The N-Terminal Juxtamembranous Domain of KCNQ1 Is Critical for Channel Surface Expression
Implications in the Romano-Ward LQT1 Syndrome
N-terminal mutations in the KCNQ1 channel are frequently linked to fatal arrhythmias in newborn children and adolescents but the cellular mechanisms involved in this dramatic issue remain, however, to be discovered. Here, we analyzed the trafficking of a series of N-terminal truncation mutants and identified a critical trafficking motif of KCNQ1. This determinant is located in the juxtamembranous region preceding the first transmembrane domain of the protein. Three mutations (Y111C, L114P and P117L) implicated in inherited Romano-Ward LQT1 syndrome, are embedded within this domain. Reexpression studies in both COS-7 cells and cardiomyocytes showed that the mutant proteins fail to exit the endoplasmic reticulum. KCNQ1 subunits harboring Y111C or L114P exert a dominant negative effect on the wild-type KCNQ1 subunit by preventing plasma membrane trafficking of heteromultimeric channels. The P117L mutation had a less pronounced effect on the trafficking of heteromultimeric channels but altered the kinetics of the current. Furthermore, we showed that the trafficking determinant in KCNQ1 is structurally and functionally conserved in other KCNQ channels and constitutes a critical trafficking determinant of the KCNQ channel family. Computed structural predictions correlated the potential structural changes introduced by the mutations with impaired protein trafficking. In conclusion, our studies unveiled a new role of the N-terminus of KCNQ channels in their trafficking and its implication in severe forms of LQT1 syndrome.
The long QT syndrome (LQTS) is a cause of sudden cardiac death and is characterized by an increased QT interval on patients’ ECG. The prolongation of the QT interval predisposes patients to cardiac arrhythmias known as torsades de pointes, eventually leading to ventricular fibrillation. Over the last decade, inherited mutations in genes encoding cardiac ionic channels or associated partners have been identified in 4 different congenital LQT syndromes (LQT1–4). For example, mutations in KCNQ1 have been associated with either autosomal dominant Romano-Ward (RW) or recessive Jervell and Lange-Nielsen (JLN) LQT1 syndromes.1–3
KCNQ1 encodes the pore forming α-subunit of a voltage gated K+ channel, which associates with an accessory subunit, KCNE1, in the heart to form channels responsible for the slow component of the delayed repolarizing K+ current (IKs).4,5 KCNQ1 mutations associated with LQT1 alter channel function through different mechanisms. Carboxy terminal mutations often disrupt channel assembly, alter regulatory subunit association and cause mistrafficking.6–9 Mutations in the pore region and transmembrane domains (TMDs), on the other hand, produce dominant negative effects on potassium permeation and KCNQ1 channel gating functions.10–12
Although more than a hundred different LQT1-causing mutations have been reported in KCNQ1, only a few are located in its cytoplasmic N-terminus.2,3 In general, these mutations cause a severe clinical phenotype, and have an early age of onset. In addition to frameshift and missense mutations leading to premature stop codons and thus, nonfunctional truncated proteins, LQT1-associated mutations appear to be clustered in 2 hotspots. One cluster is located in the middle of the N-terminus where both amino acid deletion (Δ71-AAP-73) and substitution (P73T) have been reported.3,13 The second hotspot involves the juxtamenbranous region just preceding the first TMD in which Y111C, L114P, P117L and, more recently, E115G and C122 Y substitutions have been identified.2,3,14,15
These observations and the fact that a truncated KCNQ1 splice variant (isoform 2), missing the 128 initial residues of the full length KCNQ1 (isoform 1), is not efficiently expressed on the cell surface, prompted us to investigate the role of KCNQ1 N-terminus in the trafficking of the channel.16
Here, using N-terminal serial deletions, we identified a region immediately preceding the first transmembrane domain (TMD) of the protein, which is critical for KCNQ1 trafficking. This region includes the juxtamembranous LQT1 hotspot, harboring particularly severe RW mutations. We found that these mutations cause channels to be retained in the endoplasmic reticulum (ER). Furthermore, using homology sequence analysis and mutagenesis approaches, we showed that the KCNQ1 trafficking determinant is structurally and functionally highly conserved in other KCNQ channels and thus constitutes a hallmark of the KCNQ potassium channel family. Finally, models of the KCNQ structure provide a mechanism to explain how the disease-causing mutations impair protein trafficking.
Materials and Methods
The P117L mutation has been identified as a de novo mutation in a child who died of Sudden Infant Death Syndrome.14 The Y111C mutation has been found in a 36-year-old woman with a QTc of 520 ms.2 She has had more than 30 episodes of seizures starting at 3 years of age, all precipitated by fear, panic, or physical activities. The L114P mutation has been discovered in a 7-year-old child who experienced multiple syncopes during physical training.15
Complementary DNA Constructs and Mutagenesis
Classical polymerase chain reaction based methods, described in the online data supplement available at http://circres.ahajournals.org, were used to produce the constructs used in this study: hemaglutinin (HA)-tagged KCNQ1 (HA-KCNQ1, a gift of Dr Barhanin, Valbonne, France) containing 2 HA epitopes between S3 and S4; HA-KCNQ2 and HA-KCNQ3 containing 1 HA tag in the first extracellular loop (a gift of Prof Jentsch, Hamburg, Germany)17; a VSV tagged KCNE1-KCNQ1 fusion protein (a gift of Dr Kass, NY).10,18
Primary Culture of Neonatal Mouse Cardiomyocytes
Experimental details for primary culture, transfection, and immunolabeling protocols of neonatal mouse cardiomyocytes are given in the online data supplement available at http://circres.ahajournals.org.
Cell surface detection of HA-KCNQ1 and VSV-KCNE1-KCNQ1 channels was performed on non fixed cells using mouse anti-HA (1:500 dilution, 12CA5 purified ascites, a gift of Dr LeMaout, CEA-Saclay, France) or anti-VSV antibody (1:500 dilution, Sigma-Aldrich). The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. A goat anti-KCNQ1 antibody (1:500 dilution, Santa Cruz, Calif) was used to reveal intracellular localization. In ER colocalization experiments, a CD4 construct (0.5 μg) containing the canonical carboxy-terminal di-lysine ER retention motif (CD4-KKXX) was cotransfected with KCNQ1 constructs and used to label the ER using anti-CD4 antibody (Sim2.1 ascites, 1:1000).19 After incubation with the appropriate Alexa-488 and Alexa-568 conjugated secondary antibodies (1:1000 dilution, Molecular Probes) the cells were observed using a Zeiss Axiovert microscope equipped for epifluorescence or a Leica LSM confocal microscope.
Western Blots and Immuno-Precipitation
For experimental details see the online data supplement available at http://circres.ahajournals.org. The blots were probed with anti-KCNQ1 (1:1000, Santa Cruz) or rabbit anti-HA (1:500, Santa Cruz) and revealed using an enhanced chemi-luminescence kit (NEN). Plasmalemal HA-KCNQ2 was immunoprecipitated using mouse anti-HA antibody (1:500, 12CA5 ascites) and detected by Western blotting using rabbit anti-HA antibody.
Patch Clamp Analysis
COS-7 cells were transfected with a nontagged wild-type (WT) or mutant KCNQ1 (400 ng) plus KCNE1 (800 ng) and GFP expression vector (800 ng) to visualize transfected cells. To analyze the dominant negative effect of RW mutants, the transfection mixture contained 200 ng of WT plus 200 ng of mutant-KCNQ1 complementary DNAs (cDNAs). In the latter experiments, 200 ng of WT KCNQ1 alone was used as a control. Whole-cell currents were recorded 1 or 2 days after transfection as described in the online data supplement available at http://circres.ahajournals.org. The data are presented as means ± SEM and were analyzed using a standard Student t test. KCNQ2 and KCNQ3 currents were measured in the same conditions in cells transfected with KCNQ2, KCNQ3 or L85P-KCNQ2 (1.2 μg) and GFP (0.8 μg). Heteromeric KCNQ2/3 currents were measured in cells transfected with KCNQ3 (0.6 μg) and KCNQ2 or L85P-KCNQ2 (0.6 μg).
The HA Tags Do Not Impede Channel Trafficking
Two HA tags were introduced in the S3-S4 extracellular loop of KCNQ1 isoform 1 and isoform 2 proteins to facilitate the analysis of their plasma membrane expression. These tags enabled us to detect KCNQ1 channels specifically localized at the surface of nonpermeabilized cells by immunochemistry. As illustrated in Figure 1A, the anti-HA antibody labeled HA-KCNQ1 isoform 1 present in the plasma membranes of non permeabilized CHO cells. The intracellular pools of channel proteins were revealed after cell permeabilization. The colocalization of the 2 labelings at the cell surface appears in yellow in the merge image (Figure 1A). In contrast, HA-KCNQ1 isoform 2, which lacks the initial 128 N-terminal amino acids, was not detected at the cell surface but was localized in intracellular compartments (Figure 1A). Double immunostaining performed on transiently transfected COS-7 cells showed that KCNQ1 isoform 2 remained trapped in the ER together with the cotransfected CD4-KKXX construct containing a di-lysine ER retention/retrieval motif (Figure 1B). In immunoblots, the KCNQ1-HA isoform 1 was specifically detected as a single band with the expected apparent molecular weight (75 kDa). The KCNQ1 isoform 2 appeared as a major band with the expected molecular weight (50 kDa) and 2 smaller, less intense bands that most probably correspond to degradation products (Figure 1C).
Together, these data showed that the extracellular tags do not interfere with KCNQ1 posttranslational processing, assembly and trafficking. In addition, the impaired membrane expression of the KCNQ1 isoform 2 confirmed the critical role of the N-terminal segment in KCNQ1 trafficking.
The Juxtamembranous N-terminal Sequence is Required for KCNQ1 Membrane Expression
Interestingly, sequence analysis of the KCNQ1 N-terminus revealed the presence of transport signals involved in the trafficking of other membrane proteins. In particular, KCNQ1 harbors arginine (12-RKR-14, 24-RR-25, 97-RR-98), leucine (38-LEL-40), and tyrosine (51-YAPI-54, 111-YNFL-114) based motifs together with a proline rich domain (51-P//P-89) (supplementary Table I in the online data supplement available at http://circres.ahajournals.org) that have been shown to be involved in the trafficking of other channel proteins.20–22 To investigate their role in KCNQ1 trafficking, serial deletions were made in its N-terminus and the expression of the mutant channels analyzed in stably transfected CHO cells (supplementary Table I). As illustrated in Figure 2A for Δ14, Δ50, Δ99 and Δ114-KCNQ1, Western blot analysis showed that the mutant channels were expressed at similar levels and exhibited the expected reduction of their apparent molecular weight. Immuno-histochemical analysis revealed that the deletions up to residue V106 (Δ14, Δ50, Δ84, Δ94, Δ99, and Δ106-KCNQ1; Figure 2B) did not alter channel cell surface expression. The mutant proteins were readily detected by the extracellular anti-HA antibody (green labeling in Figure 2B) and formed functional potassium channels, as illustrated for Δ106-KCNQ1 in Figure 3C. Importantly, deletion of the 114 N-terminal residues (Δ114-KCNQ1) suppressed KCNQ1 plasma membrane expression in stably transfected CHO cells (Figure 3A). Like KCNQ1 Iso2, Δ114-KCNQ1 colocalized with the ER marker CD4-KKXX and failed to induce detectable potassium currents in transiently transfected COS-7 cells (Figure 3B and C, respectively).
These data show that the initial 106 amino acids of KCNQ1 do not play a significant role in its transport to the plasma membrane. Conversely, the region immediately preceding the first transmembrane segment of the protein defines a critical trafficking determinant of the channel.
Romano-Ward LQT Syndrome Mutations Involve the Trafficking Determinant of KCNQ1
Previous reports linked the autosomal dominant RW form of the LQT1 syndrome to point mutations in the N-terminal trafficking determinant of KCNQ1 that we characterized. Y111C,2 L114P15 and P117L14 substitutions have been identified in patients diagnosed with LQT1. To explore whether the pathological mechanisms involve defects in KCNQ1 trafficking, we introduced these mutations in HA-tagged KCNQ1. Immunofluorescence experiments revealed impaired plasma membrane trafficking of Y111C, L114P, and P117L mutants regardless of expression system or culture conditions. Indeed, as illustrated in Figure 4, no surface expression was detected in either stably transfected CHO cells (Figure 4A) or transiently transfected COS-7 cells. In the latter, we showed that the mutants remained trapped in the ER together with the ER marker CD4-KKXX (eg, KCNQ1 Y111C, Figure 4A) and that culturing the cells at 26°C did not reverse ER retention (data not shown).
The ancillary subunit KCNE1 did not improve mutant channel expression. None of the mutants coexpressed with KCNE1 produced measurable potassium currents (Figure 4B). To test whether this reflected a deficient association of KCNE1 and KCNQ1 subunits, we analyzed the effect of the RW mutations on the trafficking of the KCNE1-KCNQ1 fusion protein in which the 2 subunits are concatenated. As illustrated in figure 4C, the WT VSV-tagged KCNE1-KCNQ1 fusion protein, but not the fusion-proteins containing Y111C, L114P and P117L mutations, was readily detected with extracellular anti-VSV antibody. These data demonstrate that the presence of KCNE1 is not sufficient to restore KCNQ1 trafficking to the cell surface.
Romano-Ward LQT1 Syndrome Mutations Affect KCNQ1 Trafficking in Cardiomyocytes
Next, we analyzed whether Y111C, L114P, and P117L mutations also affect KCNQ1 trafficking in cardiomyocytes. To this end, neonatal mouse cardiomyocytes in primary culture were transfected with WT or mutant VSV-KCNE1-KCNQ1 fusion proteins and cell surface expression was analyzed by immuno-fluorescence 2 days after transfection. As illustrated in Figure 5A, WT-KCNE1-KCNQ1 (green labeling) was readily detected at the surface of alpha actinin-positive (blue labeling) cardiomyocytes. On the other hand, none of the mutants were detected at the cardiomyocyte surface, even though they were efficiently synthesized as revealed by intracellular anti-KCNQ1 staining (red labeling, Figure 5A). As illustrated in Figure 5B for the Y111C mutant, the channel protein remained trapped in the ER together with the cotransfected CD4-KKXX construct.
Romano-Ward Mutations Exert Dominant Negative Effects on WT-KCNQ1
Inherited cardiac arrhythmias in patients with heterozygous RW mutations result from the strong dominant-negative effect of the mutated KCNQ1 subunit when it associates with the WT subunit produced from the unaffected allele. We tested this issue in patch-clamp experiments, using either independent or concatenated KCNE1 and KCNQ1 subunits. Whatever the system used, the 3 mutants exerted a dominant negative effect on WT-KCNQ1 (illustrated in Figure 6A and B for independent subunits). In both cases, the dominant negative effect was, however, less pronounced for L117P. Indeed, the latter needed twice the amount of plasmid in the transfection mixture to induce significant current reduction (Figure 6B and supplementary Table II in the online data supplement). In these conditions, the half activation potential (V1/2) was shifted toward positive potentials and the deactivation constant was significantly reduced (supplementary Table II).
Next, we investigated whether the mutated subunits were transported to the cell surface when coexpressed with the WT channel. We took advantage of the tagged fusion protein to analyze this possibility. Immunochemical data corroborated patch-clamp analysis. VSV-KCNE1-KCNQ1-Y111C and L114P mutants were never detected at the cell surface (data not shown). Conversely, VSV-KCNE1-KCNQ1-P117L was transported to the cell surface when cotransfected with an equal amount of WT plasmid cDNA (Figure 6C) but plasmalemal expression decreased with increasing amount of P117L. The mutant subunit was undetectable at the cell surface using 1:3 cDNA ratio (Figure 6C).
The Juxtamembranous Domain of KCNQ1 Does Not Encode ER Export or Retention-Like Motifs
Together, our data on both engineered deletion mutants and RW mutations demonstrated the critical role of the region preceding the first TMD of the protein in its cell surface expression. We next analyzed whether it encodes ER retention or export signals as described for several membrane proteins, including channels.23 In fact, the efficient membrane expression of Δ106-KCNQ1 and the ER retention of Δ114-KCNQ1 suggest that similar signals may determine KCNQ1 trafficking (Figure 3). Likewise, residues 106 to 114 may constitute an export signal, the deletion of which results in ER localization of Δ114-KCNQ1. Alternatively, residues 114 and beyond may contain a retention motif which, when exposed after the deletion of the initial 114 residues, becomes active to retain the channel in the ER. To test the former hypothesis, we inserted residues 106 to 114 (QGRVYNFL) in the N-terminus (between proline P84 and valine V85) of the transport deficient mutant KCNQ1-Y111C. A control construct was made by inserting the sequence in the same position in WT-KCNQ1. This manipulation failed to restore cell surface expression of the Y111C mutant neither did it alter the surface expression of WT-KCNQ1 in transiently transfected COS-7 cells. These observations are consistent with the idea that residues 106 to 114 do not encode an ER export signal (Figure 7). To test the second hypothesis, we fused residues 115 to 121 (ERPTGWK) at the N terminus of WT-KCNQ1. As illustrated in Figure 7, this did not alter channel cell surface expression, indicating that this amino acid stretch does contain a strong ER retention signal.
The KCNQ1 Trafficking Determinant is Conserved in the KCNQ Channel Family
Sequence analysis of the KCNQ channel family revealed that the ER trafficking domain we defined in KCNQ1 is highly conserved in other family members (KCNQ2–5, Figure 8A). To determine whether this high sequence homology underlies a conserved trafficking function, we analyzed the cell surface expression of KCNQ2 using extracellularly tagged HA-KCNQ2. As previously reported for Xenopus oocytes,17 HA-KCNQ2 was efficiently expressed at the cell surface of COS-7 cells and readily immunoprecipitated using extracellular anti-HA antibody (Figure 8B). To investigate the potential role of the trafficking determinant in KCNQ2 surface expression, we introduced in KCNQ2 the leucine to proline substitution (KCNQ2-L85P) to mimic the critical L114P mutation defined in KCNQ1. Cell surface immunoprecipitation and patch clamp experiments showed that KCNQ2-L85P failed to traffic to the plasma membrane (Figure 8B. lane 3) and to form functional channels (Figure 8C). Tail current densities measured at −40 mV were 12.9±3.3 pA/pF for KCNQ2 and below the limit of detection for KCNQ2-L85P (Figure 8C) and KCNQ3. Furthermore, KCNQ2-L85P substitution abolished the more than 15-fold potentiation of heteromeric currents observed on KCNQ2 and KCNQ3 coexpression (Figure 8C) (208±13 pA/pF n=6 and 2.5±1.2 pA/pF n=7 for KCNQ2/Q3 and KCNQ2-L85P/Q3 heteromers, respectively). It is worth noting that the low KCNQ3 current we observed is consistent with previous studies linking low KCNQ3 channel conductance to key residues in the pore region despite the presence of the same native trafficking determinant.24
Together, these data show that the trafficking determinant we defined in KCNQ1 also governs the efficient trafficking of other KCNQ channel family members.
In the present study, we identified a critical trafficking determinant in the KCNQ1 channel. Significantly, the structure is the target of several LQT1 associated mutations. The N-terminal location of the trafficking domain was unexpected because previous studies highlighted the C-terminal domain as the key determinant of subunits assembly and processing in the secretory pathway.7,8,25 Indeed, we showed that the deletion of the initial 114 residues, but not the 106 initial residues, abolished plasma membrane expression of the channel. This suggested that residues 106 to 114 may constitute an ER export signal or that the amino acid sequence beyond L114 encodes a retention motif. Studies designed to test these ideas (Figure 7) revealed that these discrete structures do not function as independent, autologous trafficking signals. Indeed, they suggest that the structural integrity of the entire region preceding the first transmembrane domain is essential for proper trafficking of the protein.
Interestingly, this region harbors 3 mutations (Y111C, L114P, and P117L) identified in patients diagnosed with RW LQTS. We found that these mutations determine ER retention of homomeric KCNQ1 in COS-7 cells and cardiomyocytes in primary culture. Furthermore, we showed that the three mutants exert a strong dominant negative effect on WT-KCNQ1. Most likely, assembled WT and mutant subunits are recognized by the ER quality control and retained until they undergo ER associated degradation (ERAD). Thus, in addition to its well-recognized involvement in inherited LQT2 and LQT3 cardiac syndromes associated to HERG and SCN5A mutations respectively, ER retention may also constitute the major cellular mechanism in the LQT1 syndrome.26
It is worth noting that even though the 3 mutants exhibited impaired trafficking when expressed as homomers, only Y111C and L114P remained trapped in the ER when cotransfected with WT- KCNQ1. Conversely, the P117L mutant was transported to the cell surface and altered both the V1/2 and the recovery from inactivation of heteromeric channels. In cardiomyocytes, these modifications of KCNQ1 channel functions would result in a decreased IKs current and are thus consistent with the phenotype observed in patients. Furthermore, increased expression of mutant subunits (up to 1:3 ratio of WT/P117L, Figure 6C) would, in addition, impair trafficking and plasma membrane expression.
To gain insight into the structural basis for the trafficking function, we performed structure prediction analysis (supplementary Figure I). The analysis predicted that this region consists in a short helix (V106-L114) followed by a loop (115-ERPTG-119) and the helical structure corresponding to the first TMD. The short initial helix (V106-L114) is lying at the membrane-cytosol interface and this conformation is stabilized by a cluster of aromatic residues involving Y111 and H126 in the first TMD.27 The adverse consequences of the 3 RW mutations on the predicted structures were evaluated. The Y111C mutation was deleterious because a cystein in position 111 failed to interact with the aromatic cluster and thus to stabilize the structure. Substitution of L114 by proline decreased the helix capacity of the fragment and ultimately hinders the interactions between Y111 and H126. In contrast to L114P, the P117L substitution increased the length of the initial helix, reduced the propensity of the 115-ERPTG-119 region to form a loop and hindered the formation of the cluster of aromatic interactions. However, G119 may still confer sufficient flexibility to the helix to bend on the membrane and allow Y111 to participate in the native interactions. Thus, one can speculate that, in the context of a multimeric complex, WT subunits could help the P117L mutant subunits to adopt the native conformation. Such complementation may rescue channel trafficking to the cell surface as previously described for ΔF508 CFTR.28 Increasing the number of mutant subunits may, however, hinder the complementation process and impair the trafficking of the channel.
What could be the impact of these mutations to trigger its recognition and retention by ER quality control? We speculate that this segment is important in channel membrane topogenesis. Its position, just preceding the first TMD, is consistent with this hypothesis. Indeed, previous studies highlighted the role of the segments flanking the TMDs in their insertion and their orientation in the lipid bilayer.29–31 Also, it is worth noting that Sato et al established the critical signal anchor function of the first TMD in a Shaker related K+ channel.32 Thus structural hindrance in this region of the protein may have a critical effect on channel folding and processing in the secretory pathway. Our analysis of other KCNQ family members also supports this idea. Indeed, structure analysis predicts that the same structural model holds for KCNQ2–5 which all harbor the key residues in similar positions. Furthermore, we showed that KCNQ2-L85P substitution, which mimics the KCNQ1-L114P mutation, also have a dominant negative defect on KCNQ2 and KCNQ2-KCNQ3 channel trafficking.
In conclusion, we have identified a critical trafficking determinant in the N-terminal domain of the KCNQ channel family. We showed that inherited KCNQ1 mutations implicated in the cardiac LQT1 syndrome potentially affect the structure of this critical region. Furthermore, even though the intimate molecular mechanisms involved remain to be unveiled, we showed that the mutations determine the ER retention of the channel proteins. Correction of the trafficking defect by chemical and molecular chaperones might open new therapeutic avenues to treat life-threatening KCNQ1 associated cardiac arrhythmias.
We thank Dr Igor Splawski and Dr Roselie Jongbloed for sharing their information concerning Y111C and L114P carriers. We thank Dr Blanche Schwappach for the gift of the CD4-KKXX construct. We also acknowledge the technical assistance of Sylvie Le Roux.
Sources of Funding
A.T. is a recipient of a tenure position at the Institut National de la Santé et de la Recherche Médicale (INSERM); P.J., I.B., and J.M. are recipients of tenure positions at the Centre National de la Recherche Scientifique (CNRS).
This work was supported by grants from Vaincre la Mucoviscidose (VLM) and the Agence Nationale de la Recherche (ANR/A05045GS).
Original received August 26, 2005; resubmission received March 30, 2006; revision resubmission received October 2, 2006; accepted October 5, 2006.
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