Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 26 January 2003 ; accepted in final form 30 January 2004
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ABSTRACT |
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potassium channel; hereditary arrhythmia; electrophysiology; protein interaction
Several types of accessory subunits exist that interact with the pore-forming subunits of K+ channels (18, 22, 23). These protein-protein interactions usually result in functional changes in channel behavior such as altered gating. Moreover, there is evidence that Kv and KChIP subunits (soluble cytoplasmic proteins) also serve a chaperone-like function by altering the abundance of channel proteins expressed at the plasma membrane (13, 16, 25, 29). The KCNE gene family encodes type 1 transmembrane proteins that are capable of associating with, and regulating, a number of voltage-gated K+ channels (1). Mutations in three of the KCNEs have been linked to human disease (2). Although regulation of channel gating by the KCNE-encoded proteins is well established, less is known about their assembly with the pore-forming subunits or their potential for chaperone-like properties.
Genetic mutations often cause disease due to misfolding and mistrafficking of the mutant proteins (34). This has been well documented for LQT2 mutations, where some HERG mutants are retained in the endoplasmic reticulum (ER) and are rapidly degraded in a process that can lead to a dominant phenotype (11, 12, 15, 44, 45). KCNE-encoded subunits play an important role in modulation of cardiac K+ channels. Therefore, studying mechanisms of channel protein coassembly and surface expression is essential to understanding the pathophysiology of LQT syndrome and other ion channel diseases. LQT mutations of KCNE1 can result in varied phenotypes: dominant suppression of IKs, altered gating of IKs, or a nondominant trafficking defect of the minK protein (6, 31). The minK-L51H mutation fails to traffic to the cell surface, thereby failing to produce effective IKs, but is functionally nondominant by electrophysiological analysis (6). The trafficking defect of minK-L51H provides a useful tool to explore KvLQT1/minK association and channel assembly. In this report we show how minK affects the KvLQT1 -subunit through the use of two minK trafficking mutants: L51H, a mutation linked to JLN, and wild-type (WT) minK with an engineered ER retrieval/retention signal at its COOH terminus (LRRRKR). We demonstrate that LQT5 mutant minK is capable of conferring trafficking defects onto its
-subunit. Moreover, our results indicate that the site of minK/KvLQT1 association/assembly is the ER and that the mechanism for minK-L51H mistrafficking differs from that of receptor-based ER retrieval/retention.
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MATERIALS AND METHODS |
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Cell culture and transfection.
Human embryonic kidney (HEK)-293 and Chinese hamster ovary (CHO) cell lines (ATCC) were cultured in RPMI 1640 (Cellgro; HEK-293) or Ham's F-12 (Cellgro; CHO) with L-glutamine supplemented with 10% fetal calf serum (Intergen) and penicillin/streptomycin (Cellgro). Cultured cells were maintained in 5% CO2-humidified air at 37 or 30°C, as indicated. For biochemical analyses, transient transfection was performed using Lipofectamine (Life Technologies). In preliminary experiments, we found that optimal expression, determined as a percentage of cells transformed and overall level of protein expression, was achieved when a total of 2 µg of DNA were combined with 12 µl of Lipofectamine for transient transfection of a 35-mm dish (data not shown). Cells were grown in 80-cm2 tissue culture flasks (Nunc) and transferred every 24 days by treatment with trypsin-EDTA when they reached a confluence of 7080%. For electrophysiology studies, CHO cells were transfected by electroporation with 2 µg of WT KvLQT cDNA, 1 µg of green fluorescent protein cDNA, and 4.5 µg of the appropriate KCNE -subunit (WT minK, minK-ER, or minK-L51H; molar ratio of
:
-subunit
1:3). Electroporation was performed on a BTX electroporation (225 V, 72
, 1,800 µF) as previously described (15, 19). Cells were studied with voltage clamp 2472 h after transfection.
Electrophysiology.
Cells were plated and grown on sterile glass coverslips within 35-mm tissue culture dishes. The coverslips were taken directly from the cell culture incubator and placed in an acrylic/polystyrene perfusion chamber (Warner Instruments) to obtain immediate electrophysiological measurements. Extracellular solution contained 150 mM NaCl, 1.8 mM CaCl2, 4 mM KCl, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.4 (titrated with HCl), at room temperature. Intracellular pipette solution contained 126 mM KCl, 4 mM K-ATP, 2 mM MgSO4, 5 mM EGTA, 0.5 mM CaCl2, and 25 mM HEPES, pH 7.2 (titrated with KOH), at room temperature. The whole cell patch-clamp configuration was used to study currents from KvLQT channels coexpressed with the various minK constructs. Patch pipettes were pulled and polished to obtain a tip resistance of 23 M in the above solutions. Pipette offset potential in these solutions was corrected to zero just before gigaseal formation. Junction potential for our experimental solutions was calculated between 3 and 4 mV (using pCLAMP9 analysis software) and was not corrected for analyses. Whole cell capacitance (generally 1025 pF) was compensated electronically through the amplifier. Whole cell series resistance of 612 M
was compensated by 7590% through amplifier circuitry such that the voltage errors for currents of 2 nA were always <6 mV. Only cells with seal resistances >10 M
were included in our analyses. As additional monitoring, we attempted postacquisition leak subtraction using pCLAMP9 software. If there were significant (>5 mV) changes in current-voltage responses, the cells were excluded. Cell capacitance was generally 720 pF and was compensated by analog circuitry. An Axopatch-1D patch-clamp amplifier was used for voltage-clamp measurements, and voltage protocols were controlled via personal computer with the use of pCLAMP9 acquisition and analysis software. Data were filtered on-line with an eight-pole Bessel filter at 2 kHz before digitization. Activation curves are derived from current values during a repolarization step to 120 mV after a series of 1.5-s depolarizations. Current densities were calculated as current (pA) divided by cell capacitance (pF). To control for transfection efficiency, current densities were normalized to the control group (KvLQT1 alone) daily on the basis of average current density of cells transfected with KvLQT and minK. Voltage activation data were plotted as peak tail current amplitudes, normalized to the maximal value, against the test potential values and were fitted to a Boltzmann function, I = 1/{1 + exp[(Vh V)/k]}, where I is the measured tail current, V is the applied membrane voltage, Vh is the voltage at half-maximal activation, and k is the slope factor.
Immunoblot and immunoprecipitation.
Cells were harvested for analysis 3648 h after transient transfection by detaching cells in RPMI supplemented with 10 mM EDTA at 37°C. Resuspended cells were pelleted by centrifugation at 300 g for 5 min at 4°C and washed with ice-cold phosphate-buffered saline (PBS). All subsequent steps were performed at 4°C. For whole cell lysate analysis, cell pellets were incubated in 50 µl of lysis buffer [25 mM Tris·HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.4% deoxycholic acid, 1% Nonidet P-40 (NP-40), and Complete EDTA-free protease inhibitor mixture tablets (Roche)] per 35-mm dish for 5 min. Lysates were then cleared by centrifugation at 16,000 g for 5 min at 4°C. The cleared supernatants were assayed for total protein content (Coomassie-Plus protein assay; Pierce), and equivalent amounts of protein were subjected to SDS-PAGE analysis. Protein samples were combined with 4x SDS-PAGE sample buffer [4% SDS (wt/vol), 40% glycerol, 20% -mercaptoethanol (vol/vol), 0.004% bromphenol blue (wt/vol), and 125 mM Tris·HCl, pH 6.8], incubated for 5 min at room temperature, separated on 7.5% SDS-PAGE, and electrophoretically transferred onto nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked with 10% nonfat dry milk and 0.05% Tween 20 in Tris-buffered saline (TBS) for 2 h at room temperature and incubated with anti-FLAG monoclonal antibody (Sigma) diluted to 1:3,000 in 5% dry milk and 0.05% Tween 20 in TBS for 4 h at room temperature. The membranes were then washed five times for 5 min each with 0.05% Tween 20-TBS and incubated in goat anti-mouse horseradish peroxidase-conjugated antibody (Pierce) diluted 1:20,0001:50,000 for 1 h at room temperature. After five washes as described above, horseradish peroxidase-bound protein was detected by chemiluminescence (SuperSignal West Pico chemiluminescent substrate; Pierce).
For coimmunoprecipitation studies, transiently transfected cells were harvested as described above, and cleared cell lysates were incubated with 30 µl of rabbit polyclonal anti-KvLQT antiserum or 3 µl of rabbit polyclonal anti-FLAG antibody (Sigma) for 4 h at 4°C. Protein-antibody complexes were precipitated with 30 µl of protein G-agarose (Pierce) for an additional 3 h. Precipitated proteins were washed with lysis buffer three times and eluted with SDS-PAGE sample buffer, separated on 12% SDS-PAGE, and subjected to immunoblot analysis.
PNGase F digestion.
Transfected HEK-293 cells were harvested, and minK proteins were immunoprecipitated with anti-FLAG antibody as described in Immunoblot and immunoprecipitation. Protein-antibody complexes precipitated with protein G-agarose were incubated with denaturing buffer (0.5% SDS and 1% -mercaptoethanol) at 100°C for 10 min. Samples were then digested by the addition of 5 µl of the endoglycosidase PNGase F (New England Biolabs) in 50 mM sodium phosphate (pH 7.5) with 1% NP-40 for 1 h at 37°C. After digestion, proteins were separated on 12% SDS-PAGE and subjected to immunoblot analysis.
Immunoprecipitation for plasma membrane and internal proteins. Intact transiently transfected HEK-293 cells were incubated with rabbit polyclonal anti-FLAG antibody (Sigma) diluted 1:2,000 in RPMI medium for 2 h at 14°C (lower temperature was selected to prevent endocytosis/internalization of membrane-attached antibody). Cells were detached from the cell culture dish surface by RPMI supplemented with 10 mM EDTA and collected by centrifugation. Resuspended cells were subsequently pelleted by centrifugation at 300 g for 5 min at 4°C and washed with ice-cold PBS. Cells were then lysed in NP-40-based lysis buffer as described above, and protein-antibody complexes were precipitated with 30 µl of protein G-agarose (Pierce) for 3 h. After incubation, the mixture was centrifuged at 16,000 g to isolate the FLAG-minK that was accessible to extracellularly applied antibody. The postimmunoprecipitation supernatant was subjected to another anti-FLAG immunoprecipitation to recover the residual FLAG-minK protein not precipitated in the first round, which represented that pool of protein not expressed on the cell surface. This allowed for separation of membrane and intracellular proteins. Precipitated proteins were eluted with SDS-PAGE sample buffer, separated on 12% SDS-PAGE, and subjected to immunoblot analysis with monoclonal mouse anti-FLAG antibody (Sigma). To control for protein immunoprecipitated from dead cells, trypan blue (0.4%) was added to transiently transfected HEK-293 cells to estimate the percentage of dead cells (always <1%).
Immunofluorescence. HEK-293 cells were transiently transfected with WT or mutant minK cDNA. Twenty-four hours after transfection, cells were replated onto sterile glass coverslips. At 48 h after transfection, culture medium was removed and cells were fixed with 4% paraformaldehyde in PBS for 20 min. After washing with PBS, cells were permeabilized with 0.3% Triton X-100 in PBS for 10 min, washed, and blocked with 5% BSA in PBS for 30 min. Slides were incubated with primary antibodies (mouse anti-FLAG and/or rabbit anti-KvLQT) and washed with PBS and 0.1% NP-40 for 1 h. After washing with PBS, the slides were incubated in the dark with the secondary antibodies goat anti-mouse AlexaFluor 568 (Molecular Probes) and/or goat anti-rabbit AlexaFluor 488 (Molecular Probes) diluted 1:500 for 1 h. For external labeling experiments, initial fixation and permeabilization steps were omitted and coverslips were directly blocked with 5% milk for 60 min. The slides were then incubated with primary antibodies (mouse anti-FLAG) for another 60 min. After extensive washing with PBS, cells were fixed with 4% paraformaldehyde for 20 min. Samples were washed with PBS and 0.1% NP-40 in PBS as above before being mounted onto slides for fluorescence microscopy examination. Images were acquired using either confocal microscopy (Bio-Rad) or an Olympus IX70 microscope with a x60 PlanApo objective and a Photometrics Censys cooled charge-coupled device. Nonconfocal images were subject to deconvolution with Powerhazebuster (Vaytek), and all images were analyzed with Adobe Photoshop.
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RESULTS |
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As an additional confirmation of altered intracellular localization of minK mutants, we examined transiently transfected cells by double-labeled immunofluorescence with antibodies for either a Golgi marker protein (GM130; a gift from Dr. Denis Shields, Albert Einstein College of Medicine, Bronx, NY) or an ER marker protein (calnexin; Affinity Bioreagents) in combination with anti-FLAG antibody to detect the FLAG-tagged minK proteins. The distribution pattern of Golgi-restricted GM130 staining was clearly distinct from that of all three minK proteins (Fig. 3). Although the staining pattern of the minK mutants was largely different from that of GM130, there was some degree of coincidence, suggesting that some small portion of these transiently transfected proteins was capable of entering the Golgi. Each of the proteins showed considerable overlap with the ER marker; however, the WT minK showed the greater distribution toward the periphery, as expected for a surface-expressed protein. That both the minK-ER and minK-L51H proteins showed a larger intracellular distribution than calnexin suggests that either calnexin has a more restricted subdomain of ER localization or the mutant minK proteins are being directed to degradation pathways.
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L51H is not dominant negative. Most LQT syndrome mutations are autosomal dominant in inheritance and exhibit dominant phenotypic mechanisms at the molecular level. The minK-L51H, however, has been linked to the JLN form of LQT syndrome with a recessive inheritance (6). We coexpressed WT minK and minK-L51H with KvLQT1 to test for a dominant or recessive effect of the mutation. Whole cell voltage-clamp experiments were used to determine whether minK-L51H could significantly reduce IKs channel current in the presence of WT minK. Currents in cells transfected with L51H and minK in equal proportions showed IKs-like kinetics without a significant reduction in maximum current density (Fig. 7).
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DISCUSSION |
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Our finding that KvLQT1 and minK physically associate in the ER is analogous to the work by Nagaya et al. (21) characterizing the biogenesis of Shaker K+ channel proteins in HEK-293 cells. Biogenesis of Shaker included -subunit tetramerization, core glycosylation, and association with Kv
2 subunits in the ER (21). Collectively, these studies underscore the importance of the ER in subunit oligomerization, glycosylation, and trafficking of K+ channels.
We show that an engineered minK-ER binds to the KvLQT1 -subunit and prevents its trafficking to the plasma membrane. The ER retention/retrieval signal attached to the minK-ER COOH terminus contains an RKR motif. This motif, noted by Zerangue et al. (43) in ATP-sensitive K+ (KATP) channel subunits, appears to function as a quality control mechanism. Partially assembled KATP channel proteins have an RKR motif that is exposed, causing ER retention (43). The cytoplasmic reticular immunofluorescence pattern of minK-L51H is similar to that of minK-ER and is consistent with ER retention. KvLQT1 binds both ER-localized minK mutants, implicating ER oligomerization of IKs channel
- and
-subunits. One difference between our results and those of Bianchi et al. (6) is that coexpression of minK-L51H in that study did not reduce KvLQT1 current expression as much as we observed. A possible explanation for the difference may be in the expression systems used. We used mammalian CHO cells, whereas the other group used Xenopus oocytes, which are now known to express considerable amounts of KCNE-encoded proteins that may alter the nondominant effect of minK-L51H (4).
MinK-L51H and minK-ER appear to have distinctly different mechanisms of ER retention. Upon immunofluorescence labeling, both mutant minK proteins exhibit identical cytoplasmic reticular patterns consistent with an ER locale. A small fraction of minK-ER, however, was consistently seen on the surface, whereas no minK-L51H was detected on the cell surface. These biochemical data were consistent with the patch-clamp measurements of whole cell current. No evidence of functional minK was ever detected with minK-L51H, whereas a small fraction (14% compared with WT minK + KvLQT1) of minK-ER with functional characteristics similar to WT minK was seen. Sequence-based receptor mechanisms for ER retrieval and retention may become saturated in an overexpression system, allowing a small amount of the minK-ER protein to escape to the cell surface where it has normal function (33, 42). Our results suggest that the minK-ER is not as avidly retained in the ER as the L51H mutant.
What is the mechanism of minK-L51H retention? On the basis of differences between minK-ER and minK-L51H function and glycosylation, it seems unlikely to be a receptor-based retention/retrieval mechanism. The substitution of a bulky, positively charged histidine for a leucine in the transmembrane segment of minK is likely to result in misfolding or altered conformation of minK-L51H. An efficient protein quality control system prevents targeting to the cell surface. Quality control mechanisms may entirely prevent the exit of misfolded proteins from the ER, rather than retrieval from the Golgi. A caveat to this interpretation is that our immunofluorescence data suggest that a small fraction of all three of the recombinant minK proteins may colocalize with the Golgi protein GM130. This may be a function of an overexpressed protein from transfected plasmids with strong viral (CMV) promoters. Our other results (differential glycosylation, absence on the cell surface, and altered current), however, point to L51H failing to transit from the ER to the Golgi.
MinK has two N-glycosylation consensus sites at N5 and N26; complex glycosylation at a single site is predicted to yielda mass of 21 kDa and at both sites,
25 kDa. The greater abundance of complex glycosylation forms of minK-ER is consistent with a protein that repeatedly enters the Golgi and is retrieved to the ER where there is constant exposure to ER or Golgi glycosylation enzymes. In contrast, minK L51H consistently demonstrated the lowest abundance of higher complex glycosylated forms of minK, suggesting that it fails to exit the ER because of protein quality control mechanisms. The predominance of low-molecular-weight minK either in whole cell lysates or on the surface probably represents limited kinetics of the glycosylation machinery. With forced overexpression of a small protein such as minK, it is reasonable to expect a rate of production and transit through the Golgi that exceeds the capacity of glycosylation.
Our results indicate that minK is not a chaperone for KvLQT1, in the strict sense, because there is no change in the immunofluorescence locale of the channel when expressed with or without WT minK. The augmented current density that minK confers on KvLQT1 is attributable to changes in single-channel behavior (28, 41). Our finding that minK-ER and minK-L51H each coprecipitate with KvLQT1 with comparable avidity to WT minK suggests that the physical interaction occurs early in the ER, before trafficking to the Golgi and plasma membrane. Alteration of the immunofluorescence pattern of KvLQT1 toward more abundance in the ER when coexpressed with minK-L51H supports this analysis. The con-sequence of this early association is the conferring of the minK-L51H trafficking defects on coexpressed KvLQT1 with reduced current density and absence of IKs-like current. This current suppression, however, was not dominant when both WT and minK-L51H were coexpressed with KvLQT1, consistent with the lack of clinical phenotype described in the original report by Bianchi et al. (6). Possible mechanisms for the nondominant trafficking defect of minK-L51H include a dominant rescue by coassociated WT minK or preferential association with WT minK. Because WT minK was able to bring KvLQT1 and the minK-L51H mutant to the plasma membrane, minK may act as a conditional chaperone in situations in which protein proofreading mechanisms attempt to retain the channel complex. Most minK LQT mutations analyzed thus far reside in extracellular or cytoplasmic portions of the molecule. These mutations have, for the most part, resulted in functional defects rather than protein trafficking defects and may underlie a possible difference between dominant and nondominant disease inheritance (24, 27, 35).
In summary, our data provide insight into mechanisms of ER retention and their importance in LQT syndrome. Our results show that minK-L51H is not a dominant-negative mutation, confirming findings in gene carriers (6). Furthermore, we provide evidence suggesting that the mechanism of ER retention differs from ER retention/retrieval and that the subunits of the IKs channel assemble at the level of the ER. Future studies that focus on mechanisms of ER retention and protein trafficking are necessary and are likely to reveal therapeutic targets to treat diseases caused by defects in protein trafficking and assembly.
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ACKNOWLEDGMENTS |
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T. McDonald is an Established Investigator of the American Heart Association.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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