Temperature and pharmacological rescue of a folding-defective, dominantl-negative KV7.2 mutation associated with neonatal seizures

Authors

  • Snezana Maljevic,

    1. Neurological Clinic and Institute of Applied Physiology, University of Ulm, Germany
    2. Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University Hospital Tübingen, Germany
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    • Contributed equally and are listed in alphabetical order.

  • Georgios Naros,

    1. Neurological Clinic and Institute of Applied Physiology, University of Ulm, Germany
    2. Department of Neurosurgery, University Hospital Tübingen, Germany
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    • Contributed equally and are listed in alphabetical order.

  • Özlem Yalçin,

    1. Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey
    2. Institute of Human Genetics, Ludwig-Maximilians-University of Munich School of Medicine, Munich, Germany
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  • Dragica Blazevic,

    1. Neurological Clinic and Institute of Applied Physiology, University of Ulm, Germany
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  • Heidi Loeffler,

    1. Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University Hospital Tübingen, Germany
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  • Hande Çağlayan,

    1. Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey
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  • Ortrud K. Steinlein,

    1. Institute of Human Genetics, Ludwig-Maximilians-University of Munich School of Medicine, Munich, Germany
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  • Holger Lerche

    Corresponding author
    1. Neurological Clinic and Institute of Applied Physiology, University of Ulm, Germany
    2. Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University Hospital Tübingen, Germany
    • Department of Neurology and Epileptology, Hertie-Institute for Clinical Brain Research, University Hospital Tübingen, D-72076 Tübingen, Germany.
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  • Communicated by Mireille Claustres

Abstract

Benign familial neonatal seizures (BFNS) are a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels KV7.2 and KV7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of KV7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-of-function with a dominant-negative effect of the mutant on WT KV7.2 and KV7.3 channels. Since single-channel recordings of KV7.3–KV7.2 and KV7.3–N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells—either at lower temperatures of <30°C or in presence of the agonist retigabine (RTG)—the N258S-derived currents increased fivefold in contrast to the WT. The obtained results represent a first example of temperature and pharmacological rescue of a KV7 mutation and suggest a folding and trafficking deficiency as the cause of reduced current amplitudes with a dominant-negative effect of N258S mutant proteins. ©2011 Wiley-Liss, Inc.

Introduction

The syndrome of benign familial neonatal seizures (BFNS) typically manifests with seizures in the first days of life, which cease spontaneously after weeks to months. It is caused by mutations in the KCNQ2 (MIM# 602235) and KCNQ3 (MIM# 602232) genes, which result in a loss-of-function of the neuronal voltage-gated potassium channels KV7.2 and KV7.3 [Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998; reviewed in Maljevic et al., 2008]. These two potassium channel subunits can assemble in homo- or heteromeric potassium channels, which open at subthreshold potentials giving rise to a slowly activating, noninactivating current, first described as the M-current, due to its suppression by muscarinic receptor agonists [Brown and Adams, 1980]. The M-current regulates neuronal excitability by stabilizing the membrane potential and impeding repetitive spike firing of neurons in response to persistent depolarizing inputs [Brown and Adams, 1980; Delmas and Brown, 2005].

A loss-of-function by a haploinsufficiency of the respective channel gene is believed to be the major pathomechanism underlying BFNS [Jentsch, 2000; Maljevic et al., 2008; Steinlein, 2004]. This is supported by the fact that the majority of so far examined mutations lead to a dramatic reduction of K currents carried by homomeric mutant channels, but causes only a small (20–30%) reduction in the maximal current when mutant and wild type (WT) KV7.2 are coexpressed with KV7.3 channels in a 1:1:2 ratio, hence mimicking the expected circumstances in a heterozygous patient. Thus far, only three KCNQ2 mutations have been reported to impair also the function of WT channels by a dominant-negative effect: two of them are localized in the S4 segment and are associated with peripheral nerve hyperexcitability [Dedek et al., 2001; Wuttke et al., 2007], the third one is a C-terminal KV7.2 mutation [Singh et al., 2003]. Most of the so far identified missense mutations in BFNS affect the KV7.2 channel protein and are dispersed within its S4 segment, pore region, or the C-terminus. Several insertions or deletions resulting in a channel truncation, sometimes associated with an incorrect elongation, have been reported. BFNS-associated mutations in KV7.3 have so far only been found in the pore region [summarized in Maljevic et al., 2008].

Here, we studied the KV7.2 mutation p.N258S found in a Turkish BFNS family [Yalçin et al., 2007], which represents the first missense mutation within the extracellular loop connecting S5 with the pore region (Fig. 1A). Using different expression systems and experimental procedures, we demonstrate a loss-of-function with reduced surface expression and a pronounced dominant-negative effect of the N258S mutation on WT channels. Furthermore, we show that these effects can be best explained by protein misfolding of the mutant channel, since channel function could be rescued by coexpression with KV7.3 and by culturing cells transfected with mutant channels at lower temperatures or in the presence of the KV7-binding drug retigabine (RTG).

Figure 1.

Predicted position of the N258S mutation within the KV7.2 channel. A: Schematic presentation of the KV7.2 and KV7.3 channel proteins, comprising six transmembrane segments and intracellular N and C-termini. S4 segment serves as a voltage sensor and pore is formed by S5–S6 regions of the four subunits assembling into a functional channel. Positions of so far reported BFNS mutations affecting KV7.2 and KV7.3 are presented. The N258S resides in the extracellular linker connecting S5 segment and a pore loop of KV7.2 channel. $, splice variant; X, stop codon; +, insertion; —, deletion; Modified from Maljevic et al., 2008; Currents of KV7.2 wild type, N258S, respective coexpressions with KV7.3 (KV7.2 + KV7.3, N258S + KV7.3) in CHO cells were recorded 40–50 hr after transfection using the whole-cell patch clamp technique. All parameters are shown as means ± SEM in Table 1. B: Families of representative raw current traces for KV7.2 and N258S recorded during voltage steps ranging from −80 mV to +60 mV. C: Mean current densities of KV7.2, N258S, KV7.2+KV7.3, N258S + KV7.3 shown as the ratio between the current amplitude at + 20mV and cell capacitance. D: N258S exerted a dominant-negative effect on KV7.2 channels when a constant amount of KV7.2 cDNA was cotransfected with N258S cDNA in a 1:1 ratio (KV7.2, 237 ± 42 pA/pF vs. KV7.2 + N258S, 100 ± 24 pA/pF vs. N258S, 19 ± 4 pA/pF, n = 11–14). E: Conductance–voltage relationships of KV7.2, N258S, KV7.2 + KV7.3, and N258S + KV7.3. Lines represent fits to the sum of two Boltzmann functions with parameters given in Table 1. F: Kinetics of activation. A second-order exponential was fit to the activation time course of KV7.2, KV7.2 + KV7.3, and N258S + KV7.3. Shown is the faster time constant, which had relative amplitude of 40–60%. Statistically significant differences are indicated by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001. G and H: Representative single-channel recordings of concatemeric KV7.3–KV7.2 and KV7.3–N258S channels at 0 mV (top). The resting potential of the cells was set to zero using a high potassium bathing solution. Single-channel amplitude plotted against patch membrane potential: the slope conductances (mean ± SEM) were 8.9 ± 0.3 pS (n = 8) for concatemeric KV7.3–KV7.2 channels and 8.6 ± 0.2 pS (n = 7) for KV7.3–N258S (G). Open probabilities (PO) measured at different potentials for WT and mutant dimers (H).

Materials and Methods

Mutagenesis

KV7.2 and KV7.3 cDNAs inserted into the pTLN vector were kindly provided by Prof. Thomas Jentsch. The missense mutation N258S was inserted into KV7.2 using the Quick Change mutagenesis kit (Stratagene, La Jolla, CA). For the expression in mammalian cells, WT and mutant cDNAs were subcloned into the pcDNA3.1hygro vector (Invitrogen, Carlsbad, CA). Using an overlap PCR strategy, we inserted a FLAG-tag (DYKDDDDK) into the S1–S2 loop of the WT and mutant channel and created concatemeric fusion proteins of human KV7.3 and WT or mutant KV7.2. The HA-tagged KV7.2 construct [Schwake et al., 2000] was kindly provided by Dr. M. Schwake. To link the C-terminus of KV7.3 to the N-terminus of KV7.2, we removed the stop codon in KV7.3 and inserted a short amino acid sequence containing a His tag and the FCYENE motif representing an endoplasmatic reticulum (ER)-export signal [Ma et al., 2001].

Oocyte Preparation and Cell Culture

The use of animals and all experimental procedures were approved by local authorities (Regierungspraesidium Tuebingen, Tuebingen, Germany). Preparation and injection of oocytes were essentially performed as described previously [Maljevic et al., 2003] and are presented as Supporting Information.

Chinese hamster ovary (CHO) cells were grown in F12 medium (Biochrom) supplemented with 10% fetal calf serum in a humidified incubator at 37°C (5% CO2) and passaged every 2 days. For experiments, we plated cells on 35-mm dishes and transfected them using Lipofectamine (Invitrogen, Carlsbad, CA). Cotransfection of cDNA encoding CD8 receptor was used to sort out transfected cells using anti-CD8 antibody-coated microbeads (Invitrogen). KV7.2 and KV7.3 were coexpressed in a 1:1 ratio with the same amount of total DNA as in homomeric experiments. To test for a dominant-negative effect, mutant and WT KV7.2 encoding cDNAs were cotransfected in a 1:1 ratio, with the amount of the WT cDNA being the same as for homomeric expressions. Experiments were performed 40–50 hr after transfection.

Whole-Cell Recordings

All recordings of potassium currents in transfected CHO cells were performed at room temperature (20–22°C) using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). For whole-cell recordings, signals were filtered at 1–2 and sampled at 4–5 kHz. Data were acquired using pClamp 8.2 software (Axon Instruments). The patch pipettes were pulled from thin wall borosilicate glass pipettes with filament (Science Products, Hofheim, Germany) and had a resistance of 1–2 MΩ when filled with the following intracellular solution (in mM): 130 KCl, 1 MgCl2, 5 K2ATP, 5 EGTA, and 10 HEPES, adjusted with KOH to pH 7.4. The external solution contained (in mM): 140 NaCl, 4 KCl, 1.8 CaCl2, 1.2 MgCl2, 11 glucose, and 5.5 HEPES, adjusted with NaOH to pH 7.4 [Peretz et al., 2005]. Leakage and capacitive currents were subtracted using a −P/4 protocol, and series resistance was compensated (approx. 90%) and monitored regularly.

Cell-Attached Single-Channel Recordings

Single-channel activity was recorded 40–50 hr after transfection in the cell-attached configuration, sampled at 5 and low-pass filtered at 1 kHz. Pipettes had resistances of 8–12 MΩ and were filled with the following external solution (in mM): 150 NaCl, 5 KCl, 1 MgCl2, and 10 HEPES (pH 7.4 was adjusted with NaOH). The bath solution contained (in mM): 175 KCl, 4 MgCl2, and 10 HEPES, pH 7.4 adjusted with KOH. The resting membrane voltage was assumed to be 0 mV under these conditions [Li et al., 2005].

Description of electrophysiologiocal data analysis, immunocytochemistry, and western blot analysis of total and membrane protein is provided in Supporting Information.

Results

The N258S Mutation Diminishes K+ Currents in Different Expression Systems

We performed a detailed functional analysis in CHO cells using single-channel and whole-cell patch clamping. Whole-cell currents of KV7.2 WT, N258S, and their respective coexpressions with KV7.3 were recorded 40–50 hr after transfection. Cells were held at −80 mV and depolarized in 10-mV steps up to +60 mV followed by a 500-ms pulse to −120 mV. N258S produced very small currents (Fig. 1B and C) and upon coexpression with KV7.3 the amplitude of these currents was increased by 17-fold, indicating the formation of a heteromeric functional pore with KV7.3. N258S/KV7.3 had similar gating properties as KV7.2/KV7.3 WT currents, but their amplitude was reduced by almost 50% (Fig. 1C). When the usual amount of KV7.2 cDNA was co-transfected in a 1:1 ratio with additional N258S cDNA (n = 14), we observed a reduction of the current amplitude to about 50% indicating the dominant-negative effect on KV7.2 WT channels, since a slight increase instead of a reduction in current size would have been expected without an interaction between the two subunits (Fig. 1D).

Similar effects on current amplitudes were also observed in Xenopus laevis oocytes (Supp. Fig. S1). Coexpression of N258S and KV7.2 with KV7.3 channels in a 1:1:2 ratio, mimicking the presumed situation in a BFNS patient, reduced current by almost 50% instead of expected 25% shown previously for other mutations [Schroeder et al., 1998; Singh et al., 2003], confirming a dominant-negative effect of the N258S mutation (Supp. Fig. S1A). Additional coexpression experiments by adding mutant to a constant amount of WT KV7.2 cRNA in a 1:1 or a 1:2 ratio (thus yielding the double or triple total amount of cRNA compared to the other experiments) reduced current amplitudes by 60% or 80%, respectively (Supp. Fig. S1B).

There were no significant changes between the WT and N258S conductance–voltage relationships, but as previously reported [Selyanko et al., 2000], coexpression of both clones with KV7.3 in CHO cells led to a slight hyperpolarizing shift of the activation curves (Fig. 1E, Table 1). However, the activation time course was significantly slowed for N258S/KV7.3 coexpression compared to KV7.2/KV7.3 or homomeric KV7.2 channels (Fig. 1F, Table 1). The deactivation time constant was not significantly changed (Table 1). Currents of homomeric N258S channels were too small for a reliable evaluation of gating kinetics.

Table 1. Gating Parameters for Mutant and WT Channels Obtained from Whole and Single Channel Recordings
 Current densitiesG-V parametersTime course parametersSingle channel parameters
 I (pA/pF)NV11/2 (mV)k1V21/2 (mV)k2nτact fasta (ms)τact slowa (ms)τdeactb (ms)nC (pS)POn
  1. aat 0 mV.

  2. bat −110 mV.

KV7.2237 ± 4212−25 ± 2−6.2 ± 0.33 ± 3−11 ± 1994 ± 12340 ± 3320 ± 167.3 ± 0.30.36 ± 0.095
N258S19 ± 411−24 ± 4−6.2 ± 0.615 ± 7−13 ± 26       
KV7.2 + KV7.3642 ± 9314−33 ± 1−5.5 ± 0.3−3 ± 4−12 ± 28104 ± 5316 ± 24340 ± 3368.0 ± 0.40.38 ± 0.144
N258S + KV7.3328 ± 7312−29 ± 1−6.3 ± 0.31 ± 3−11 ± 25130 ± 17500 ± 1421 ± 458 ± 10.52 ± 0.023
KV7.3–KV7.2457 ± 487−28 ± 1−5.8 ± 0.33 ± 2−14 ± 17134 ± 11493 ± 3823 ± 278.9 ± 0.30.56 ± 0.078
KV7.3–N258S172 ± 3312−26 ± 1−5.3 ± 0.48 ± 4−14 ± 19139 ± 11516 ± 4823 ± 358.6 ± 0.20.49 ± 0.087
KV7.2 (29°C)164 ± 258−34 ± 1−6.6 ± 0.51 ± 5−13 ± 15       
KV7.2 (XE991)188 ± 704−31 ± 2−6.3 ± 0.15 ± 12−16 ± 33       
KV7.2 (RTG)210 ± 687−30 ± 3−6 ± 19 ± 5−16 ± 25       
N258S (29°C)75 ± 1411−26 ± 2−7.0 ± 0.45 ± 3−8 ± 26       
N258S (XE991)19 ± 23            
N258S (RTG)70 ± 1410−29 ± 2−5 ± 1−2 ± 5−11 ± 27       

Single-Channel Recordings in CHO Cells

The N258S mutation resides in the pore region, which motivated us to investigate whether its single-channel properties could account for the observed current reduction. Since coexpression of two subunits assembling into heterotetrameric channels can result in 16 different channel configurations [MacKinnon, 1991], we created concatemers comprising one KV7.3 and one KV7.2 channel subunit (KV7.3–KV7.2), so that all tetramers should be composed of two KV7.2 and two KV7.3 subunits. These WT dimers yielded functional channels (Supp. Fig. S2) with similar gating properties in whole-cell experiments compared to the coexpression of both WT subunits, although with a slight reduction in current amplitude (Supp. Fig. S2) and a slowing of the activation time course (Table 1). Similar to coexpression experiments, mutant KV7.3–N258S concatemers revealed a reduction in current amplitude compared to the respective WT concatemers (Supp. Fig. S2B).

Single-channel recordings in the cell-attached mode were performed in CHO cells expressing WT or mutant dimers (Fig. 1G and H). Their analysis revealed similar slope conductances for concatemeric KV7.3–KV7.2 and KV7.3–N258S channels (WT: 8.9 ± 0.3 pS, mutant: 8.6 ± 0.2 pS, n = 8) (Fig. 1G), and also similar open probabilities (0.6 ± 0.1 vs. 0.5 ± 0.1, n = 8) (Fig. 1H). Thus, the whole-cell and single-channel data recorded from concatemeric channels ruled out the possibility that altered single-channel properties were responsible for the reduction of current amplitudes observed for channels comprising the N258S mutation. Therefore, we expected that mutant channels are improperly folded and that a reduced number reaches the surface membrane, which was evaluated by further experiments.

The N258S Mutation Shows Reduced Surface Expression

We analyzed the surface expression in the nonpermeabilized Xenopus oocytes expressing extracellularly HA-tagged KV7.2 channels [Schwake et al. 2000] and observed a clear expression of the KV7.2 WT in the surface membrane, which was not found for the N258S mutation (Fig. 2A). Western blot analysis using an anti-KV7.2 antibody [Maljevic et al., 2007] showed the expected 92 kDa band of the same intensity for both WT and N258S expressed in Xenopus oocytes (data not shown) and in CHO cells (Fig. 2B) suggesting that protein production and stability were not affected by the N258S mutation in neither of the expression systems. However, in membrane protein fraction from CHO cells the amount of detected mutant protein was reduced by about 50% compared to the WT, which was also found in a biotinylation assay of surface protein (Fig. 2B). This clearly reduced surface expression however could not fully explain the even larger reduction in current amplitudes. Channel trafficking was further examined by immunostainings of transfected CHO cells. Although we were able to detect abundant expression of homomeric channels in the endoplasmatic reticulum using the anti-KV7.2 antibody (Fig. 2C), we did not observe WT or mutant channels in the surface membrane, probably due to a limited number of homomeric channels reaching the surface. The concomitant expression with KV7.3 revealed a detectable signal for both KV7.2 WT and mutant channels in the plasma membrane, but the differences between the two fluorescent signals were indiscernible. Using an antibody directed against a FLAG sequence inserted in the extracellular KV7.2 S1–S2 loop, we stained nonpermeabilized transfected CHO cells and observed a patchy surface expression pattern, which was stronger for WT than for mutant channels (Fig. 2D), whereas the signal in permealized cells resembled the one obtained with the anti-KV7.2 antibody (Supp. Fig. S3).

Figure 2.

Surface expression of WT and mutant channels. A: Nonpermeabilized oocytes expressing KV7.2 WT or N258S, containing an HA-tag in the S1–S2 extracellular loop, were stained using an anti-HA antibody. Pronounced staining of the surface membrane was observed only for the WT channels. Scale bar 100 µm. B: Western blot analysis of CHO cells transfected with KV7.2 or N258S, using an anti-KV7.2 antibody revealed similar bands of the expected size (92 kDa) for both WT and mutant in total cell lysates (left). In the membrane protein fraction, the amount of mutant channel protein is reduced by about 50% (middle left), which was not observed when the cells were preincubated at 29°C (middle right). A biotinylation assay showed a reduction of mutant protein in the surface membrane by about 40% compared to the WT (right). Shown are representative Western blots and relative expression levels pooled from three independent experiments as mean ± SD; *p < 0.01. C: CHO cells expressing KV7.2 or N258S or their respective coexpressions with KV7.3 were stained using the anti-KV7.2 antibody: surface membrane staining was observed only for coexpressions with KV7.3. D: WT and mutant channel containing a FLAG-tag in the S1–S2 extracellular loop were expressed in CHO cells and stained without permeabilization using an anti-FLAG antibody. The same procedure was used to stain CHO cells transfected with the mutant channel and incubated at <30°C to produce a temperature rescue (lower right). Scale bars 10 µm; NT, nontransfected CHO cells (C and D).

A Trafficking Defect of the N258S Mutation Revealed by Temperature and Pharmacological Rescue

Different studies previously reported a rescuing effect on surface expression of trafficking-deficient channels by a lowered incubation temperature, channel-interacting drugs, or modulating proteins [Gong et al. 2004; Rusconi et al., 2007, 2009; Zhou et al., 1999]. To strengthen the evidence for a trafficking defect of the N258S mutation, cells expressing KV7.2 WT or N258S channels were incubated at <30°C for 24 hr prior to whole-cell recordings. We observed a fivefold increase of N258S-derived current density but no significant changes for the WT (Fig. 3A, Supp. Fig. S4). Interestingly, in membrane protein fractions obtained from CHO cells preincubated at <30°C, the level of expression of WT and mutant channels was similar (Fig. 2B). Moreover, in stainings of nonpermeabilized CHO cells using the anti-FLAG antibody, we could detect increased surface expression for the N258S (Fig. 2D). We next tried to provoke a pharmacological rescue by a 24-hour preincubation with RTG (500 µM), a drug that enhances channel activity by binding in the pore region of neuronal KV7 channels [Lange et al., 2009; Schenzer et al., 2005; Wuttke et al., 2005]. N258S-derived current density was again increased by fivefold without a significant change for the WT (Fig. 3C, Supp. Fig. S4). To rule out a direct channel activation by RTG, extensive washing out for 1 hr prior to recordings was performed and complete washout confirmed by the lack of a hyperpolarizing shift of the activation curve as a proof of RTG action [Rundfeldt and Netzer, 2000], which was still inducible by instantaneous re-application of 10 µM RTG (Fig. 3D). In contrast to RTG, incubating with the specific channel blocker XE991 did not change current amplitudes (Fig. 3B).

Figure 3.

Temperature and pharmacological rescue of N258S channels in CHO cells. A: The mean current density at +20 mV (mean ± SEM) for N258S channels was fivefold increased after incubation at 29°C for 24-hr prior to whole-cell recordings, but no significant (n.s.) change of wild type (KV7.2[29°C]) mean current density has been observed. B: N258S-mediated currents after incubation with 100 µM of XE991 24 hr prior to recordings were hardly measurable, and there was also no significant change of wild type (KV7.2[XE991]) mean current density. C: After 24-hr incubation with 500 µM Retigabine (RTG) followed by a 1-hr washout prior to recordings N258S currents (N258S[RTG)]) were fivefold increased, whereas no significant change of wild type (KV7.2[RTG]) mean current density was found. D: There was no change in the conductance–voltage relationship when RTG was washed out 1 hr prior to measurements (N258S [RTG]). However, an instantaneous application of 10 µM RTG during measurements induced a large hyperpolarizing shift of the mutation's activation curve. All the parameters are shown as mean ± SEM in Table 1. Statistically significant differences are indicated by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001.

Discussion

The N258S is the first missense mutation found in the S5–H5 linker of KV7.2 and its functional effects clearly confirm a KV7.2/7.3 loss-of-function as the pathophysiological concept of BFNS. Gating was only slightly affected by this mutation revealing a slowing of the activation time course, which may provide a minor contribution to the decreased seizure threshold of patients carrying this mutation. On the biophysical level, this could be explained by a structural rearrangement of the pore region, since this channel part contains the activation gate [Yellen, 2002]. On the other hand, single-channel conductance and open probability were not affected by the mutation, confirming that a defect of channel permeability or gating of mutant heteromeric channels was not the reason for the largely diminished potassium currents. Western blots suggested unaffected protein stability without degradation of mutant channels, indicating that improper folding and trafficking are the crucial pathomechanisms responsible for the reduced current amplitudes.

Using a specific antibody against an external HA-epitope, we showed a lack of membrane staining in oocytes injected with only mutant cRNA, whereas the surface membrane of those injected with KV7.2 WT cRNA was nicely labeled. In CHO cells however, both WT and mutant homomeric channels were widely distributed within the endoplasmatic reticulum but could not be detected reliably in the surface membrane using an anti-KV7.2 antibody, whereas using the same antibody upon coexpression with KV7.3 revealed nice membrane staining, though without a clear difference between mutant and WT channels. Stainings with an anti-flag antibody in nonpermeabilized cells suggested a difference between WT and mutant channels, but the results were heterogeneous and could not be well quantified. Obviously, there was a low efficiency to detect homomers in the surface membrane using immunofluorescence despite robust currents in electrophysiological experiments.

We therefore used immunoblotting for a semiquantitative analysis of mutant and WT channels in the surface membrane, revealing a reduction of about 40% for mutant compared to WT protein. Nevertheless, this significant difference alone cannot account for the almost complete loss of currents observed for mutant homomeric channels. Hence, we suggest that a misfolding of those mutant proteins not only affects trafficking to the surface but also their functionality, meaning that only a minority of mutant channels reaching the surface mediates the recorded potassium currents.

To substantiate our hypothesis about misfolding of the KV7.2 channel harboring the N258S mutation, we performed pharmacological and temperature rescue experiments. A similar rescue of misfolded, trafficking-defective hERG channel mutations causing long QT syndrome [Gong et al. 2004, 2006; Zhou et al., 1999], or two NaV1.1 mutations associated with epilepsy [Rusconi et al., 2007, 2009] have previously been demonstrated. Such rescuing procedures have been discussed to stabilize folding intermediates or more general channel integrity [Robertson and January, 2006]. The incubation of N258S-expressing cells at lower temperatures or with RTG, a specific opener of neuronal KV7 channels, both provided a rescue of functional channels. In contrast to RTG, which has been suggested to bind near the internal pore vestibule [Lange et al., 2009; Schenzer et al., 2005; Wuttke et al., 2005], the KV7 channel blocker XE991, supposed to bind extracellularly [Costa and Brown, 1997; Lamas et al., 1997], caused no such effect. These observations suggest (1) that a structural rearrangement within the pore region is the reason for the loss-of-function of N258S mutant channels that can be retrieved by RTG binding, incubation at lower temperature or KV7.3 coexpression and (2) that stabilization of the pore region is generally a crucial step for rescue by interacting subunits (KV7.3) or drugs. The differential effect of the two KV7.2 binding drugs rules out the possibility of an unspecific pharmacological rescuing effect of RTG. Interestingly, the temperature rescue does not seem to be an intrinsic KV7.2 channel property in this case, since experiments in Xenopus oocytes, which were commonly incubated at lower temperatures (16°C), failed to increase the number of functional channels. A different cellular milieu, including presence of particular chaperones, obviously plays an important role in this process.

It has been proposed that N-glycosylation may affect the channel stability and promote trafficking of voltage-gated potassium channels to the surface [Watanabe et al., 2004]. In KV7.1, for example, a robust increase of current amplitudes seems to be due to N-glycosylation of an asparagine in the S5–H loop [Schenzer et al., 2005]. However, this asparagine is not conserved in the sequence of the KV7.2 channel and the mutated N at position 258 is not predicted to be a part of the consensus sequence for N-glycosylation (http://www.cbs.dtu.dk/services/NetNGlyc/).

Improper folding could also explain the dominant-negative effect on KV7.2 WT and heteromeric KV7.2/KV7.3 channels that we observed in both expression systems. Since the tetramerization with WT subunits is not severely disrupted and we observed a clear interaction with KV7.3, association of mutant with WT subunits could lead to improperly folded heteromers that would either be partially retained in intracellular compartments, thereby reducing the surface expression of WT subunits or could be nonfunctional despite reaching the plasma membrane. Both mechanisms could cause the dominant negative effect.

Carriers of the N258S mutation showed typical brief seizures in the first days after birth without persistent seizures later in life or other neurological symptoms [Yalçin et al., 2007]. Peripheral nerve hypexcitability, which has been observed for carriers of two other dominant negative mutations [Dedek et al., 2001; Wuttke et al., 2007], was also not reported for N258S carriers. Our experiments on different rescue phenomena suggest an unstable state of the mutant channel protein, which can be at least partially compensated by small interventions, such as posttranslational processing or chaperons. A different genetic background, which may induce a better compensation of defective KV7.2 channels could also explain why members of the family carrying the N258S mutation, as well as those carrying a C-terminal BFNS mutation with a relatively weak dominant-negative effect [Singh et al., 2003], do not exhibit a more severe phenotype, which could be expected from the dominant negative effect observed in vitro.

Acknowledgements

We thank Prof. Thomas Jentsch for providing the human KV7.2 and KV7.3 clones, and Dr. Michael Schwake for the extracellularly-tagged KV7.2 channel.

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