The novel C-terminal KCNQ1 mutation M520R alters protein trafficking

doi:10.1016/j.bbrc.2007.04.127

Biochemical and Biophysical Research Communications

Volume 358, Issue 1, 22 June 2007, Pages 304-310

https://doi.org/10.1016/j.bbrc.2007.04.127 Get rights and content

Abstract

The long QT-syndrome is characterized by a prolongation of the QT-interval and tachyarrhythmias causing syncopes and sudden death. We identified the missense mutation M520R in the calmodulin binding domain of the Kv7.1 channel from a German family with long QT-syndrome. Heterologous expression of the mutant did not reveal any whole-cell currents independent of the auxiliary subunit KCNE1. Co-expression of the wild-type Kv7.1 channels and the mutant showed that the mutant did not have a dominant negative effect. In immunocytochemical assays of transfected COS-1 cells wild-type Kv7.1 showed an immunopositive labeling of the plasma membrane. For M520R no plasma membrane staining was visible, instead a strong signal in the ER was observed. These results indicate that the LQT1 mutation M520R leads to ER-retention and dysfunctional trafficking of the mutant channel resulting in haploinsufficiency.

Section snippets

Materials and methods

Molecular biology. For DNA diagnosis, informed consent according to the ethics committee of the University of Münster was obtained from all family members. DNA was isolated from venous EDTA blood and direct sequencing of the complete coding sequences of the LQT1 (KCNQ1) and the LQT2 (KCNH2) genes were performed. The mutation M520R of human Kv7.1 (NM_000218) was constructed using site-directed PCR mutagenesis and subsequently cloned into the pcDNA3 and pGEM-HE vectors for expression in mammalian

Case presentation

A family of 7 living members was referred for clinical and genetic consultation (Fig. 1); the 34-year-old female proband (III-1) was asymptomatic but had a prolongation of the heart-rate corrected QT-interval (QTc: 460–490 ms^1/2). At a heart-rate of 54 bpm, the T-wave was small-based and tall, together with an isoelectric ST-segment. Transthoracic echocardiography revealed a discrete mitral valve prolaps. Other examinations including routine physical examination, exercise ECG, EEG, programmed

Discussion

We identified the novel KCNQ1 M520R mutation that co-segregates in a patient diagnosed with LQT1-syndrome. Analysis of family members showed that the heterozygous mutation carriers are characterized by a mild QTc prolongation, an isoelectric ST-segment together with a heart-rate in the lower normal range and a mitral valve prolaps indicating an intermediate probability for familial LQT-syndrome. Our heterologous ion channel expression data show that homomeric mutant channels failed to conduct

Acknowledgments

E.S.-B was funded by German Research Foundation, Germany (DFG Schu 1082/3-1) and the Ernst und Berta Grimmke-Stiftung, Germany. N.S. and K.C. were supported by The John and Birthe Meyer Foundation, the Velux foundation, and the Danish National Research Foundation, Denmark. N.H.N. received a research fellowship from the Danish Cardiovascular Research Academy (DaCRA).

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Cardiac Delayed Rectifier Potassium Channels in Health and Disease
2016, Cardiac Electrophysiology Clinics
A phosphoinositide 3-Kinase (PI3K)-serum- and glucocorticoid-inducible Kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein
2013, Journal of Biological Chemistry
Citation Excerpt :
In its recessive form, the Jervell and Lange-Nielsen syndrome (13), the disease additionally leads to hearing loss due to disturbances in the flow of potassium in the inner ear. The mechanism underlying the LQT syndrome is reflected in a loss of Kv7.1 function, frequently originating from trafficking disorders, and hence a decrease in number of channels in the plasma membrane (14–16). Nevertheless, the molecular and cellular mechanisms controlling the cell surface expression of Kv7.1 in cardiomyocytes and epithelial cells are still largely unknown.
Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.
Structure of a Ca<sup>2 +</sup>/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M current modulation
2013, Journal of Molecular Biology
Citation Excerpt :
The Kv7.1 mutant M520R51 corresponds to anchor position (14) in the 1–14 motif, Leu540 in Kv7.4 (Fig. 4b and d). In accordance with the buried nature of this position, the M520R change has been reported to disrupt both channel function and CaM binding.51 Two Kv7.2 mutations, R533Q26,52 and K562N,53 correspond to Kv7.4 positions Arg547 and Lys548 (Fig. 4b) that make electrostatic interactions with Ca2 +/N-lobe residues Glu54 and Asp50, respectively (Fig. 4e), and would disrupt these electrostatic contacts.
Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I_KS currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca^2 +/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca^2 +/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca^2 +/CaM:Kv7.4 B segment complex shows that Ca^2 +/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca^2 +/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca^2 +/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.
The rate-dependent biophysical properties of the LQT1 H258R mutant are counteracted by a dominant negative effect on channel trafficking
2010, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
This indicates that the reduced current level is due to an impaired translocation to the plasma membrane of channel complexes carrying a mutant H258R subunit and that the H258R mutation has a dominant negative effect on the trafficking efficiency of the resulting mutant IKs channel complex. Reduced ion channel trafficking by itself can indeed lead to cardiac arrhythmias and is a well-known mechanism for LQT2 [46] but has recently also been described for the LQT1 isoform [47–54]. While the current density at + 60 mV is a convenient measure for functional expression, the cardiac action potential reaches this voltage for less than a millisecond during the initial peak and the subsequent plateau is situated between + 10 mV and − 30/− 40 mV depending on the size of the early repolarization (notch).
The long QT syndrome (LQTS) is a cardiac disorder caused by a prolonged ventricular repolarization. The co-assembly of the pore-forming human KCNQ1 α-subunits with the modulating hKCNE1 β-subunits generates I_Ksin vivo, explaining why mutations in the hKCNQ1 gene underlie the LQT1 form of congenital LQT. Here we describe the functional defects of the LQT1 mutation H258R located in the S4–S5 linker, a segment important for channel gating. Mutant subunits with this arginine substitution generated no or barely detectable currents in a homotetrameric condition, but did generate I_Ks-like currents in association with hKCNE1. Compared to the WT hKCNQ1/hKCNE1 complex, the H258R/hKCNE1 complex displayed accelerated activation kinetics, slowed channel closure and a hyperpolarizing shift of the voltage-dependence of activation, thus predicting an increased K⁺ current. However, current density analysis combined with subcellular localization indicated that the H258R subunit exerted a dominant negative effect on channel trafficking to the plasma membrane. The co-expression hKCNQ1/H258R/hKCNE1, mimicking the heterozygous state of a patient, displayed similar properties. During repetitive stimulation the mutant yielded more current compared to WT at 1 Hz but this effect was counteracted by the trafficking defect at faster frequencies. These rate-dependent effects may be relevant given the larger contribution of I_Ks to the “repolarization reserve” at higher action potential rates. The combination of complex kinetics that counteract the trafficking problem represents a particular mechanism underlying LQT1.
Novel mechanisms of trafficking defect caused by KCNQ1 mutations found in long QT syndrome
2009, Journal of Biological Chemistry
Citation Excerpt :
There are several other mutations not affecting the pore structure but causing functional impairment, most of which were identified in the C-terminal cytoplasmic region. Although the cytoplasmic region contained several functional domains (9–12), molecular mechanisms of KCNQ1 mutations in the cytoplasmic domain have not been fully elucidated. On the other hand, more information is available about the functional alterations caused by mutations in KCNH2 that encodes pore-forming α-subunits of another voltage-gated cardiac potassium channel, HERG.
Long QT syndrome (LQTS) is a hereditary arrhythmia caused by mutations in genes for cardiac ion channels, including a potassium channel, KvLQT1. Inheritance of LQTS is usually autosomal-dominant, but autosomal-recessive inheritance can be observed in patients with LQTS accompanied by hearing loss. In this study, we investigated the functional alterations caused by KCNQ1 mutations, a deletion (delV595) and a frameshift (P631fs/19), which were identified in compound heterozygous state in two patients with autosomal-recessive LQTS not accompanied by hearing loss. Functional analyses showed that both mutations impaired cell surface expression due to trafficking defects. The mutations severely affected outward potassium currents without apparent dominant negative effects. It was found that delV595 impaired subunit binding, whereas P631fs/19 was retained in endoplasmic reticulum due to the newly added 19-amino acid sequence containing two retention motifs (R⁶³³GR and R⁶⁴⁶LR). This is the first report of novel mechanisms for trafficking abnormality of cardiac ion channels, providing us new insights into the molecular mechanisms of LQTS.
Trafficking-deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: Evidence for clinically significant I<inf>Kr</inf>-I<inf>Ks</inf> α-subunit interaction
2009, Heart Rhythm
Citation Excerpt :
Cellular processing and membrane trafficking is also a crucial determinant of KCNQ1 function. A variety of KCNQ1 mutations induce LQTS by preventing effective membrane trafficking.7,18–22 Critical domains for KCNQ1 trafficking have been identified in both the N- and C-terminal regions.18,21
KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I_Ks. Previous work showed a KCNH2 and KCNQ1 α-subunit interaction that increases KCNH2 membrane localization and function.
We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers.
Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed.
KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I_KCNH2 versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 α-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I_KCNH2 upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I_Kr in isolated cardiomyocytes.
The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I_KCNH2, which would be expected to decrease repolarization reserve and synergize with reduced I_KCNQ1 caused directly by the mutation, potentially explaining the malignant clinical phenotype in affected patients.

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¹: These authors joint first authorship.

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The novel C-terminal KCNQ1 mutation M520R alters protein trafficking

Abstract

Section snippets

Materials and methods

Case presentation

Discussion

Acknowledgments

Am. Heart J.

Genet. Med.

J. Biol. Chem.

J. Mol. Cell Cardiol.

J. Biol. Chem.

Biophys. J.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Curr. Opin. Neurobiol.

Heart Rhythm

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Cell Calcium

Cell Calcium

Mutant caveolin-3 induces persistent late sodium current and is associated with long-QT syndrome

Circulation

A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly

EMBO J.

Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias

Nat. Genet.