KCNJ10 Mutations Disrupt Function in Patients with EAST SyndromeFreudenthal B.a · Kulaveerasingam D.b · Lingappa L.c · Shah M.A.c · Brueton L.d · Wassmer E.e · Ognjanovic M.f · Dorison N.g · Reichold M.h · Bockenhauer D.a, i, j · Kleta R.a, b, i, j · Zdebik A.A.a, b
aCentre for Nephrology, University College London, Royal Free Hospital, and bDepartment of Neuroscience, Physiology and Pharmacology, University College London, London, UK; cRainbow Children‘s Hospital and Perinatal Centre, Hyderabad, India; dClinical Genetics Unit, Birmingham Women’s Hospital, eBirmingham Children’s Hospital, Birmingham, and fGreat North Children‘s Hospital, Newcastle upon Tyne, UK; gNeuropédiatrie, Hôpital Trousseau, Paris, France; hMedical Cell Biology, University of Regensburg, Regensburg, Germany; iInstitute of Child Health, University College London, and jGreat Ormond Street Hospital for Children, London, UK Corresponding Author
Background/Aims: Mutations in the inwardly-rectifying K+ channel KCNJ10/Kir4.1 cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (EAST syndrome). KCNJ10 is expressed in the kidney distal convoluted tubule, cochlear stria vascularis and brain glial cells. Patients clinically diagnosed with EAST syndrome were genotyped to identify and study mutations in KCNJ10. Methods: Patient DNA was sequenced and new mutations identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by two-electrode voltage clamping. Results: Three new mutations in KCNJ10 (p.R65C, p.F75L and p.V259fs259X) were identified, and mutation p.R297C, previously only seen in a compound heterozygous patient, was found in a homozygous state. Wild-type human KCNJ10-expressing oocytes showed strongly inwardly-rectified currents, which by comparison were significantly reduced in all the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated residual function in all mutant channels (p < 0.05) but V259X. Conclusion: This study confirms that EAST syndrome can be caused by many different mutations in KCNJ10 that significantly reduce K+ conductance. EAST syndrome should be considered in any patient with a renal Gitelman-like phenotype with additional neurological signs and symptoms like ataxia, epilepsy or sensorineural deafness.
Copyright © 2011 S. Karger AG, Basel
Defects in renal ion transport systems can lead to distinct salt-losing nephropathies, which demonstrate the kidneys’ crucial role in fluid and electrolyte homeostasis. For example, Bartter syndrome, caused by mutations in various ion transport proteins in the thick ascending limb of the loop of Henle, and Gitelman syndrome, caused by mutations in a NaCl cotransporter in the distal convoluted tubule, are characterized by distinct electrolyte abnormalities .
Recent studies showed that mutations in the inwardly-rectifying potassium channel KCNJ10/Kir4.1 cause an autosomal recessive renal salt-losing tubulopathy similar to Gitelman syndrome with salt-wasting, activation of the renin-angiotensin-aldosterone system, hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria [2,3]. KCNJ10 is expressed not only in the kidney, but also in brain, inner ear and eye, and mutations in this gene lead to a combination of features we called EAST syndrome: epilepsy, ataxia, sensorineural deafness, and renal tubulopathy .
In mouse kidney, KCNJ10 is expressed in the distal convoluted, connecting tubule, and cortical collecting duct. In some mouse strains and humans there is also expression in the cortical thick ascending limb of the loop of Henle [2,4,5]. When KCNJ10 is expressed in MDCK cells, it is found to localize to the basolateral membrane . It functions either as a homotetramer or possibly as part of a heterotetramer with the related channel KCNJ16 . In the kidney tubule, KCNJ10 is localized to the basolateral membrane , where it enables ‘K+ recycling’, supplying K+ to the extracellular K+ site of the basolateral Na+/K+-ATPase that drives transcellular electrolyte transport. This process has been termed pump-leak coupling [9,10]. KCNJ10 activity is also necessary to maintain the driving force for basolateral Cl– exit by hyperpolarizing the basolateral membrane, and in conjunction with apical ion conductances, maintains a lumen-positive transepithelial potential that may drive para- and transcellular reabsorption of Ca2+ and Mg2+, respectively . Consequently, patients with mutations in this gene and KCNJ10–/– mice display deficits of renal reabsorptive function , even though KCNJ10 is not the only K+ channel expressed in this segment.
KCNJ10 is also expressed in the stria vascularis that winds along the lateral wall of the cochlea of the inner ear, where it functions to secrete K+ into the endolymph . It is instrumental in generating the high ‘endocochlear potential’ of around +100 mV, largely a K+ diffusion potential across the apical membrane of strial intermediate cells, which is essential for hearing .
Brain astroglia and retinal Müller cells, which also express KCNJ10, function to maintain the ionic environment of the extracellular space by providing ‘spatial buffering’ of K+, siphoning K+ from regions of high [K+] o, resulting from neuronal excitation, to places of low [K+] o such as neighboring blood vessels . Some speculate that inwardly-rectifying K+ channels facilitate uptake of K+ into depolarized glial cells as well as its release, though the former is a matter of some debate [15,16].
Apart from hearing impairment due to loss of the endocochlear potential, KCNJ10–/– mice showed marked motor impairment and premature death, a consequence of hypomyelination and degeneration of the spinal cord , and KCNJ10–/– mice showed a loss of the slow PIII response of the light-evoked electroretinogram, which is generated by K+ fluxes in Müller cells also expressing KCNJ10 . In fact, we could demonstrate similar findings in patients with EAST syndrome .
A conditional knockout mouse with specific deletion of KCNJ10 in astroglia caused mice to die prematurely and display severe ataxia and stress-induced seizures . In this model, glial cells were strongly depolarized, supporting the role of KCNJ10 in clearing K+ from the extracellular space.
Mutations previously discovered in the coding region of KCNJ10 include the homozygous missense mutations c.194G>C (p.R65P), c.229G>C (p.G77R), c.418T>C (p.C140R) and c.491C>T (p.T164I) [2,3]. Compound heterozygous missense/nonsense mutations c.194G>C (p.R65P) and c.595C>T (p.R199X) and compound heterozygous missense mutations c.500C>T (p.A167V) and c.889C>T (p.R297C) were found in two other kindreds .
Heterologous expression of all the aforementioned KCNJ10 mutants in Xenopus laevis oocytes had shown highly reduced channel currents in comparison to wild type (WT) [2,21,22]. Further patch-clamp experiments on transfected mammalian cells showed highly reduced currents (R65P and R175Q), or currents abolished to levels indistinguishable from mock-transfected cells (G77R and R199X) . The comparative effects of these KCNJ10 mutations were replicated at the single-channel level, with strongly reduced open probability in R65P and R175Q and almost absent channel activity in G77R . In excised patches, a dramatic shift in pH sensitivity of the mutant channels R65P and R175Q was found, with greater residual channel activity at higher than normal pH. This change in pH sensitivity was thought to cause their decrease in conductance by causing perturbed pH gating . In a study of heterologous expression in oocytes, all mutated channels were found to have less residual conductance when intracellular pH was reduced from 7.4 to 6.8, a change in pH that did not affect WT KCNJ10 . Due to undetectable currents, the truncating mutant R199X could not be investigated in isolation in this study.
Here we describe mutations in further patients clinically diagnosed with EAST syndrome. All mutations identified were heterologously expressed in X. laevis oocytes to study the function of mutant KCNJ10 by two-electrode voltage clamping.
Materials and Methods
Genetic studies were approved by the Institute of Child Health-Great Ormond Street Hospital Research Ethics Committee, after obtaining written consent. The entire single coding exon and adjacent intronic sequence of KCNJ10 was sequenced by the Sanger method using a Beckman Coulter CEQ8000 sequencer. PCR primer sequences can be supplied on request. Ensembl gene transcript ENST00000368089 was used as reference.
All 6 patients described invariably showed epilepsy from infancy, debilitating ataxia from an early age with difficulties in walking, sensorineural deafness and hypokalemic metabolic alkalosis with variable hypomagnesemia. The patient in family 1 is the second male child of consanguineous unaffected Algerian parents. The patients in family 2 are male and female siblings from healthy non-consanguineous Afro-Caribbean parents. Family 3 also had 1 affected male and female sibling from consanguineous unaffected Indian parents. Family 4 has an affected boy from unaffected consanguineous Iranian parents.
WT and mutant KCNJ10 were cloned by ligating a genomic PCR amplificon of the single coding exon into the pTLB oocyte expression vector (a kind gift of T.J. Jentsch) with primers introducing XhoI and XbaI restriction sites, maintaining a Kozak sequence . Cloned plasmids were transformed into XL1 blue bacteria (Stratagene), and DNA was purified using NucleoSpin® columns (Macherey-Nagel, Düren, Germany) following the manufacturer’s protocol. The cDNA inserts of all plasmids were sequence-verified. For N-terminal GFP fusions, inserts were cloned into pEGFP-C1 (Stratagene) and sequence-verified.
Cloned plasmids were linearized by MluI digestion (Fermentas, UK), and cRNA was synthesized using the mMESSAGE mMACHINE® SP6 Kit (Ambion, UK). Adult female X. laevis (University of Portsmouth) were sacrificed under Home Office-licensed Schedule 1 of the Animals (Scientific Procedures) Act 1986, and oocytes were harvested by ovariectomy. Oocytes were incubated in ND96, containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2·2H2O, 1 MgCl2·6H2O, 5 HEPES, pH 7.5 (NaOH). Healthy-looking stage V and VI oocytes  were separated by digestion with collagenase (Worthington, Lakewood, N.J., USA) for injection with 2.5 ng WT or mutant KCNJ10 cRNA and then incubated for 1–2 days at 17°C.
Membrane fractions were extracted from ten of each mutant, WT and control-injected oocytes by trituration and two centrifugation steps at 3,000 g to remove yolk and lipid granules. The intermediate phase was used for denaturation and Western blotting according to standard procedures. Plasmids containing eGFP fused to the N-termini of KCNJ10 WT, R65C, and V259X were transfected into CHO cells using polyethyleneimine and harvested for membrane preparations after 36 h.
Membranes were stained with 0.2% Ponceau’s stain in 5% acetic acid for 5 min, washed and imaged using a Dyversity gel analyzer and Genesnap software (Syngene, UK). The membrane was blocked and incubated with the KCNJ10 monoclonal antibody 1C11 (Sigma, UK) or a monoclonal anti-GFP antibody (Roche UK, a kind gift of Trevor Smart). Bound primary antibodies were detected by a secondary anti-mouse antibody coupled to peroxidase and enhanced chemoluminescence using homemade reagents.
Voltage clamping was performed with a TEC01C amplifier (NPI Electronic GmbH, Tamm, Germany). Oocytes were held in an RC-1Z Oocyte Chamber (Harvard Apparatus, UK). Microelectrodes were pulled from 1.5-mm glass capillaries with filament (Harvard Apparatus, UK) using a DMZ-universal puller (Zeitz, Augsburg, Germany), and had a resistance of ∼1 MΩ when filled with 3 M KCl. Recordings were controlled using Strathclyde electrophysiology software and a PCI-6025 interface (National Instruments), programmed at clamp steps from –100 to +100 mV in 20-mV increments, each lasting 500 ms. The baseline holding voltage was held close to EK+ at –40 mV, with the addition of 20 mM K+ (total 22 mM) to standard ND96 in order to increase specific currents as Kir channel conductance increases with [K+] o [25,26]. Oocytes will be unaffected over several minutes by this slightly higher bath osmolality as they have low diffusional water permeability, owing to little endogenous aquaporin expression . Uninjected and water-injected controls were also measured. 100 µM BaCl2 was used as a reversible specific inhibitor of inwardly-rectifying K+ channels . Recordings were analyzed and plotted using Strathclyde electrophysiology software and Origin (OriginLab, Northampton, USA).
Statistical analysis was performed using Origin. Testing was performed by one- and two-sided Student’s t tests as appropriate for paired and unpaired data and p < 0.05 was considered significant. All data are given in mean values ± SEM.
Four single nucleotide mutations were identified in the coding region of KCNJ10 amongst the EAST patients from four families whose DNA was submitted for this study (fig. 1a). In family 1, a homozygous missense mutation p.R65C (c.193C>T) was identified in 1 patient, and both parents were heterozygous carriers. In family 2, a homozygous missense mutation p.F75L (c.225T>G) was identified in 2 affected siblings. DNA from their parents was not available. In family 3, a single nucleotide deletion was present (c.775delG), causing a frame-shift mutation p.V259fsX259 (shortened to V259X from hereon) with generation of a premature stop codon. Two affected siblings were homozygous for the mutation, and their parents were both heterozygous carriers. In family 4, a homozygous missense mutation p.R297C (c.889C>T) was identified in 1 patient, and both parents were heterozygous carriers.
|Fig. 1.a Sequence chromatograms showing mutation p.R65C (c.193C>T), mutation p.F75L (c.225T>G), mutation p.V259fsX259 (c.775delG), and mutation p.R297C (c.889C>T), all with WT for comparison. b ClustalW protein-alignment (homology) plot of the first transmembrane region and two sections of the carboxy-terminus of KCNJ10 homologues in 12 vertebrate species in the NCBI Protein database, showing conservation of residues R65, F75 and R297, and conserved continuation of the protein after V259, marked with arrows. c Localization of mutations R65C and F75L in the first membrane-spanning domain, and V259X and R297C in the cytoplasmic carboxy-terminus .|
p.R297C was previously identified in an EAST syndrome patient heterozygous for mutations p.R297C and p.A167V , and was never reported before in a homozygous state. All three other mutations have not been reported to our knowledge. None of the four mutations is a known single nucleotide polymorphism (SNP) listed in the NCBI SNP database. All four mutations were located in domains of the KCNJ10 protein highly conserved across species as shown in figure 1b. R65 and F75 both lie within the first transmembrane domain of KCNJ10, and V259 and R297 both lie within the carboxy-terminal region that may be important for multimerization and also contributes to a cytoplasmic extension of the ion pathway (fig. 1c) . Mutation V259X produces a significantly truncated protein. In all species compared, the translated protein is at least 375 amino acids long, and the normal human protein length is 379 amino acids.
Whole-cell currents of voltage-clamped Xenopus oocytes expressing WT KCNJ10 showed strong inward rectification (fig. 2a, c). Mean current when voltage-clamped at –100 mV was –10.0 ± 0.73 µA (n = 36). K+ conductance in all mutant channels was significantly reduced in comparison with WT (p < 0.001) (table 1; fig. 2b, d, e). WT and mutant KCNJ10 were expressed in three batches of oocytes except for V259X which was investigated in one batch.
|Table 1. Voltage-clamped oocyte whole-cell currents expressing WT and mutant KCNJ10 and controls; oocytes are voltage-clamped at –100 mV|
|Fig. 2.a–d Whole-cell currents in oocytes overexpressing WT KCNJ10 showing typical inward rectification. Voltage-clamp steps were from –100 to +100 mV in 20-mV increments (a, c). Whole-cell currents in oocytes overexpressing KCNJ10 F75L showing highly reduced K+ conductance (b, d). e Effects of KCNJ10 mutations on whole-cell currents normalized as percentages of WT currents (significant comparisons with WT are marked by asterisks). f Summary showing the percentage effect of barium inhibition on whole-cell currents (asterisks denote p < 0.05 for Ba2+ inhibition; paired t test).|
Specific reversible inhibition of KCNJ10 conductance was achieved by addition of 100 µM BaCl2, which nearly abolished conductance in WT KCNJ10-expressing oocytes. Barium inhibition determines how much of the membrane current (Im) is due to KCNJ10 channels as opposed to membrane leak or endogenous channels for other ions (e.g. chloride or sodium), confirming that the currents measured are due to inwardly-rectifying potassium channel conductance. Barium had no significant effect on uninjected or water-injected controls. Whole-cell currents of each oocyte exposed to barium were compared to the averaged current from before and after barium application. Barium had a significant inhibiting effect on current carried by three of the KCNJ10 mutants (R65C > F75L & R297C) but had no significant effect on V259X (table 2; fig. 2f). This suggests that there is residual channel function in mutations R65C, F75L and R297C.
|Table 2. Inhibition by 100 µM barium chloride of whole-cell currents of oocytes expressing WT and mutant KCNJ10 and controls, voltage-clamped at –100 mV|
Western blot of the membrane fraction protein extracted from oocytes used in the experiments confirmed expression of KCNJ10 protein in both WT and three mutants (fig. 3a). KCNJ10 V259X cannot be detected by the 1C11 antibody as it recognizes an epitope located in the truncated carboxy-terminal region. N-terminal antibodies were not available for detection of KCNJ10. Higher bands in the KCNJ10 blots are likely due to its strong tendency to form multimers and complexes with glycoproteins . However, N-terminal fusions of GFP to truncated, R65C and WT KCNJ10 detected with an antibody against GFP showed comparable expression levels (fig. 3b).
|Fig. 3.a Western blot showing multiple bands stained for KCNJ10 protein extracted from oocytes expressing WT and mutant KCNJ10-V259X is a truncated protein, which lacks the epitope for the anti-KCNJ10 antibody used. b Western blot of membrane fractions from untransfected (con) and HEK cells expressing GFP-tagged WT and mutant KCNJ10. Detection for GFP shows that GFP-V259X is truncated and therefore runs at a lower molecular weight, but is expressed at comparable level.|
Mutations of the human KCNJ10 gene have been shown to cause EAST syndrome, an autosomal recessive syndrome featuring seizures, sensorineural deafness, ataxia and a salt-losing tubulopathy. Seven different disease-causing KCNJ10 mutations have been previously described in patients with EAST syndrome. These mutations have been recently shown in heterologous expression experiments to have residual function (R65P > R175Q > G77R) [2,22]. R199X, a nonsense mutation, displayed complete loss of function , but had been only identified in a compound heterozygous patient.
In the present study, one mutation (R297C) was seen for the first time in a homozygous state, and three new mutations are described. Heterologous expression of these mutated channels showed that they severely impaired (R65C, F75L, R297C) or nearly abolished (V259X) channel function. Although we observed currents significantly different from uninjected oocytes for V259X, barium inhibition experiments suggested that only three of these mutated channels had residual function (R65C, F75L and R297C), the magnitude of which was dependent on the mutation. Similar to R65P, the new mutation R65C had most residual current of all mutations studied here. KCNJ10 V259X showed no significant evidence of barium inhibition, which is consistent with the findings from mutation R199X, producing a similarly truncated protein. We conclude that leak currents due to injection and/or endogenous currents induced by expression of a non-functional protein (which might not be sensitive to the low concentrations of barium we used) may account for the difference to currents observed in non-injected oocytes. Although unlikely given the position of the mutation, we cannot exclude that KCNJ10 V259X lost barium sensitivity, and the resolution of our technique may exclude the detection of small specific currents. This supports the notion that mutants with very low or absent KCNJ10 function can be compatible with life in man. The expression of other potassium channels, including KCNJ16, in the same tubule segments may explain phenotypic variability within the same families .
The results of this study provide further evidence that mutations in KCNJ10 cause EAST syndrome in various populations, and may be more frequent than previously thought. It should be considered as a differential diagnosis in patients with a Gitelman-like phenotype whenever hearing is impaired and/or other neurological symptoms coexist.
This study will also further improve our understanding of the structure and function at the molecular level of KCNJ10 and other Kir channels. Human KCNJ10 has not been crystallized, but the structure of KirBac1.1  and a chimera between KirBac3.1 and mouse Kir3.1  provide a framework to understand molecular disease mechanisms and will enhance the development of drugs activating partially functional mutants.
It is noteworthy that mutations R65C and F75L are in close proximity to a lysine residue at position 67 that has been shown to determine the characteristic pH sensitivity of Kir channels including KCNJ10 , a critical part of H+-sensing machinery in the first membrane-spanning domain that determines inward rectification and ion selectivity via a proposed intra-subunit interaction that transduces the pH-dependent movement of the cytoplasmic domains into pore closure [34,35]. The dysfunction of KCNJ10 R65P has been shown to be largely due to a shift in pH sensitivity , and F75 may therefore also contribute to pH sensitivity.
For R297C, the shift in the activation curve towards more alkaline pH is thought to be due to disruption of the intersubunit salt bridge Glu288–Arg297 . A mutation in the carboxy-terminus has also been shown to decrease affinity for PIP2 (phosphatidylinositol 4,5-bisphosphate) , a known regulator of channel activity [37,38].
Channel dysfunction could be due to a change in subunit association. However, the pattern of oligomerization reflected by the idiosyncratic appearance of KCNJ10 in Western blots is consistent between the mutants we studied here.
Mutations in KCNJ10 could destabilize both RNA and protein. Patch-clamp experiments performed on mammalian cells had shown that KCNJ10 R65P and mutations at positions distinct from the ones described here trafficked to the cell membrane, as they could be activated by alkaline pH . Western blots performed on oocytes showed comparable expression levels to WT for all new missense mutants. However, the trafficking behavior of mammalian cells may more closely resemble the human in vivo situation than Xenopus oocytes. We therefore performed Western blotting for KCNJ10 fused to GFP and expressed in HEK cells. These experiments showed a similar expression pattern and similar expression levels for KCNJ10 R65C, WT, and V259X, suggesting that these mutations did not affect trafficking or stability. Cell surface biotinylation assays in one recent study indicate significant plasma membrane expression of all mutant KCNJ10 channels analyzed . However, the lack of an intracellular control makes these results difficult to interpret. We were unable to biotinylate KCNJ10 at the cell surface, possibly due to the extremely short extracellular loop. Future studies will have to address whether the loss of function we observed in mutated KCNJ10 is due to reduced surface expression, single-channel conductance or open probability.
In conclusion, we show further diversity in specific mutations affecting KCNJ10 function causing EAST syndrome .
B.F. received support from the UCLH/UCL Comprehensive Biomedical Research Centre and the Bnai Brith Leo Baeck Lodge. D.B. was supported by the Special Trustees of Great Ormond Street Hospital. A.Z. received support from the Peter Samuel Trust Fund and St. Peter’s Trust for Kidney, Bladder & Prostate Research.
Anselm A. Zdebik
University College London
Royal Free Hospital Medical School, 1-702
Rowland Hill Street, London NW3 2PF (UK)
Tel. +44 75 3432 9860, E-Mail email@example.com
Received: April 3, 2011
Accepted: June 17, 2011
Published online: August 18, 2011
Number of Print Pages : 9
Number of Figures : 3, Number of Tables : 2, Number of References : 41
Vol. 119, No. 3, Year 2011 (Cover Date: October 2011)
Journal Editor: Kleta R. (London)
ISSN: 1660-2137 (Print), eISSN: 1660-2137 (Online)
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