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Cardiovascular Research 2007 75(4):748-757; doi:10.1016/j.cardiores.2007.05.010
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Copyright © 2007, European Society of Cardiology

Impaired interaction between the slide helix and the C-terminus of Kir2.1: A novel mechanism of Andersen syndrome

Niels Decherb,c,*,1, Vijay Reniguntac,1, Marylou Zuzartec, Malle Soomd, Stefan H. Heinemannd, Katherine W. Timothya, Mark T. Keatinga, Jürgen Dautc, Michael C. Sanguinettib and Igor Splawskia

aChildren's Hospital Department of Cardiology, Harvard Medical School and HHMI, Boston, MA 02115, United States
bNora Eccles Harrison CVRTI and Department of Physiology, University of Utah, Salt Lake City, UT 84112, United States
cInstitute of Physiology and Pathophysiology, University of Marburg, D-35037 Marburg, Germany
dDepartment of Biophysics, Friedrich Schiller University Jena, D-07747 Jena, Germany

* Corresponding author. Philipps-University Marburg, Institute of Physiology and Pathophysiology, Deutschhausstra{alpha}e 1-2, 35037 Marburg, Germany. Tel.: +49 6421 28 62148; fax: +49 6421 28 68960. decher{at}staff.uni-marburg.de

Received 17 March 2007; revised 20 April 2007; accepted 7 May 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary data
 References
 
Objective Andersen syndrome (AS) is a rare genetic disease caused by mutations of the potassium channel Kir2.1 (KCNJ2). We identified two unrelated patients with mutations in the slide helix of Kir2.1 leading to AS. The functional consequences of these two mutations, Y68D and D78Y, were studied and compared with previously reported slide helix mutations.

Methods Channel function and surface expression were studied by voltage clamp recordings and a chemiluminescence assay in Xenopus laevis oocytes and by patch clamp recordings and fluorescence microscopy in HEK293 cells. In addition, a phosphatidylinositol bisphosphate (PIP2) binding assay and a yeast-two-hybrid assay were used to characterize the molecular mechanisms by which slide helix mutations cause AS.

Results Neither mutant channel produced any current, but both had dominant negative effects on Kir2.2, Kir2.3, and Kir2.4 channels. We show that Y68D, D78Y, and previously reported AS mutations are clustered on the hydrophilic, cytosolic side of the slide helix and traffic normally to the plasma membrane. The in vitro lipid binding assay indicated that Y68D or D78Y N-terminal peptides bind PIP2 similar to wild-type peptides. Yeast-two-hybrid assays showed that AS-associated mutations disturb the interaction between the slide helix and the C-terminal domain of the channel protein.

Conclusion Our experiments indicate a new disease-causing mechanism independent of trafficking and PIP2 binding defects. Our findings suggest that the hydrophilic side of the slide helix interacts with a specific domain of the C-terminus facing the membrane. This interaction, which may be required for normal gating both in homomeric and heteromeric Kir2 channels, is disturbed by several mutations causing AS.

KEYWORDS Andersen syndrome; LQT syndrome; Arrhythmia; Potassium channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary data
 References
 
Andersen syndrome (also called Andersen–Tawil syndrome) is a rare genetic disorder characterized by periodic paralysis, cardiac arrhythmia and dysmorphic features [1–3]. In most cases, it is caused by loss-of-function mutations in KCNJ2, the gene encoding the Kir2.1 inward rectifier potassium channel [5–7]. The severity of skeletal muscle, cardiac and developmental phenotypes in AS varies considerably. Developmental problems manifest as low-set ears, short stature, and abnormalities in the limbs such as clinodactyly, syndactyly or brachydactyly [2–4]. Periodic paralysis of AS patients can be associated with hyper-, hypo-, or normokalemia. Ventricular arrhythmias and prolongation of the QT interval are the most common cardiac phenotypes [2,5]. Loss of Kir2.1 function has been attributed to defective trafficking or decreased affinity for PIP2 [8–10].

It has been controversial for many years whether Kir2.x subunits can co-assemble to form heteromeric channels [11–13]. Recently, evidence was provided that heteromerization of mutant Kir2.1 channels with wt Kir2.2 and Kir2.3 can occur and that the differential capability of different Kir2.1 mutants to co-assemble with wt Kir2.1, Kir2.2 or Kir2.3 subunits may contribute to the extraordinary pleiotropy of AS [14]. In addition, functional heteromerization of Kir2.1 and Kir2.4 may occur in various tissues [15], but whether AS-associated mutants of Kir2.1 can also cause dominant-negative suppression of Kir2.1/Kir2.4 heteromultimers has not been demonstrated.

AS point mutations of Kir2.1 are located near the pore helix or scattered throughout the C-terminus of the channel subunit. Several AS mutations have also been found near the cytosolic part of the M1 domain of Kir2.1 in a 10 amino acid region called the slide helix (Fig. 1) [16]. The slide helix was first identified when the structure of the KirBac1.1 channel was solved and was proposed to be involved in gating [16]; however, its precise role in channel function is unknown. Mutation of the slide helix residues R67, D71, T75 were previously shown to cause AS [7,10,17,18]. More recently, several novel slide helix mutations associated with AS were reported, including D78G and T74A [19,20], Y68D [19] and D78Y [21].


Figure 1
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  		Fig. 1 Kir2.1 missense mutations cause Andersen syndrome. AS pedigrees showing occurrence of the disease phenotype and inheritance of the missense mutations Y68D (A) and D78Y (B) Circles and squares indicate females and males, respectively. Filled and empty symbols denote affected and unaffected individuals. Amino acid sequence alignments show conservation of the mutated amino acids in multiple members of the Kir channel family. (C) Location of the mutations Y68D and D78Y on the Kir2.1 homology model based on the crystal structure of KirBac1.1 [16]. (D) Positions of known N-terminal AS mutations [7,17,18,20]. (E) Helical wheel plot of the Kir2.1 slide helix (amino acids 67 to 78). Hydrophilic and hydrophobic residues are shown in black and white, respectively; other residues are shown in grey circles. Asterisks mark amino acids mutated in AS.

 
In the present study we report two further cases of AS with slide helix mutations Y68D and D78Y. We used these mutants, which have not been functionally characterized so far, to study mechanisms by which slide helix mutations cause Andersen syndrome. In addition, we studied the possible heteromerization of these and other Kir2.1 mutants with Kir2.4 subunits. Finally, we examined the effect of several slide helix mutations on the surface expression of Kir2.1 and measured PIP2 binding affinity. We conclude from our experiments that slide helix mutations give rise to a gating defect that is presumably caused by a disrupted interaction of the N- and C-termini of the channels. Our results highlight the importance of the slide helix for Kir2 channel gating.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary data
 References
 
The investigation conforms with the guide for the Care and Use of laboratory Animals (NIH Publication 85-23).

2.1 Subject ascertainment and phenotypic analysis
Informed consent or assent was obtained from all individuals or their guardians according to standards established by local institutional review boards. Phenotypic analyses included history, physical examination, and electrocardiography.

2.2 Molecular biology
QuikChange (Stratagene) was used to introduce mutations into human Kir2.1 (KCNJ2) cDNA. The constructs for oocyte recordings and the chemiluminescence assay were cloned into a pKSII vector and cRNA was prepared with T7 Capscribe (Roche) after linearization with XhoI. For Kir2.x subunit coexpression experiments, all constructs were subcloned into the pSGEM expression vector. Complementary RNA (cRNA) for these constructs was prepared with T7 Capscribe (Roche) after linearization with NheI. A detailed description of the methods used for the lipid binding assay, yeast two-hybrid assay, the genotypic and sequence analyses as well as the chemiluminescence assay is given in the Supplementary Material.

2.3 Expression of Kir channels in oocytes
Ovarian lobes were dissected from mature Xenopus laevis anesthetized with tricaine and treated with collagenase (1 mg/ml, Worthington, type II) in OR2 solution (in mM: NaCl 82.5, KCl 2, MgCl2 1, HEPES 5, pH 7.4) for 120 min. Isolated oocytes were stored at 18 °C in ND96 recording solution (in mM: NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4) plus Na-pyruvate (275 mg/l), theophylline (90 mg/l) and gentamicin (50 mg/l). Stage IV and V oocytes were injected with Kir2.x cRNA (1.25, 2.5 or 5.0 ng). Standard two-microelectrode voltage-clamp experiments were performed at room temperature (21–22 °C) 2–4 days after injection of oocytes with cRNA. Microelectrodes were fabricated from glass pipettes filled with 3-M KCl and had a resistance of 0.2–1.0 M{Omega}. Currents were elicited by 200-ms pulses applied in 10 mV increments to potentials ranging from –140 mV to +30 mV from a holding potential of –80 mV.

2.4 Patch clamp of HEK 293 cells
Human embryonic kidney (HEK) 293 cells were grown and transfected as previously described [22]. Current recordings were conducted in the whole cell configuration at room temperature (22 °C). Pipettes had a tip resistance of 1.5–2.5 M{Omega} when filled with the pipette solution containing (in mM): 140 KCl, 2 MgCl2, 1 EGTA, and 10 HEPES, pH 7.3. Twenty-four to 36 h after transfection, cells were bathed in a solution containing (in mM): 135 NaCl, 5 KCl, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.3. Currents were elicited by 200-ms pulses applied in 10 mV increments to potentials ranging from –120 mV to +30 mV from a holding potential of –80 mV.

2.5 Fluorescence imaging
HEK, COS7 or OK cells were grown to about 50% confluency on 35 mm glass bottom Petri dishes (Wellco) supplemented with DMEM medium containing 10% fetal bovine serum (Invitrogen). Twenty-four hours later, the cells were transfected with EGFP- and/or DsRed-tagged Kir2.1 constructs in pIRES using Fugene 6 (Roche). The cells were maintained at 37 °C for 24–48 h in an incubator supplied with 5% CO2/95% air. Microscopy and imaging were performed with an Olympus IX71 microscope equipped with a 60x N.A. 1.3 PL APO objective or a 100x N.A. 1. 4 PL APO objective (Olympus), standard EGFP/Texas Red filter sets and a cooled 12-bit CCD camera (SensiCam QE). During imaging, the cells were maintained at 37 °C using an objective heater (Bioptechs). Digital images were processed using Image-Pro® Plus 4.5 (MediaCybernetics).

2.6 Data analyses
Results are reported as mean±S.E.M. (n=number of oocytes). Statistical differences between wild-type (WT) and mutant channels were evaluated by Student's unpaired t-test. Significance was assumed for P<0.05, indicated in the figures by an asterisk (*); "NS" indicates non-significant changes.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary data
 References
 
3.1 Identification of Kir2.1 slide helix mutations in Andersen syndrome
Two unrelated probands were diagnosed with AS (Fig. 1). In kindred 1, the proband is a 30-year-old female who presented with premature ventricular contractions (PVCs) and periodic paralysis (PP). Sequencing of KCNJ2 revealed a coding change (T202G) predicted to result in a Tyr to Asp mutation in residue 68 (Y68D). The proband's 29-year-old brother was diagnosed with long QT syndrome (LQT) and periodic paralysis and harbors the same mutation. It was inherited from their mother who was diagnosed with bigeminy (BiG) and long QT syndrome (Fig. 1A). In kindred II, the proband is a 25-year-old female diagnosed with bigeminy, ventricular tachycardia (VT), long QT syndrome, periodic paralysis, and the facial features of AS. We identified a de novo mutation (G232T) predicted to result in substitution of an Asp with a Tyr at residue 78 (D78Y). A paternity test with 6 highly polymorphic markers confirmed that the mutation arose de novo. The proband's Her 2-year-old daughter, who inherited the mutation, has periodic paralysis, facial dysmorphic features (Dys) and a cleft palate (Cleft), but no arrhythmia (Fig. 1B). The probands of both kindreds were also screened for mutations in the known LQT genes (KVLQT1, HERG, SCN5A, KCNE1, KCNE2, and CACNA1C) but no additional mutations were identified. The Y68D and D78Y mutations were not observed in 180 ethnically matched control samples.

Y68 and D78 are located in a N-terminal, {alpha}-helical domain of the Kir2.1 channel subunit called the slide helix. Based on the crystal structure of a related bacterial channel (KirBac1.1) the slide helix is predicted to be connected to the M1 transmembrane domain and oriented parallel to the inner side of the plasma membrane [16] (Fig. 1C). Y68D, D78Y and four other AS-associated mutations (R67W, D71V/N, T74A and T75R/M/A) are located on the same side of the amphipathic slide helix (Fig. 1D). This can be visualized most clearly in a helical wheel plot shown in Fig. 1E. In this plot, hydrophilic residues are shown in black circles, hydrophobic residues in white circles; residues with grey circles do not strictly fall into one of these two categories. Andersen syndrome mutations (marked with an asterisk) are specifically located on the hydrophilic side of the amphipathic slide helix.

3.2 Characterization of WT and mutant Kir2.1 channels in Xenopus oocytes
As previously reported [25,26], Kir2.1 channels expressed in Xenopus oocytes activate rapidly and the current–voltage (I–V) relationship exhibits strong inward rectification. Injection of oocytes with 2.5 ng WT KCNJ2 cRNA induced currents that were approximately twice as large as currents induced by injection of 1.25 ng of cRNA (Fig. 2A), whereas endogenous inward currents recorded from oocytes injected with H2O were very small (Fig. 2B). Y68D and D78Y Kir2.1 channels did not functionally express. Oocytes injected with 2.5 ng of mutant subunit cRNA had currents indistinguishable from H2O-injected oocytes (Fig. 2B, upper panels). Co-expression of WT and mutant Kir2.1 subunits by injection of 1.25 ng of each cRNA reduced current amplitude to less than half of that measured after injection of 1.25 ng WT Kir2.1 cRNA alone (Fig. 2B, lower panels). The I–V relationships for all the experiments described above are plotted in Fig. 2C. Currents activated between –60 and –30 mV are most important for repolarization of cardiac action potentials [27]. The current–voltage relationship for this range of test potentials is shown in Fig. 2D. Obvious outward currents were only detected in oocytes expressing WT Kir2.1 channels. Together, these findings indicate that the Y68D and D78Y mutations caused loss of function and a dominant negative suppression of Kir2.1 channel function.


Figure 2
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  		Fig. 2 Mutations in KCNJ2 cause loss of function and dominant-negative suppression of Kir2 currents in Xenopus oocytes. (A) Representative currents induced by injection of individual oocytes with 1.25 ng or 2.5 ng WT KCNJ2 cRNA (n=12–13). (B) Currents induced by injection of individual oocytes with the indicated amounts of mutant cRNA alone (n=7), WT plus mutant cRNA (n=10–11), or H2O (n=5). In panels (A) and (B), capacitive spikes were truncated to facilitate display. (C) I–V relationships for oocytes injected with the indicated WT or mutant KCNJ2 cRNA. Statistical significance is indicated for comparison between 1.25 ng WT alone and 1.25 ng WT co-expressed with various AS mutations. (D) Expanded view of the current–voltage relationship boxed by dotted lines in panel C. Statistical comparisons were performed as in panel C. (E) Surface expression of Kir2.1 analyzed with a chemiluminescence assay in Xenopus oocytes. 2.5 ng cRNA of WT or mutant subunits with an attached extracellular HA-epitope were injected into Xenopus oocytes. Two days after injection surface expression was analyzed photometrically with a horseradish peroxidase-linked immunoassay. Relative light units from 30–35 oocytes from three independent batches were averaged. The right panel shows the total Kir2.1 protein expressions analyzed by Western blotting. (F) Relative current amplitudes at – 120 mV in oocytes injected with 2.5 ng Kir2.1 cRNA alone or mixed with 2.5 ng of mutant subunit cRNA. The previously described mutations D71V, S136F, N216H and R218Q [5,7,35,36] were also co-expressed for comparison. (G–I) Relative current amplitudes of Kir2.2, Kir2.3 and Kir2.4 expressed alone (2.5 ng cRNA) or co-expressed with mutant Kir2.1 subunits (2.5 ng cRNA). (F–I) Statistical significance is indicated for comparison between WT Kir2.1 alone and WT Kir2.1 co-expressed with various AS mutations.

 
3.3 Cell surface expression of mutant Kir2.1 channels in Xenopus oocytes
A dominant negative suppression of Kir2.x current amplitude by mutant subunits could be caused by either a trafficking or a gating defect. To analyze cell surface expression of WT and mutant Kir2.1 channels in Xenopus oocytes we introduced an extracellular HA-epitope into all the KCNJ2 constructs and assayed surface expression with a single-oocyte chemiluminescence assay [23]. Two days after injection of 2.5 ng WT or mutant channel cRNA, the mutant channels showed a similar chemiluminescence as WT (expressed in relative light units; Fig. 2E). Western blots from Xenopus oocytes indicated a similar level of total protein expression (Fig. 2E, right panel). These data indicate that the loss of function and dominant negative effect of these mutations is caused by a defect in gating of Kir2.1 channels localized in the plasma membrane and not by abnormal channel trafficking.

3.4 Andersen syndrome mutations in Kir2.1 subunits cause a dominant negative suppression of Kir2.4 channels
Next we analyzed whether the Kir2.1 mutations also exhibit dominant negative effects on Kir2.4 channels. Oocytes were injected with 2.5 ng cRNA encoding WT Kir2.1, Kir2.2, Kir2.3 or Kir2.4 subunits either alone or together with equal quantities of cRNA encoding Kir2.1 subunits containing AS mutations. Y68D, D78Y and N216H caused a dominant negative effect on Kir2.2 and Kir2.3 current amplitudes (Figs. 2F–H), similar to the other mutations studied by Preisig-Müller et al. [14]. In addition, we found that AS mutations suppress Kir2.4 currents (Fig. 2I). These findings support a previous report that Kir2.1 channels might form functional heteromers with Kir2.4 [15]. Thus, although Kir2.4 subunits are not expressed in the heart, loss of Kir2.1/Kir2.4 heteromer function might contribute to non-cardiac symptoms (e.g., developmental abnormalities) of AS.

3.5 Characterization of WT and mutant Kir2.1 channels in HEK293 cells
Mutant proteins with deficient trafficking in mammalian cells can sometimes be transported normally to the cell membrane in oocytes or at lower temperatures in mammalian cells (e.g., mutations in CFTR or hERG [28–30]). Therefore, we also expressed the mutant Kir2.1 channels in HEK293 cells cultured at 37 °C. For these experiments we used a bi-cistronic pIRES vector described previously [8]. EGFP-N1-tagged WT Kir2.1 or mutant channels were cloned into the multi-cloning site (MCS) A and co-expressed with a DsRed2-N1-tagged WT Kir2.1 channel cloned into MCS B of the pIRES vector. Cells transfected with the construct containing WT-EGFP/WT-DsRed had an average current amplitude of –3.38±0.34 nA at –120 mV (n=8). When mutant Kir2.1 channels were cloned into MCS A and co-expressed with WT Kir2.1 (cloned into MCS B), currents were suppressed in a dominant negative manner (Fig. 3). Currents recorded after transfection with Y68D-EGFP/WT-DsRed or D78Y-EGFP/WT-DsRed constructs had amplitudes of –0.44±0.15 nA (n=8) and –0.54±0.16 nA (n=7) at –120 mV, respectively (Fig. 3). Currents recorded after transfection of the mutants alone (Y68D-EGFP or D78Y-EGFP) had similar amplitudes as control recordings with the pIRES vector (Fig. 3).


Figure 3
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  		Fig. 3 Dominant-negative effect of the mutants Y68D and D78Y in HEK 293 cells. Depicted are I–V relationships for currents in transfected HEK293 cells. Statistical significance is indicated for comparison between WT-EGFP/WT-DsRed2 and AS-mutants-EGFP/WT-DsRed2.

 
3.6 Mutant Kir2.1 channels reach the plasma membrane of mammalian cells
To confirm the data obtained in Xenopus oocytes which suggested normal trafficking of the mutant Kir2.1 subunits to the plasma membrane, we transfected HEK293 cells with mutant cDNAs cloned into the pEGPF-C1 vector (Clontech). After 24 h, surface protein expression was detected by fluorescence microscopy for WT, Y68D and D78Y Kir2.1 channels (Fig. 4A). In addition, we found that channels with previously described mutations of the slide helix and tagged with EGFP-C1 also reached the plasma membrane (Fig. 4B). In live cell imaging, plasma membrane fluorescence was observed for all these constructs over a time period of 8 to 36 h after transfection (not shown). We also observed plasma membrane fluorescence after transfection of Y68D and D78Y into COS7 and OK cells (not shown), indicating that intracellular transport of these mutant channels is normal in different mammalian cell lines.


Figure 4
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  		Fig. 4 Plasma membrane expression of AS mutations in HEK293 cells. (A) Fluorescence microscopy images taken 24 h after transfection of EGFP alone, WT or mutant (Y68D or D78Y) pEGFP-C1 constructs into HEK293 cells. Inset shows the phase contrast image of the cell. A close-up of the plasma membrane (dashed box) is shown underneath each panel. (B) Fluorescence images of cells transfected with AS-associated slide helix mutations R67W, D71N, D71V and T75R.

 
3.7 Co-localization of mutant and WT Kir2.1 channels in the plasma membrane
For these experiments we used the pIRES constructs described above. In Fig. 5, EGFP and DsRed fluorescence are depicted in green and magenta, respectively, so that co-localization merges to white (to assist color blind). Twenty-four hours after transfection of HEK293 cells, WT subunits and Y68D (Fig. 5A) or D78Y subunits (Fig. 5B) co-localized in the plasma membrane. Thus, Y68D and D78Y mutant channels appear to co-assemble with WT Kir2.1 channels and traffic to the plasma membrane of mammalian cells where they exert a dominant negative effect on the channel tetramer.


Figure 5
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  		Fig. 5 Co-localization of WT and mutant Kir2.1 proteins in the plasma membrane of HEK293 cells. Fluorescence images of cells transfected with the mutants (A) Y68D or (B) D78Y. HEK293 cells were transfected with pIRES constructs containing mutant-EGFP in the first and WT-DsRed in the second multiple cloning site. The EGFP signal is depicted in green and DsRed in magenta, so that co-localization merges to white. Close-up of the plasma membrane (dashed box) are shown on the right.

 
3.8 Slide helix mutations Y68D and D78Y do not impair PIP2 binding
We then addressed the question whether either mutation has a reduced PIP2 binding affinity. We performed a lipid binding assay using Kir2.1 GST-fusion proteins and fluorescently labelled liposomes with different concentrations of incorporated PI(4,5)P2, as previously described [24]. The Kir2.1 GST-fusion proteins included the N-terminus from amino acids 33 to 83. This shortened fragment was chosen because the first 32 amino acids or the amino acids after position 83 strongly reduced protein solubility. Fig. 6 illustrates the PIP2 binding affinity, expressed as relative arbitrary units, of the GST-protein alone or the N-terminal Kir2.1-fusion proteins for 5 mol% and 10 mol% PI(4,5)P2. Using 10 mol% PI(4,5)P2 no changes in affinity were observed for the D78Y and Y68D mutants, while with 5 mol% the fluorescence signal of the Y68D mutant was reduced by 23%. This minor reduction in PIP2 binding affinity for the Y68D mutation might be caused by impairment of electrostatic PIP2 interaction of the neighboring residue R67, a known PIP2 binding site [10]. Thus, there is a minor or no reduction in PIP2 affinity for Y68D and D78Y Kir2.1 peptides, respectively.


Figure 6
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  		Fig. 6 In vitro PIP2 binding assay of N-terminal Kir2.1 fragments. Average binding data of GST-alone and GST-Kir2.1-fusion proteins (amino acids 33–82) of WT and mutant channel subunits. Relative arbitrary units of PIP2 binding are shown for liposomes containing 5 mol% and 10 mol% PI(4,5)P2. Kir2.1 GST fusion proteins had significantly increased PIP2 binding compared to GST-alone (not indicated). PIP2 binding of GST-alone was not significantly different for 5 mol% and 10 mol% PIP2 (not indicated).

 
3.9 Disrupted interaction between the slide helix and the C-termini of Kir2.1 in Andersen syndrome
A recent publication showed that some carboxy terminal AS mutations affect the structure and the proper assembly of the cytoplasmic domains of Kir2.1 [31]. Analogously, slide helix mutations in AS might affect Kir2.1 channel function by disturbing a possible interaction between N- and C-terminal domains. Most AS mutations are located on the interface between the C-termini (e.g., E303K, R312C) or on the part of the C-terminus that faces the plasma membrane (Fig. 7A). The latter mutations and AS-associated mutations on the slide helix are localized on a potential interaction face between the N- and C-termini (Fig. 7B). Utilizing a yeast two hybrid direct interaction assay we tested for an interaction of the N-terminus of human Kir2.1 with its own N- or C-termini (Fig. 8). In this assay the N-terminus of the channel interacted with the C-terminus, but not with the N-terminus. The interaction between the N- and the C-terminus was disrupted after introducing the AS mutation Y68D, D71V or D78Y (Fig. 8). Similar results were obtained for N-terminal constructs harbouring both AS mutations (Y68D and D78Y) and a construct that lacked the entire slide helix (data not shown). The Andersen mutation R218W in the C-terminus facing the plasma membrane also disrupted the interaction of the N-terminus with the C-terminus (Fig. 8). The double mutant G300V/V302M (two AS mutations) located on the same interface also prevented interaction of the two domains (Fig. 8). These results suggest that the interaction of Kir2.1 N- and C-termini might be important for channel gating and might depend on an intact slide helix.


Figure 7
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  		Fig. 7 Localization of AS mutations in the slide helix and in the C-terminus. (A) Structure of the C-terminal domains of human Kir2.1 as viewed from the plasma membrane. Residues mutated in AS [5,7,10,37] are labeled and shown in space-fill. (B) Side view of two Kir2.1 subunits (KirBac1.1, upper structures) and four C-termini (hKir2.1, lower structures), illustrating how AS-associated mutations in the slide helix (labeled) and in the upper face of the C-termini might be oriented towards one another.

 

Figure 8
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  		Fig. 8 Andersen syndrome mutations in the slide helix and C-terminus prevent interaction of Kir2.1 N- and C-termini. Yeast-two-hybrid assay of the interaction between the amino (Kir2.1 N) and carboxy terminus (Kir2.1 C) of Kir2.1. –LW, spotted on selective medium lacking leucine and tryptophan, indicating transformation of both vectors; –LWH, spotted on selective medium lacking leucine, tryptophan, and histidine, indicating protein–protein interaction). Protein interactions are marked with a + sign.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary data
 References
 
In 60–70% of the cases, Andersen syndrome is caused by mutations in KCNJ2 [5], but the mechanisms causing the pleiotropic phenotype of the disease might be quite diverse. Of the disease-causing KCNJ2 mutations known so far, some have been reported to cause trafficking defects [8], whereas others have been reported to cause gating defects mediated by loss of PIP2 interaction [9,10]. Furthermore, it has been shown that heteromerization of Kir2.1 channels with Kir2.2, Kir2.3 and Kir2.4 can occur [14,15] and that the differential capability of different Kir2.1 mutants to co-assemble with wt Kir2.1, Kir2.2 or Kir2.3 subunits may contribute to the pleiotropy of AS. We have confirmed and extended these findings by demonstrating that the Andersen mutants Y68D, D78Y and N216H have a dominant-negative effect not only on Kir2.2 and Kir2.3 but also on Kir2.4.

Here we report a novel disease-causing mechanism for Andersen syndrome: disruption of the interaction between the slide helix and the C-terminus of Kir2.1. The two mutants Y68D and D78Y, whose disease causing mechanism has been studied for the first time here, did not produce any functional currents in heterologous expression systems. The dominant-negative effect of the mutants expressed in Xenopus oocytes suggests that the assembly of the channels was not disturbed by the mutations. Furthermore, our lipid binding assay suggests that the interaction of the N-terminus of Kir2.1 with PIP2 was not impaired by the mutations. Our fluorescence imaging studies showed that the trafficking of the channels to the surface membrane was also unaffected by the mutations. Our homology modeling suggested that the two mutations Y68D and D78Y are localized to the hydrophilic side of the slide helix facing the C-terminal domain of the channel, which forms the cytosolic pore. Our yeast-two hybrid direct-interaction assay showed that the N-terminus of human Kir2.1 interacts with the C-terminus of Kir2.1, and that this interaction was disrupted after introducing the AS mutations Y68D or D78Y (or both). Taken together, these findings suggest that the interaction between the N-terminal slide helix and the C-terminus plays an important role in the gating of Kir2.1 and that the gating is disrupted by the two mutations studied here.

Having arrived at this conclusion we tested whether the functional effects of other known Andersen mutants can be explained by the same hypothesis. Indeed, we found that all N-terminal AS mutations reported so far are exclusively located on the hydrophilic side of the slide helix, while the remaining N-terminus was never affected. In addition, many C-terminal AS-associated mutations are localized at a potential interaction face with the slide helix (Fig. 7). Therefore we speculate that disruption of the interaction between N- and C-terminal domains of Kir2.1 may be a general mechanism underlying Andersen syndrome and that the associated gating defects are caused by disturbances of the intramolecular conformation. In agreement with this hypothesis we found that four additional mutations localized at the putative interface between N- and C-terminus (D71V, R218W, G300V/V302M) also disrupted the interaction between the two domains in our yeast-two-hybrid assay.

Can we extrapolate the hypothesis that the interaction between the N- and C-terminal domains is important for channel gating to other channels of the Kir family? Interestingly, slide helix mutations in Kir1.1 at positions equivalent to T75 and D78 were reported to cause Bartter syndrome [32,33]. Furthermore, disease-causing slide helix mutations also cluster along the slide helix of Kir6.2 channels [38]. Thus, the mechanism described here could be of more general significance. Additionally, amino acids positioned at the interface between the C-termini might interact with one another, and this may also be of functional importance for heteromerization and gating of Kir2.1 in AS [31]. These considerations suggest that the phenotype of different AS mutations may be attributable to a wide variety of mechanisms which are not mutually exclusive: (i) a dominant negative effect of mutant Kir2.1 channels on Kir2.2, Kir2.3 and Kir2.4; (ii) impaired trafficking of mutant homomeric Kir2.1 or heteromeric Kir2.1/Kir2.x channels; (iii) deficiency in PIP2 binding, (iv) impaired interaction of the C-termini and finally, as shown here, (v) a gating defect caused by the impaired interaction between the slide helix and the C-terminus. Interaction between slide helix and C-terminus may be a conserved mechanism that is relevant for the gating of other inward rectifier channels as well, for example Kir1.1 and Kir6.2.

In conclusion, we have shown that mutation of residues in the hydrophilic side of the slide helix can cause Andersen syndrome without disrupting the trafficking of Kir2.1 or the binding of PIP2. Our findings suggest that the observed gating defect of Y68D, D78Y, and perhaps many other AS-associated mutations, may be due to the disruption of an interaction between the slide helix and C-terminus of Kir2.1.


   Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.05.010.

Time for primary review 23 days


   Acknowledgements
 
The authors thank R. Klinger and C. Kirsch for help with the lipid binding assay and Susanne Rinné for help with the chemiluminescence assay. We are grateful to Said Bendahhou for the gift of pIRES constructs. This work was supported by NIH (NHLBI) grant HL046401 to MTK and MCS, by the DFG grant SFB593 TPA4 to JD and by the P.E. Kempkes Stiftung 09/05 to ND.


   Notes
 
1 These authors contributed equally. Back


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

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