing RWS reported to date on the LQTS databases (HUGO mutation database initiative, Long QT syndrome database (LQTS database): http://www.ssi.dk/en/forskning/lqtsdb/kvlqt1.htm; The European Society of Cardiology database: http://pc4.fsm.it:81/cardmoc), the majority are predicted to have a localised effect on the protein. These are mainly missense, with some in-frame deletions and splice mutations (detailed analysis of which predicts translation of an in-frame protein [25]), and only a few cause frameshift. As RWS is a more common condition than JLNS, there has been more opportunity to study the functional effects of mutations associated with RWS, which have been shown to act in a strong dominant negative manner. So far, only three JLNS mutations (R243H, W305S, 544) have been studied functionally [11,15,18,26]. There is no clear consensus on how these mutations cause their functional effects. In this study, we report the functional effects of eight JLNS mutations.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Site-directed mutagenesis and cRNA synthesis
Two human KCNQ1 cDNA clones were used in the present study: short KCNQ1 (s-KCNQ1) [23] and long KCNQ1 (l-KCNQ1) [15]. Human synthetic KCNE1 clone was obtained from Dr Richard Swanson. Vectors pSP64, pGEM3ZHE and pGEMA were used for the expression of s-KCNQ1, l-KCNQ1 and KCNE1 cDNA clones, respectively. In this study we largely examined s-KCNQ1 as this expressed robust currents in Xenopus laevis oocytes after cRNA injection. However reasonable current expression from the l-KCNQ1 cDNA was achieved after synthesis and injection of cRNA generated after subcloning into the pGEMHE vector [27]. The pGEMHE vector contains the 5' and 3' untranslated regions from the Xenopus laevis beta globin gene and this can significantly increase currents. KCNQ1 and KCNE1 mutations were introduced into wild type (WT) s-KCNQ1, l-KCNQ1 and KCNE1 gene using QuickChange Kit (Stratagene). All the mutants were characterised by restriction mapping and DNA sequencing. An in-vitro transcription kit (Stratagene) was used to synthesize cRNAs from WT and mutant s-KCNQ1 (SP6 kit), l-KCNQ1 (T7 kit) and KCNE1 (T7 kit) clones. Size, integrity and quantity of cRNAs were verified by electrophoresis on formaldehyde/agarose gels together with standards of known size and quantity. Kir 2.1 was used as previously described [28].

2.2 Isolation of Xenopus laevis oocytes and electrophysiology oocytes
Xenopus laevis were anaesthetised by immersion in 0.1% Tricaine (Sigma) for 30–60 min. After removal of oocytes the Xenopus laevis were then humanely sacrificed according to UK Home Office Schedule I guidelines. Oocytes were prepared as previously described [28]. Injected oocytes were incubated in ND96 (in mmol/l, 96 NaCl, 2 KCl, 1 MgCl₂, 5 HEPES, 1.8 CaCl₂, pH 7.5 at 16°C. Equimolar amounts of KCNQ1 and KCNE1 cRNAs were injected. To study the dominant-negative effects, different cRNA ratios between WT and mutant were used. At a 1:1 ratio, equimolar amounts of cRNAs of mutant and WT protein were injected. A 1:3 ratio was equivalent to three times more mutant than wild type. Experiments were analysed within a batch or pooled from a number of batches as indicated.

Recordings were made 1–5 days after injection at room temperature (22–25°C) in a modified ND96 (mmol/l, 96 NaCl, 2 KCl, 2 MgCl₂, 0.1 CaCl₂, 5 HEPES, pH 7.6). Currents were recorded with a standard two-electrode voltage clamp technique using the TEV-200 (Dagan, USA). Glass microelectrodes were filled with 3 M KCl and had 0.3–0.8-M resistance. pClamp5 software (Axon Instruments) was used to generate voltage-clamp commands and collect experimental data. The membrane potential was held at –80 mV between test pulses. Currents were recorded during 3.5-s pulses from a holding potential of –80 mV to test potentials ranging from –80 to +60 mV (10-mV intervals). Deactivating tail currents were elicited by repolarisation to –50 mV. The inter-pulse interval was 5 s. To determine the voltage-dependence of the channel activation, oocytes were depolarised to potentials ranging from –80 +120 mV (10-mV intervals) for 3.5 s before repolarising to –50 mV. The voltage dependence of channel deactivation time constant was determined by repolarising the oocytes to potentials ranging from –20 to –90 mV (10-mV intervals) for 5 s after a 3.5-s pulse to +60 mV.

Data analysis was carried out using pCLAMP 6.0.4, Microsoft Excel 97 and Origin 6.0. Time constants were determined by fitting currents to a single exponential decay function. The voltage dependence of the channel activation was determined by fitting the normalised amplitude of the peak tail currents (y/y_max) versus test voltage potential (V_t) with a Boltzmann function (y/y_max=1/(1+exp[(V_0.5–V_t)/k])). Data were represented as mean±S.E.M. and statistical comparisons were made using a two-tailed Student's t-test.

2.3 Family data
All homozygotes with JLNS were severely to profoundly deaf and had prolonged QTc intervals; two families (UK80 and UK7B) were ascertained through syncope in the proband, and hearing was reported as normal in all individuals in these two families. A series of criteria have been presented for the diagnosis of long QT syndrome [29]. As a generalisation a QTc greater than 440 ms in males and greater than 460 ms in females is considered prolonged in an individual who has symptoms or a family history of sudden early cardiac death. For asymptomatic individuals, QTc should be greater than 470 ms in order for designation as ‘affected’ with long QT syndrome [30]. The family data together with relevant references [31–34] are summarised in Table 1.

View this table: [in this window] [in a new window]   Table 1 Summary of clinical details of heterozygotes studied

 

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The location and nature of the mutations in KCNQ1 and KCNE1 examined in this study are shown in Fig. 1. Site-directed mutagenesis was used to introduce these into the respective cDNAs, and cRNA was synthesised and injected into oocytes for electrophysiological study using two-electrode voltage clamp (Methods).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Mutations in KCNQ1 and KCNE1 used in this study [9,32].

 
3.1 Functional expression of currents
In agreement with previous studies [22,23], injection of WT KCNQ1 cRNA alone led to the expression of low levels of a rapidly activating potassium current (0.40±0.05 µA at +60 mV, n=6) and KCNE1 cRNA alone led to low-level expression of a slowly activating potassium selective current (1.26±0.09 µA at +60 mV, n=4). The latter is due to the low level expression in Xenopus laevis of an endogenous KCNQ1-like protein [22]. However, coinjection of equimolar KCNQ1 and KCNE1 cRNAs led to the prominent expression of a slowly activating potassium selective current (7.02±0.48 µA at +60 mV, n=11). Mutant KCNQ1 cRNAs were coinjected together with WT KCNE1 cRNA. None of the mutants produced any significant current in the presence of WT KCNE1 (Fig. 2). Furthermore, co-expression of the mutant KCNE1 (T59P/L60P) and WT KCNQ1 cRNAs also did not induce any significant current (Fig. 2).

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Expression of KCNQ1 and KCNE1 mutations alone did not produce significant currents. (A) Currents recorded in oocytes expressing WT KCNQ1+WT KCNE1, R243H+WT KCNE1, E261D+WT KCNE1 and WT KCNQ1+T59P/L60P. Voltage protocol is shown in the inset. Oocytes were depolarised for 3.5 s from a holding potential of –80 mV to test potentials ranging from +60 to –80 mV (10-mV intervals). Deactivating tail currents were elicited by repolarisation to –50 mV. Note that the currents induced by coinjection of WT KCNQ1 and T59P/L60P are similar to that induced by WT KCNQ1 alone. (B) Current–voltage relationships of traces shown in A: WT KCNQ1+WT KCNE1, R243H+WT KCNE1, E261D+WT KCNE1, WT KCNQ1+T59P/L60P. (C) Bar graph of current amplitudes measured at +60 mV in oocytes co-expressing mutant KCNQ1 and KCNE1 in the presence of WT KCNE1 and WT KCNQ1, respectively: Data are represented as mean±S.E.M. n numbers are in brackets above the bar graph. *P<0.05, **P<0.01 in two-tailed Student's t-test, analysed within the same batch of oocytes.

 
3.2 Dominant negative effects
Potassium channels are tetrameric proteins [35] and thus there is the potential for the presence of a single (or more) mutant subunit in a tetramer to inactivate the function of the remaining wildtype subunits. Consequently we examined the tendency for the above mutations to behave in this way. Before undertaking these studies we first established the amount of KCNQ1 cRNA (together with equimolar KCNE1 cRNA) that gave half-maximal current expression. This ensured that we achieved measurable currents but did not saturate the oocyte translation machinery. To further control for non-specific effects, the mutants were injected together with a potassium channel (Kir 2.1) from a totally distinct family [28].

KCNQ1 or KCNE1 mutants were coinjected at a 1:1 (Fig. 3) and 1:3 injection ratio (Fig. 4, WT:mutant). E261D gave a pronounced reduction in current compared to control (65% at 1:1 ratio) and R243H a statistically significant but more moderate effect (25% at 1:1 ratio) at both injection ratios. The KCNQ1 mutants (572–576del, 1008delC, R518X, Q530X and R594Q) did not give a pronounced dominant negative effect at a 1:1 injection ratio but did so at a 1:3 injection ratio. The KCNE1 mutant T59P/L60P did not significantly reduce the current when co-expressed at either ratio with WT KCNE1 (in the presence of KCNQ1). The dominant-negative effect of the KCNQ1 mutants at high injection ratios is unlikely to be due to non-specific effects as injection of the mutants E261D or R243H at an amount equivalent to a 1:9 ratio did not reduce Kir 2.1 current amplitudes. Current amplitudes measured at –100 mV were –8.80±1.21 µA (Kir 2.1, n=5), –8.84±0.48 µA (E261D+Kir 2.1, n=5) and –8.91±0.59 µA (R243H+Kir 2.1, n=5), respectively.

View larger version (60K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Dominant negative effects at 1:1 injection ratio. (A) Currents recorded using the same voltage protocol as in Fig. 2 in oocytes expressing WT KCNQ1+WT KCNE1, WT KCNQ1+572–576del+WT KCNE1, WT KCNQ1+R243H+WT KCNE1, WT KCNQ1+E261D+WT KCNE1 and WT KCNE1+T59P/L60P+WT KCNQ1. Note that the deactivation tail current in the presence of R243H is faster than control. (B) Bar graph of current amplitudes measured at +60 mV in oocytes co-expressing KCNQ1 and KCNE1 mutants at 1:1 ratio in the presence of WT KCNQ1 and WT KCNE1. Data are analysed within the same batch of oocytes except for R243H and E261D in which they are pooled from three and two batches, respectively. Data are represented as mean±S.E.M. *P<0.05, **P<0.01.

 

View larger version (61K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Dominant negative effects at 1:3 injection ratio. (A) Currents recorded using the same voltage protocol as in Fig. 2 in oocytes expressing WT KCNQ1+WT KCNE1, WT KCNQ1+R243H+WT KCNE1, WT KCNQ1+572–576del+WT KCNE1, and WT KCNE1+T59P/L60P+WT KCNQ1. Note that the deactivation tail current in the presence of R243H is much faster than control. (B) Bar graph of current amplitudes measured at +60 mV in oocytes co-expressing KCNQ1 and KCNE1 mutants at 1:3 ratio in the presence of WT KCNQ1 and WT KCNE1. (C) Increasing the injection dose of WT KCNE1 cRNA did not affect the dominant-negative effect induced by 572–576del at 1:3 injection ratio. Data are represented as mean±S.E.M. *P<0.05, **P<0.01, analysed within the same batch.

 
One mechanistic possibility for the dominant negative effect at a higher mutant to WT ratio is that KCNQ1 mutants could interact with WT KCNE1 protein. Thus there would be less free KCNE1 protein to co-assemble with WT KCNQ1 subunits to potentiate current. To test this hypothesis, the amount of WT KCNE1 cRNAs was increased four-fold, i.e the molar ratio between WT KCNQ1 and KCNE1 became 1:4. Under these conditions a prominent dominant negative effect with 572–576del was still observed (Fig. 4C).

We compared our observations on the dominant negative effects of these mutations in JLNS with a mutation (L273F) we have studied in a family with RWS. We found that at a 1:1 injection ratio there was a small but non-significant reduction in current (WT KCNQ1+WT KCNE1=4.29±0.40 µA (n=21), WT KCNQ1+WT KCNE1+L273F=3.75±0.46 µA (n=12), P>0.05). A statistically significant dominant negative effect occurred at a 1:3 injection ratio (WT KCNQ1+WT KCNE1=2.0±0.0.38 µA (n=8), WT KCNQ1+WT KCNE1+L273F=0.66±0.06 µA (n=6), P<0.01).

3.3 Effect of R243H and other mutants on channel kinetics
It was apparent from simple visual inspection of the recordings that in coinjection experiments with WT KCNQ1 (+equimolar KCNE1) and the R243H mutation at both 1:1 and 1:3 ratios, there was a change in the deactivation rate (Figs. 3A and 4A). We thus decided to characterise this at a 1:1 injection ratio. It is apparent from Fig. 5B that there is a significant rightward shift and change in slope of the voltage activation curve. A kinetic analysis revealed that the deactivation rate was increased but the activation rate was not affected (Fig. 5C and D). These findings prompted us to investigate whether the other KCNQ1 or KCNE1 mutants affect channel kinetics. None of the other KCNQ1 or KCNE1 mutants affected the time constants of channel activation/deactivation (Table 2). Due to the small currents with the E261D mutation at a 1:1 injection ratio time constants were not determined.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 The change of channel kinetics induced by R243H at 1:1 ratio. (A) Currents induced by injection of WT KCNQ1+WT KCNE1 and WT KCNQ1+R243H (1:1)+WT KCNE1 using the voltage protocol shown on the top. Oocytes were repolarised from a 3.5-s pulse at +60 mV to potentials ranging from –20 to –90 mV (10-mV interval) for 5 s. (B) Relative activation curves were determined using the voltage protocol shown in the inset. Oocytes were depolarised to potentials ranging from –80 to +120 mV (10-mV interval) for 3.5 s before repolarising to –50 mV. The current amplitudes were measured at the beginning of the deactivating tail current at the indicated test potential and the data were normalised and fitted to a Boltzmann function. WT (): V0.5=28.01±1.35 mV, k=24.42±1.20 mV (n=13); R243H (): V0.5=53.25±1.44 mV, k=30.24±1.36 mV (n=14). (C) Voltage-dependent channel deactivation time constant was determined by fitting the tail currents induced by the voltage protocol shown in A to a single exponential function. A faster deactivation rate in the presence of R243H was observed at all potentials (–20 to –70 mV). WT (): n=14; R243H (): n=14. (D) Voltage-dependent activation time constants were determined by fitting the currents induced by the voltage protocol shown in Fig. 2 to a single exponential function. WT (): n=14; R243H (); n=14. Data are represented as mean±S.E.M. **P<0.01.

 

View this table: [in this window] [in a new window]   Table 2 Time constants of channel activation and deactivation in the presence of KCNQ1 and KCNE1 mutants at 1:1 injection ratio

 
3.4 Studies on l-KCNQ1
The experimental work described above was performed on the short isoform of KCNQ1 (Methods). We performed a comparable set of experiments on the long isoform of KCNQ1 examining expression, dominant negative effects at 1:1 injection ratio and changes in kinetics with R243H, and saw a similar pattern of behaviour (not shown).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Our objective was to study the mechanism by which disease-causing mutations in KCNQ1 and KCNE1 lead to changes in I_Ks and repolarisation abnormalities in patients and carriers of JLNS mutations. In particular we sought to understand why carriers of JLNS mutations are so rarely symptomatic in contrast with carriers of RWS mutations who are much more commonly symptomatic. We tried to mimic the situation that is likely to occur in the patients (homozygotes) and in the carriers (heterozygotes). The simplest hypothesis is that RWS mutations cause a dominant negative effect in addition to the simple loss of function, whereas JLNS mutations do not. Dominant negative effects may arise because of the tetrameric nature of potassium channels and reflect the ability of a single mutant (or more) subunit in a tetramer to inactivate the function of the three (or less) normal subunits. We used the Xenopus laevis expression system for these studies. Whilst it is a heavily used and productive technique, there are some issues concerning its use. For example, KCNQ1 is natively expressed at low levels [22,23], high levels of cRNA injection can induce native anion currents [36] and protein trafficking can be temperature dependent such as is the case for mutant cystic fibrosis transmembrane conductance regulator [37,38].

All these mutations studied here inactivate the function of KCNQ1 as a homomultimer. In addition, we coinjected KCNQ1 with KCNE1 T59P/L60P. Proline is a known helix breaker and the occurrence of two such mutations in the transmembrane domain might severely impair function. Indeed we failed to observe the characteristic enhancement of current seen normally with KCNE1. Interestingly, the KCNQ1 current was rapidly activating as occurs when it is expressed in the absence of KCNE1. The failure to see a dominant negative effect supports the idea that little stable protein is synthesised.

The injection of mutant and WT cRNAs at 1:1 ratio mimics the condition of heterozygous carriers with one mutant and one WT allele. At 1:1 ratios, most JLNS mutations did not cause a dominant negative effect. The results suggest that carriers of the mutations observed in six out of eight JLNS families (572–576del, 1008delC, R518X, Q530X, R594Q, KCNE1 T59P/L60P) would have little additional impairment of I_Ks current under normal circumstances other than that due to the loss of a potentially functional allele. However, surprisingly at a 1:3 ratio the KCNQ1 mutations did have a specific dominant negative effect. It is possible that such effects might have clinical sequelae. Indeed, one heterozygous carrier of R518X, who presented with cardiac syncope diagnosed with RWS had prolonged QTc although carriers were asymptomatic in four other families with JLNS. Similarly one heterozygous carrier of 572–576del had RWS with cardiac syncope in an extended JLNS family. The only true loss of function mutation was the KCNE1 T59P/L60P in which there was no dominant negative effect.

Two of these mutations did produce more pronounced dominant negative effects. The strong dominant-negative effect of E261D implies that heterozygous carriers may have a long QT. One carrier had a normal QTc of 410 ms although the extended family data are not available. Though the R243H mutation was moderate in its effect we compared it with a known mutation L273F in RWS (also present in a family we have studied). We found that the dominant negative effect was at best equivalent to that occurring with R243H. The functional effects of a number of other mutations have been described in RWS and generally they lead to a more pronounced dominant negative effect [15].

The data therefore suggest some interesting conclusions. The observation that the mutations may act in a dominant negative manner at high injection ratios but not in 1:1 ratios with wild type may explain why most carriers of JLNS mutations are asymptomatic but why occasionally they may have symptoms. In our group of families whose mutations were analysed because of presentation through a proband with JLNS, only one obligate carrier of 45 had symptoms (obligate carriers being parents of an index case with JLNS or relatives who have been molecularly proven to be heterozygous for the mutation segregating in a family). If one includes the individuals heterozygous for the R518X mutation but in whom the proband was ascertained with RWS, this becomes two symptomatic heterozygotes out of 46. This supports the concept of a degree of myocardial repolarisation reserve [29,39]. In other words it is possible to tolerate a degree of I_Ks reduction without clinical sequelae and it is likely that this degree varies between individuals. Such ideas may explain why some individuals are particularly predisposed to drug induced long QT syndrome [40] and why we see a spectrum of functional effects for these mutations in JLNS. It is also consistent with the variable penetrance of some mutations in RWS in some families [29]. It is clear that there has to be total abolition of K⁺ flux into the endolymph for hearing loss to occur. What might the cellular mechanisms of reserve be? It is quite possible that there are variations in the current density of I_Kr and I_Ks between individuals determined by genetic factors such as promoter polymorphisms [41,42]. The spatial variation of currents between different ventricular regions and the response to adrenergic stimulation are also likely to be important factors [29].

Our data also have implications for channel function. It has been reported that there is a domain between amino acids 590 and 620 in the KCNQ1 C terminus that determines assembly [26]. Certainly both mutants that showed a dominant-negative effect (E261D and R243H) are missense mutations in S4–S5 linker with an intact C terminus. Thus it is possible for the mutants to co-assemble with WT KCNQ1 subunits, whereas the other KCNQ1 mutants are non-sense (R518X, Q530X) and frameshift mutations (572–576del, 1008del) with a truncated C terminus or a missense mutation (R594Q) with an amino acid change within the proposed assembly domain. Such mutants are unlikely to co-assemble with WT KCNQ1. However at higher injection doses, a dominant-negative effect was found for all the KCNQ1 mutants. These observations imply that another assembly domain exists and the effect of 572–576del indicates that it lies before S2–S3. It is established that voltage-gated channels of the K_v family have assembly domains in the N-terminus and S1 [43].

Our data suggest an important role for the S4–S5 linker in channel function and gating. E261D is a very conservative mutation but this mutant was non-functional and led to a pronounced dominant negative effect. R243H was also non-functional but when co-expressed with WT KCNQ1 and WT KCNE1 cRNAs, it caused a +25-mV rightward shift of the midpoint (V_0.5) in channel activation due to a faster deactivation rate. Other mutations in the S4–S5 linker have been reported to change the gating of KCNQ1 [44] and some functional data has been reported on the R243H mutation [45]. Our data suggest that some R243H heteromultimers are functional.

In summary we have studied the functional effects of eight JLNS mutations and our data indicate a spectrum of functional effects for these mutations in keeping with the variable but generally mild clinical effects on heterozygous carriers. The fact that the functional effect may be altered by the relative amounts of mutant and wild type protein may suggest why predicting phenotype is not straightforward and why the effects of a mutation may differ between individuals of the same or different families.

Time for primary review 28 days.

	Acknowledgements

 
This work was supported by the British Heart Foundation (Dr Lingqian Huang), Medical Research Council (Dr Maria Bitner-Glindzicz) and the Wellcome Trust (Dr Andrew Tinker). We would like to thank for Dr Jess Tyson for her help throughout the course of the study. We thank Professor Mark Keating for the gift of s-KCNQ1 cDNA, Dr Jacques Barhanin for the gift of l-KCNQ1 cDNA, Dr Richard Swanson for the gift of KCNE1 and Dr E. Liman for the gift of pGEMHE.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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