Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6, Canada
* Author for correspondence (e-mail: alvin.shrier{at}mcgill.ca)
Accepted 6 April 2005
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Summary |
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Key words: Cyclic-nucleotide-binding domain, Hyperpolarization-activated cyclic-nucleotide-modulated channel, Human ether-a-go-go-related gene, Long QT syndrome
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Introduction |
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The human ether-a-go-go-related gene HERG encodes a CNBD-containing potassium channel involved in the repolarization of cardiac action potential (Sanguinetti et al., 1995). Abnormal HERG function is a mechanism for one form of the long QT syndrome, LQT2, a cardiac disorder associated with ventricular arrhythmias and sudden death (Curran et al., 1995
; Sanguinetti et al., 1995
). Loss of HERG function has been reported for LQT2 missense mutations (Satler et al., 1996
) or truncations (Kupershmidt et al., 1998
; Aydar and Palmer, 2001
) in the C-terminus of HERG. Zhou et al. (Zhou et al., 1998
) provide evidence that the LQT2 mutant V822M lacks functional channel expression because of improper trafficking. By contrast, Cui et al. (Cui et al., 2001
) reported that V822M and another LQT2 mutant that fall in the C-terminus of HERG (R823W) exhibit cell-surface localization. However, Ficker et al. (Ficker et al., 2002
) provide evidence that the C-terminal LQT2 mutants R823W and F805C are misprocessed and improperly trafficked. This is consistent with reports by Ficker et al. (Ficker et al., 2000
) and Akhavan et al. (Akhaven et al., 2003) that LQT2 mutants R752W and N861I, both of which are within the C-terminus of HERG, are trafficking defective. These results and those of Ficker et al. (Ficker et al., 2002
) tend to support that original contention by Zhou et al. (Zhou et al., 1998
) that the C-terminal V822M mutant is a trafficking-deficient mutant. Furthermore, this might suggest that the absence of HERG currents for two other LQT2 mutations in the C-terminus of HERG (F805S and S818L) is also due to misprocessing. It has been previously noted that some of the above C-terminal mutations appear to be localized within a region of the C-terminus that bears a high degree of homology to the cyclic-nucleotide-binding domain (CNBD) of catabolite gene activator protein (CAP) (Warmke and Ganetzky, 1994
).
In the current study, we perform a sequence alignment to determine which LQT2 mutants fall within the CNBD of HERG. We then evaluated diverse mutations in the CNBD to test the notion that an intact CNBD is crucial for HERG trafficking and that all LQT2 mutations in this domain result from defective channel trafficking and loss of function. We reassessed the trafficking properties of mutants that have not been fully characterized and those for which there have been conflicting reports. We also evaluated the consequence of deleting the conserved structural motifs or the entire CNBD. Importantly, we examine the possible significance of the CNBD for the trafficking of distinct ion channels including ERG3 and HCN2. We provide evidence that disruption of the CNBD can have critical consequences on the intracellular trafficking of ion channels that might underlie LQT2 and perhaps other inherited channel disorders.
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Materials and Methods |
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Myc-tagged HCN2 was a generous gift from M. Biel (Ludwig-Maximilians University, Munich, Germany). HA-tagged ERG3 was generated by PCR amplification of ERG3 cDNA (kindly provided by D. McKinnon, State University of New York, Stony Brook, NY) using 5' and 3' primers with BglII and NotI recognition sequences, respectively. The amplified product was digested with BglII and NotI restriction enzymes and subcloned in frame into the HA fusion vector pHA-CMV (Clontech). The HCN2 cassette was generated by excising the KpnI fragment from the wild-type clone followed by religation of the vector. The amplified and mutated fragment was religated with the KpnI insert generated from restriction digestion of the wild-type clone. The ERG3 cassette was generated by excising the XhoI-NotI fragment followed by religation into pHA-CMV. The amplified and mutated cassette was digested with XhoI and NotI, and religated into HA-tagged ERG3.
Cell biology and biochemistry
All biochemical experiments were performed in HEK-293T cells unless otherwise indicated. HEK, PC-12, M2, COS-7 and HL-1 mouse cardiac-muscle cells were maintained as previously described (Akhavan et al., 2003). Transfections were performed using Lipofectamine (Invitrogen). Proteasomal inhibitors and tunicamycin treatments were performed 1 day after transfection. Cells in culture were exposed to dimethylsulfoxide (100 µM), lactacystin (50 µM), ALLN (N-acetyl-leucinal-leucinal-norleucinal; 100 µM), MG132 (GBZ-Leu-Leu-Leucinol; 50 µM) or tunicamycin (5 µg ml1) for 6-8 hours and cell lysates were analysed by immunoblotting. For Fig. 2A, cells were lysed in 2x sample buffer and, in Fig. 5A, cell lysates were subjected to high-speed centrifugation (150,000 g, 45 minutes). In all other cases, cell lysates were prepared using mild detergent conditions and insoluble material was sedimented at 16,000 g for 30 minutes (Akhavan et al., 2003
). Membranes were probed with anti-HA antibodies unless otherwise indicated. Double blots were performed by simultaneous incubation of membranes with two antibodies. In all experiments, immunoblots shown on the same membrane were subject to identical conditions and all treatments were performed using master mix solutions.
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For immunolocalization, HEK293 cells transfected on glass coverslips coated with poly-L-lysine (Sigma) were fixed in paraformaldehyde and permeabilized using 0.1% Triton X-100. Nonspecific antibody binding was blocked with 10% goat serum followed by incubation with monoclonal anti-HA antibodies for 1 hour. The coverslips were then washed and incubated with Oregon-Green/Cy3-conjugated secondary antibody for 45 minutes. After extensive washing with PBS, the coverslips were mounted onto glass slides using ImmunoFloure Mounting Medium (ICN Biomedicals). Images were analysed using a Bio-Rad Microradiance confocal laser-scanning microscope mounted on a Zeiss 200IM inverted microscope.
Proteinase K (PK) experiments were performed 1 day after transfection on 100% confluent cells. After PK treatment for 20-30 minutes at 37°C, cells were lifted as a sheet and placed in PK-blocking solution containing 10 mM HEPES, 25 mM EDTA, 20 mM Pefablock SC (Roche). In all cases, PK quantification was performed relative to the CASK (calcium/calmodulin-dependent serine protein kinase) signal.
For pulse-chase experiments, cells were labeled with 200 µCi ml1 [35S]-methionine/cysteine (Perkin-Elmer Life Sciences) for 1 hour and chased with Dulbecco's modified Eagle's medium containing 2 mM unlabeled methionine and cysteine. Equal amounts of protein were immunoprecipitated with anti-HA antibody, subjected to SDS-PAGE and visualized by autoradiography.
Sucrose-gradient analysis was performed on cell lysates subjected to high-speed centrifugation (150,000 g, 45 minutes). The supernatants were layered on top of a 5-40% continuous sucrose-density gradient. Molecular-weight protein standards (alcohol dehydrogenase and thyroglobulin, 150 kDa and 669 kDa, respectively) were layered on a separate 5-40% sucrose gradient. Samples were centrifuged for 16 hours at 220,000 g (4°C) and fractions were collected and analysed as previously described (Manganas et al., 2001; Akhavan et al., 2003
).
Densitometric analysis was used for quantification of western blots as previously described (Akhavan et al., 2003). All quantifications are based on four to 11 independent experiments. The means and standard errors were calculated, and the statistical significance of the observed differences was determined using the Student's t-test.
Sequence alignment analysis was performed using free software provided by the European Bioinformatics Institute website (http://www.ebi.ac.uk/clustalw/).
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Results |
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We next tested the consequence of other known LQT2 missense mutations that fall within the putative CNBD. We found that all of these mutants generate only the immature species (Fig. 3A, lanes 2,3,5,7), which is of particular significance because this represents the first demonstration that HERGF805S and HERGS818L are nonfunctional owing to retention in the ER. These results also show that HERGR823W is not normally processed, consistent with the report of Ficker et al. (Ficker et al., 2002) and contrary to Cui et al. (Cui et al., 2001
). The normal trafficking phenotype can, however, be rescued by introducing a conservative mutation for R823 (HERGR823K) (Bordo and Argos, 1991
), as indicated by the appearance of a mature PK-sensitive HERGR823K band (Fig. 3A,B). This observation is similar to that previously reported for HERGwt (Akhavan et al., 2003
) and serves as a positive control. Furthermore, all LQT2 missense mutants that fall in the CNBD display currents similar to those found in mock-transfected cells, demonstrating that the absence of cell-surface localization is reflected by a lack of HERG current (Fig. 3C). However, consistent with its cell-surface localization, the HERGR823K mutant was functional with similar biophysical properties to that of HERGwt (data not shown).
Disruption or deletion of the CNBD abolishes HERG trafficking
Previously, we reported that residues 860-899 of HERG (Fig. 4A, downward arrows) constitute a crucial trafficking segment (Akhavan et al., 2003). In view of the alignment of the CNBD presented above, ER retention associated with deletion of this segment might be a consequence of a disruption of the CNBD. To test this possibility, we generated a series of truncation constructs that incorporated a progressively larger fraction of the CNBD as indicated in Fig. 4A. Truncations extending to the terminal border of the CNBD at residue 870 (HERG870X) showed the presence of the slower-migrating band (Fig. 4B, lane 2) that was sensitive to PNGase (peptide N-glycanase) glycosidase digestion but not EndoH (Fig. 4C). All truncations extending to residue 860 (HERG860X) and beyond caused a loss of this band leaving only the more rapidly migrating band. This is consistent with the notion that truncations extending into the CNBD disrupt channel trafficking. To rule out the possibility that the trafficking-sensitive portion of the CNBD is relegated only to the 860-870 segment, we deleted all of the CNBD except for these residues (HERG
750-860). As shown in Fig. 4D, the deletion construct only generates a single rapidly migrating species. Taken together, these results indicate that HERG trafficking can be disrupted by deletion of multiple regions of the CNBD. To test this notion further, we systematically deleted each secondary structural motif of HERG, as predicted from the alignment in Fig. 1. As shown in Fig. 4E, deletion of any of the three
helices or eight ß sheets resulted in ER retention of the ion channel.
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To test whether abnormal trafficking is a general consequence of any domain deletion, we analysed the effect of removing the PAS domain (HERG26-135) from the N-terminus of HERG. HERG
26-135 generates two prominent bands (Fig. 5E), indicating that removal of this domain does not prevent ER exit.
CNBD is crucial for trafficking of other channels
Trafficking of ERG3
ERG3 is a CNBD-containing isoform of HERG (Shi et al., 1997) whose biochemical properties have not been previously characterized. Here, we demonstrate that recombinant wild-type ERG3 (ERG3wt) produced in HEK cells generates two prominent bands (Fig. 6A). To determine whether ERG3wt is a glycoprotein, we tested the sensitivity of the recombinant protein to glycosidase digestion. As shown in Fig. 6A, PNGase treatment resulted in a shift of both immunoreactive species, whereas EndoH shifted only the more rapidly migrating band. In addition, tunicamycin treatment of cells producing ERG3wt inhibited the generation of the more-slowly migrating form of ERG3wt (Fig. 6A). Pulse-chase experiments (Fig. 6B) demonstrate that ERG3wt is initially synthesized as a single immunoreactive species with a molecular weight corresponding to that of the immature protein (Fig. 6B, see chase times 0 and 0.5). Progressively longer chase times show that the disappearance of the lower molecular mass band is directly related to the appearance of the higher-molecular-mass band. Taken together, these results demonstrate that ERG3wt is indeed a glycoprotein that acquires differential glycosylation in the ER and Golgi.
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Trafficking of HCN2
The HERG and ERG3 channels are closely related and so we decided to investigate the role of the CNBD in a distinct channel, HCN2. We confirmed that HCN2wt is a glycoprotein with a SDS-PAGE migration pattern associated with ER and Golgi glycosylation (not shown) (Much et al., 2003). As shown in Fig. 7A, the slower migrating band of HCN2wt is sensitive to PK in a dose-dependent manner, whereas the more rapidly migrating species is not. This result indicates that the higher-molecular-mass form is the mature species processed in the Golgi and localized to the cell surface. It has been reported that the
B region of CNBD is crucial for HCN2 trafficking to the plasma membrane (Proenza et al., 2002b
). Our analysis with HERG channel indicates that multiple segments throughout the entire CNBD are important for normal trafficking. Consequently, we tested the effect of missense mutations at the highly conserved residues G560 (HCN2G560W) and P619 (HCN2P619M) of HCN2 that fall within the CNBD but outside the
B region (gray arrows in Fig. 1). As shown in Fig. 7B, in both cases, the mutations result in the disappearance of the more slowly migrating species. A similar result was obtained with deletion of the entire CNBD (HCN2
525-644) (Fig. 7B).
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Discussion |
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We extended our analysis to other channel types and showed that ERG3wt and HCN2wt also require an intact CNBD, broadly implicating this domain in channel trafficking. Our findings are consistent with those of Proenza et al. (Proenza et al., 2002b), who reported that the
B of the CNBD is indispensable for cell-surface localization and function of HCN2 channels (Proenza et al., 2002b
). Moreover, the analysis of a point mutation upstream of
B in HCN2 argues that multiple regions within the CNBD can also be important for its trafficking.
Our results suggest that channel misassembly is not the cause of exit block of CNBD and that ER retention is the likely mechanism associated with lack of surface localization of HERG and HCN2 mutants. This is consistent with the observation that a mutant of HCN2 that lacks the CNBD is able to form heteromers with wild-type HCN1 (Proenza et al., 2002b
). However, this does not provide direct evidence that the channels are folded properly. In the case of Shaker potassium channels, channel assembly does not require prefolded monomers (Schulteis et al., 1998
). Thus, it is fully conceivable that
CNBD mutants are misfolded channels that nonetheless can form tetramers. Alternatively, it is possible that the CNBD might function as an ER export signal without affecting overall structure and/or assembly of the ion channel (Ma et al., 2001
).
Defective trafficking associated with some LQT2 mutations in the putative CNBD of HERG (Ficker et al., 2000; Ficker et al., 2002
) can be rescued by lowering temperature, ostensibly by stabilizing misfolded proteins (for a review, see Kopito, 1999
). This suggests that these mutations cause structural defects that, in turn, lead to ER retention. However, we did not notice any change in the trafficking of
CNBD HERG (HERG
750-870) at permissive temperatures. A similar finding has been reported for an HCN2 mutant channel harboring a truncation that completely eliminates the CNBD (Proenza et al., 2002a
). Thus, different mechanisms might account for mistrafficking of channels that contain mutations within the CNBD versus those that completely lack a CNBD. Alternatively, complete removal of the CNBD could result in gross structural changes that are not amenable to rescue at permissive temperatures.
Regardless of the mechanism involved, it seems that the trafficking of channels is particularly sensitive to mutations in the CNBD. Is this a unique feature of CNBD or will other highly structured domains have similar effects? It has been recently shown that a mutation in the PAS domain causes ER retention (Paulussen et al., 2002). However, it has also been reported that other mutations in the PAS domain or N-terminal truncations that eliminate this domain do not always preclude generation of functional HERG channels (Morais Cabral et al., 1998
; Chen et al., 1999
; Wang et al., 1998
; Viloria et al., 2000
; Gomez-Varela et al., 2002
). Our results indicate that complete deletion of PAS is well tolerated for proper trafficking (Fig. 5E). These observations suggest that the CNBD has a more profound effect upon ion channel processing and trafficking than other highly structured modules. With this in mind, it will be very interesting to test whether binding of nucleotides is a prerequisite for proper folding and ER exit of CNBD-harboring ion channels. The accessibility of nascent proteins to the cytoplasmic environment (Hegde and Lingappa, 1996
) and the involvement of trans-acting factors (Hedge et al., 1998) are becoming recognized as crucial criteria for early steps of protein biogenesis at the ER. Based on these observations, it is entirely possible that the CNBD is engaged with the cytosolic cyclic nucleotides in the vicinity of the ER membrane, which might promote proper folding of nascent channels. Consistent with this view, misfolded proteins that are retained in the ER are often rescued by specific ligands (Rajamani et al., 2002
).
A conserved proline in the CNBD of ERG3, HCN2 and HERG (Fig. 1) at residues 849, 619 and 846, respectively, is required for normal trafficking of these channels. We found that mutation of the conserved proline to methionine in the CNBD of ERG3 (ERG3P819M) and HCN2 (HCN2P619M) led to ER retention and an absence of cell-surface expression of these channels. Interestingly, mutation of the proline of HCN2 to an alanine does not affect the functional expression of the HCN2 channel (Zahynacz et al., 2003). This might suggest that alanine and proline can be `safely' substituted (Bordo and Argos, 1991
), in a fashion analogous to the observation that HERGR823W results in ER retention, whereas the conservative mutant HERGR823K restores ER exit (Fig. 3A).
Many mutations that cause hereditary cone-photoreceptor disorders or achromatopsia fall within the CNBD of distinct CNG subunits (Kohl et al., 1998; Sundin et al., 2000
; Wissinger et al., 2001
). Considering the role of this domain in channel gating, it has been assumed that these mutations result in permanent closure of channels owing to their inability to bind cyclic nucleotides (Kohl et al., 1998
). However, based on our findings, we speculate that some of these mutants might be associated with an absence of channels from the plasma membrane. Furthermore, we propose that abnormal trafficking caused by mutations in the putative CNBD might underlie diverse genetic ion channel disorders.
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Acknowledgments |
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