JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M206638200 on September 20, 2002

J. Biol. Chem., Vol. 277, Issue 49, 47779-47785, December 6, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/49/47779    most recent
M206638200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Roti, E. C. R.
Right arrow Articles by Robertson, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roti, E. C. R.
Right arrow Articles by Robertson, G. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Interaction with GM130 during HERG Ion Channel Trafficking

DISRUPTION BY TYPE 2 CONGENITAL LONG QT SYNDROME MUTATIONS*

Elon C. Roti RotiDagger , Cena D. MyersDagger §, Rebecca A. AyersDagger , Dorothy E. BoatmanDagger , Samantha A. DelfosseDagger , Edward K. L. Chan, Michael J. Ackerman||, Craig T. JanuaryDagger **, and Gail A. RobertsonDagger DaggerDagger

From the Dagger  Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, the  Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, the || Departments of Internal Medicine, Pediatrics, and Molecular Pharmacology, Divisions of Cardiovascular Diseases and Pediatric Cardiology, Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905, and ** Section of Cardiovascular Medicine, the Department of Medicine, University of Wisconsin, Madison, Wisconsin 53792

Received for publication, July 3, 2002, and in revised form, September 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many mutations in the Human Ether-à-go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Among the mechanisms underlying long QT syndrome (LQTS),1 failed trafficking of potassium channels encoded by the Human Ether-à-go-go-Related Gene (HERG) is increasingly recognized as a leading cause of disease (1-5). Under normal conditions, HERG channels give rise to cardiac IKr (6, 7), a current contributing to the repolarization of the ventricular action potential (8). Inherited mutations in HERG associated with type 2 Long QT Syndrome (LQT2) disrupt IKr (9), leading to delayed repolarization and a prolonged QT interval. Untreated, LQTS can result in potentially fatal torsades de pointes arrhythmias (10, 11). The mechanisms by which LQT2 mutations disrupt HERG channel trafficking are unknown.

More than 90 different LQT2-causing HERG mutations have been reported, including deletions, insertions, and missense mutations scattered throughout the gene (3). Many give rise to defects in channel trafficking, manifest as a reduction in membrane current density, accumulation of the HERG protein in intracellular compartments and a failure of terminal glycosylation associated with the mature protein expressed at the membrane (1, 2). Even some mutant channels with altered gating properties exhibit significantly reduced current density, indicating that failure to traffic may contribute to the mechanism of disease in these cases as well (12-16). Given the large number and varied distribution of LQT2-causing mutations, we hypothesize that some HERG mutations might disrupt interactions with protein machinery required for normal trafficking or processing.

To identify proteins involved in normal HERG function and transport, we carried out a yeast two-hybrid screen to look for human cardiac proteins that interact with the large C-terminal region of HERG. Here we report that GM130 (Golgin-95), a Golgi-associated protein (17, 18), interacts with HERG as the channel is transported between the endoplasmic reticulum and the plasma membrane. Previously, GM130 has been associated with vesicular traffic to the cis-Golgi apparatus from the compartment known as the vesicular tubular or endoplasmic reticulum-Golgi intermediate compartment (VTC or ERGIC, respectively; Ref. 18). By using yeast two-hybrid interaction assays, immunocytochemistry, biochemistry, and electrophysiology, we found that HERG interacts with GM130 in the Golgi apparatus. This interaction was disrupted by specific LQT2-causing mutations in HERG. We propose that the HERG C terminus facilitates the transport and targeting of the ion channel by its interaction with GM130, which plays a previously undefined role in cargo recognition.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screen-- The C terminus of HERG was used to probe for interacting proteins expressed from a human heart library using the yeast two-hybrid technique. A cDNA fragment corresponding to amino acids (aa) 669-1159 of HERG was amplified by PCR using primers that also introduced BamHI sites at either end (5' TTG GGA TCC GCA CAG CCC GCT ACC ACA CAC 3' and 5' TTT TGG ATC CTC CGA GCC GTG TCT GTG C 3'). The PCR product and the Gal4-binding domain (Gal4-BD) vector, pAS2-1 (BD Biosciences and Clontech), were digested with BamHI and assembled using T4 DNA ligase. Appropriate synthesis of all DNA constructs was confirmed by sequencing on an ABI377 sequencer at the University of Wisconsin Biotechnology Center DNA Facility. Stable expression of the HERG C-terminal construct was verified by Western blot analysis using Clontech's Gal4 DNA-BD monoclonal antibody according to the manufacturer's protocols.

We screened 2.2 × 106 independent clones from a human heart cDNA library consisting of recombinant plasmids fused to the Gal4-activation domain (Gal4-AD) in the pACT2 vector (Clontech, catalog number HL4042AH, lot number 8030758). A large scale co-transformation in yeast strain PJ69-4A (19), a modified cell line that introduced the more stringent adenine selection (ade2 reporter), was carried out with 100 µg of HERG C terminus/Gal4-BD plasmid and 50 µg of library cDNA. Positive colonies (Leu+, Trp+, His+, Ade+) were further screened for beta -galactosidase (lacZ reporter) activity.

To ensure the heart cDNA clones represented true HERG-interacting proteins, an additional set of yeast transformations was established to test pairwise interactions between the HERG C terminus and the interacting proteins (Table I). In addition, complementary controls for GM130 were carried out (Table I). Only those clones recapitulating the positive interaction with the HERG C terminus and negative results with control plasmids were selected. Among the cDNAs representing five proteins produced at the end of this screen were full-length GM130 and three partial GM130 fragments. Such pairwise or binary tests were also used for mapping interaction domains between HERG and GM130 and for testing interactions between HERG LQT2 mutants and GM130.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Yeast two hybrid binary tests
pAS2-1 encodes the Gal4 binding domain (Gal4-BD) fusion protein. pACT2 encodes the Gal4 activation domain (Gal4-AD) fusion protein. pLam5'-1 encodes the human lamin C sequence fused to the Gal4-BD. pTD1-1 encodes the SV40 large T-antigen sequence fused to the Gal4-AD. + indicates growth on selective media. Blue indicates activation of the beta -galactosidase reporter.

LQT2 mutation V822M was generated previously (1). S818P and R823P were created using Gene Editor (Promega) in HERG-pCDNA-3 per the manufacturer's instructions. Two other point mutations were generated in pGH19 using PCR-based site-directed mutagenesis and internal HERG restriction sites for subcloning (5'gAg ATC ACC TTC ATC CTg CgA gAT ACC AAC3' and 5'gTT ggT ATC TCg CAg GAT gAA ggT gAT CTC3' for N861I and 5'CgC ACg gAC ACg gAC ACg gAg C3' and 5'gCT CCg TgT CCg TgT CCg TgC g3' for K897T). The mutant C-terminal fragments (aa 669-1159) resulting from either approach were amplified by the primers indicated above and inserted into pAS2-1 for binary tests with GM130. All constructs were sequenced at the University of Wisconsin Biotechnology Center DNA Facility. For specificity tests, KvLQT1 N and C termini (aa 1-143 and 367-690, respectively) and Shaker N and C termini (aa 1-123 and 487-656, respectively) were cloned into the yeast vectors and tested in reciprocal, pairwise combinations with GM130 and Tara (20) (aa 362-593) according to the procedure described above. Stable expression of each clone in pACT2 and pAS2-1 was confirmed by Western blot analysis using Clontech's Gal4-AD and Gal4-BD monoclonal antibodies, respectively, per the manufacturer's instructions.

Co-immunoprecipitation-- Protein A beads (Seize X, Pierce) were prepared by incubating 200 µl of beads with 10 µl of rabbit anti-GM130 antibody (17) for 2 h at 4 °C. The antibody was then cross-linked to the beads according to the manufacturer's instructions. Beads were stored in BupH TBS (25 mM Tris-HCl, 0.15 M NaCl, pH 7.2) + 0.025% sodium azide. Samples were prepared by incubating HEK-293 cells stably transfected with HERG (HERG/HEK-293 cells) at 18 °C for 2 h in minimum Eagle's medium with Hanks' salts (Invitrogen), with pH maintained between 7.2 and 7.4. For each experimental condition, cells at 70% confluency from 3 × 100-mm dishes were lysed by incubating for 15 min in lysis buffer (150 mM NaCl, 10 mM Tris base, pH 7.5, 1.5 mM MgCl2, 1% Nonidet P-40, 0.7 µg/µl pepstatin, and 1 minitab of protease inhibitors from Roche Molecular Biochemicals). The lysate was then sonicated twice for 10 s each. The insoluble fraction was pelleted by centrifuging at 14,000 rpm for 15 min at 4 °C. 2 ml of supernatant was incubated with 200 µl of prepared beads for 2 h, rotating, at 4 °C. Beads were washed 2× in BupH TBS per the manufacturer's instructions and then washed 4× in 0.5% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 50 mM Tris base, pH 7.5, 0.1% SDS, 0.1% deoxycholic acid (17). Sample was eluted in 190 µl of elution buffer (Pierce, Seize X). Control samples were prepared in parallel from HEK-293 cells lacking HERG.

Western Blot Analysis-- Western blots were performed according to published protocols (21, 22). A HERG antibody was raised against a C-terminal peptide corresponding to HERG aa residues 883-901 (RQRKRKLSFRRRTDKDTEQ; Zymed Laboratories Inc., San Diego). Total lysate or immunoprecipitated eluate (30 µl each) were loaded in each lane. Proteins were separated by SDS-PAGE (7.5% gel) under reducing conditions (0.1 M dithiothreitol) and transferred to polyvinylidene difluoride membrane (Immuno-blot, Bio-Rad). For each experiment, two blots were run in parallel. One was probed with the HERG C-terminal antibody at a 1:10,000 dilution and the other with an anti-GM130 antibody (17) at 1:5000 dilution. Blots were incubated in the primary antibody at 4 °C overnight followed by a 1-h incubation at RT with horseradish peroxidase-conjugated, anti-rabbit secondary antibodies at 1:7000 dilution (Amersham Biosciences). The blots were developed using enhanced chemiluminescence (ECL, Amersham Biosciences). The blot was imaged by exposure to film for varying lengths of time.

Immunocytochemistry-- Rat ventricular myocytes were kindly provided by the laboratories of Drs. Timothy Kamp and Jeffery Walker (University of Wisconsin, Madison). Myocytes were isolated from the hearts of adult male Sprague-Dawley rats (200-250 g) euthanized by inhalation of metofane for 3 min, according to previously published protocols approved by the University of Wisconsin Animal Care and Use Committee (23). Isolated myocytes were washed, pelleted, and resuspended in 0.5 mM Ca2+ Ringer's solution at RT at a density of ~105 cells ml-1. Myocytes with rod-shaped morphology and clear striations were then permeabilized with 100 µg/ml saponin for 5 min at RT in a relaxing solution composed of (in mM) 100 KCl, 1 MgCl2, 2 EGTA, 4.5 ATP, 10 imidazole, pH 7.0. The cells were washed and resuspended in relaxing solution, blocked 30 min with 5% goat serum, and washed again. Primary and secondary antibodies were diluted to the appropriate concentrations in the relaxing solution with 20% goat serum. Primary antibodies for HERG and GM130 (17) and the secondary antibody (Alexa 488, Molecular Probes) were diluted 1:1000. In one experiment a monoclonal GM130 antibody (Amersham Biosciences) was used at a dilution of 1:250. Myocytes were incubated overnight at 4 °C in primary antibody, washed 3× in the relaxing solution, and incubated at RT for 1 h in secondary antibody. Myocytes were again washed 4× in the relaxing solution and placed between two size 1 glass coverslips for viewing under confocal microscopy.

For immunocytochemistry in HEK-293 cells, we used a HERG-FLAG/HEK stable cell line, which made it possible to co-localize HERG (using anti-FLAG 1o Ab and anti-mouse 2o Ab; see below) and GM130 (anti-GM130 1o Ab and anti-rabbit 2o Ab). The FLAG tag was inserted in-frame at the C terminus of the full-length protein. The stably transfected cell line was selected by its G418 resistance and identified by the presence of HERG protein on Western blot; the biophysical properties of the expressed currents were indistinguishable from those recorded from HERG wild type stable cell lines.2 HEK-293 and HERG-FLAG/HEK cells were plated in MatTek Glass Bottom Number 0 dishes (P35GC-0-14-C) at low density. After 24 h growth in 1 ml of complete media (1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 0.1 µg of penicillin, 2 mM L-glutamate, 50 ml of fetal bovine serum), cells were fixed with 4% paraformaldehyde for 45 min at RT, pre-blocked with PBS + 0.05% Tween 20 + 5% goat serum for 30 min, then washed 2× with PBST. Cells were then incubated 2 h at RT with primary antibodies against GM130 (17) at 1:2000 dilution or the FLAG epitope (Sigma) at 1:5000 dilution in PBST + 20% goat serum, washed, and incubated with secondary antibody (Texas Red or FITC, The Jackson Laboratories, at 1:200 or 1:1000 dilution, respectively) for 1 h at RT. Cells were washed, and Prolong antifade (Molecular Probes) was applied to each dish per the manufacturer's instructions. Cells were viewed on a Bio-Rad MRC 1024 laser scanning confocal microscope equipped with a mixed gas (Ar/Kr) laser operated by 24-bit LaserSharp software.

Electrophysiology-- Oocytes were isolated from anesthetized female frogs and collagenase-treated according to protocols approved by the University of Wisconsin Animal Use and Care Committee and published previously (6, 24, 25). All HERG constructs were cloned into a pGEM-derived vector containing untranslated Xenopus beta -globin sequences for RNA stability. GM130 was cloned into pcDNA3-mycB (Invitrogen). Purified cRNA (mMessage mMachine, Ambion) was quantified and injected using a Drummond calibrated oocyte injector. Oocytes were incubated at 18 °C for 12-24 h. Recordings were carried out at room temperature within 10 min after retrieval from the incubator.

Two-electrode voltage clamp experiments were carried out as described previously (6). Currents were recorded in a solution containing 5 mM KCl, 93 mM NaCl, 1 mM MgCl2, 0.3 mM CaCl2, and 5 mM HEPES, pH 7.4. PClamp (Axon Instruments) and Origin 4.1 (Microcal Software, Inc.) were used for data analysis and plotting. Time constants (tau s) for deactivation were measured in Clampfit with a Chebyshev fit to the deactivating tail current using the equation y = A0 + A1e-t/tau 1 + A2e-t/tau 2. Current amplitudes were measured as tail current peaks or extrapolated values from fits to the deactivating traces. All amplitudes are represented as means ± S.E. A Student's t test was used to determine the significance of unpaired observations. Where appropriate, fitting was done using a nonlinear least squares regression analysis (pCLAMP, Axon Instruments; Microcal Origin).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Vivo Interactions of HERG with GM130 in Yeast-- GM130 was isolated in a yeast two-hybrid screen of a human heart library using a C-terminal fragment of HERG as bait (aa 667-1159; Fig. 1A). The interaction was robust, providing four overlapping but distinct isolates (Fig. 1B). The smallest of these clones, termed H8, delimits the minimal domain required for interaction to a region within the most C-terminal 181 amino acids of GM130 (aa 810-990).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1.   A, schematic of HERG ion channel protein showing the region used for bait in yeast two-hybrid screen. The filled region is the hydrophobic core comprising the membrane spanning domains S1-S6. The entire C terminus was used as the bait. B, Schematic of GM130 isolates in alignment. CC, predicted coiled coil domains. Full-length GM130 is shown on top. C, HERG C terminus (aa 667-1159) showing location of LQT2-causing mutations, and one common polymorphism, evaluated in binary yeast two-hybrid tests with HERG. CNBD, putative cyclic nucleotide-binding domain.

Finer mapping was accomplished by subdividing the H8 fragment. At the end of H8 are 14 amino acids previously shown to link GM130 to the Golgi apparatus via interactions with GRASP-65 (26-28). Immediately upstream is a region of 92 residues with 80% identity to Golgin 67, a Golgi-resident protein of unknown function (29, 30). The similarity diminishes dramatically for the most N-terminal 75 amino acids of H8. Splitting H8 roughly in half, we generated one fragment corresponding to the more divergent sequences of the N terminus and the other encompassing the region of homology with Golgin 67 and the GRASP-65-binding domain (Fig. 1). In binary yeast two-hybrid assays, interactions with HERG were observed for the N-terminal region of H8 (GM130 aa 810-883) but not the C-terminal region (aa 887-990). A slightly smaller N-terminal fragment (aa 810-859) also interacted (data not shown). Thus, HERG appears to bind to a domain of GM130 upstream from the GRASP-65 interaction domain and the adjacent region of homology that predicts a shared function for GM130 and Golgin 67.

To test the specificity of the GM130-HERG interactions in yeast, we carried out binary tests of GM130 with the human cardiac potassium channel KvLQT1 (31) and the Drosophila Shaker potassium channel (32). GM130 did not interact with the N or C terminus of KvLQT1. KvLQT1 interacted with another HERG-interacting protein, Tara3 (20), demonstrating that its failure to interact with GM130 was not due to gross misfolding of the KvLQT1 fragment.

LQT2 Mutations Disrupt HERG-GM130 Interactions-- Because some trafficking defects caused by LQT2 mutations might be due to the disruption of interactions with proteins along the transport pathway, we looked for a loss of interactions between GM130 and HERG LQT2 mutants in the yeast two-hybrid assay. Such pairwise tests revealed that GM130 interactions with HERG are disrupted by LQT2 mutations in the putative cyclic nucleotide-binding domain (CNBD) including V822M (33, 34), S818P (34), and R823W (3) (Fig. 1C). At the extreme C terminus, deleting amino acids 1063-1159 (Delta 1063-1159) also disrupted interactions with GM130. Situated between these regions, LQT2 mutation N861I (3) and the common polymorphism K897T (35) failed to disrupt interactions with GM130. These findings indicate that GM130 interactions require the integrity of non-contiguous regions of the HERG C terminus, minimally including the putative cyclic nucleotide-binding domain and the last ~100 amino acids of the C terminus. Interactions with Tara were not disrupted by V822M, suggesting that the binding domains for GM130 may be disrupted without a gross alteration of C-terminal structure.

Biochemical Interactions between HERG and GM130 in HEK-293 Cells-- To confirm the interactions identified in the yeast screen, we immunoprecipitated GM130 from a HERG/HEK-293 cell line and looked for an association with HERG. Cells were incubated at 18 °C to enhance the formation of the VTC intermediate compartment and accumulate HERG cargo predicted to associate with GM130 there (36-38). GM130 was precipitated from cell lysates, separated on a 7.5% SDS-PAGE gel, transferred to a membrane, and probed with either anti-HERG or anti-GM130 antibodies (see "Experimental Procedures"). Two bands of the size expected for HERG (21), and similar to those in the cell lysate, were observed (Fig. 2). Consistent with the cycling of GM130 between the VTC and the cis-Golgi (39), the co-immunoprecipitation of both the immature band at 135 kDa and the fully glycosylated band at 155 kDa suggests that GM130 interacts with HERG before and during its occupancy in the Golgi compartment in which the maturation of the HERG protein occurs.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2.   Co-immunoprecipitation of HERG and GM130 from HERG/HEK-293 cells. A, blot probed with anti-HERG antibodies. Lane 1, HERG/HEK-293 cell lysate. Lane 2, HEK-293 (no HERG) lysates. Lane 3, eluate from HERG/HEK-293 cell lysates incubated with Protein A-Sepharose beads conjugated to GM130 antibodies. Lane 4, eluate from HEK-293 cell lysates not expressing HERG incubated with beads conjugated to antibody. Lane 5, eluate from HERG/HEK-293 cell lysates incubated with Protein A-Sepharose beads NOT conjugated to antibody. B, parallel blot probed with GM130 antibodies. Lanes 6 and 7, native GM130 present in HERG/HEK-293 and HEK-293 cell lysates, respectively. Lanes 8 and 9, native GM130 immunoprecipitated from HERG/HEK-293 and HEK-293 cell lysates, respectively. Lane 10, no immunoprecipitation of GM130 from HERG/HEK-293 cells when beads are used without antibody. All lanes are from the same blot; lane 1 is inserted from a shorter film exposure.

GM130 and HERG Co-localize to the Golgi Apparatus-- As shown for other cell types (17), GM130 antibodies localized to a restricted perinuclear region in rat cardiac myocytes consistent with the location of the cis-Golgi apparatus (40, 41). Fig. 3 shows a differential interference contrast image of a myocyte (Fig. 3A), followed by a confocal image of GM130 in the same cell (Fig. 3B). In Fig. 3C, the aperture was fully opened to allow the background autofluorescence of the cytoplasm to silhouette the nucleus. No other signal was observed using the GM130 antibodies. A monoclonal GM130 antibody showed the same perinuclear staining pattern (data not shown).


View larger version (52K):
[in this window]
[in a new window]
  		Fig. 3.   Localization of rat-ERG and GM130 to the Golgi apparatus in rat cardiac myocytes. A, differential interference contrast image of an isolated rat cardiac myocyte. B, confocal immunofluorescent image of the same cell probed with GM130 primary and FITC secondary antibodies. C, outline of nucleus emerges from background fluorescence by increasing the aperture, showing characteristic positioning of the Golgi apparatus at the nuclear poles. GM130 is a marker for the cis-Golgi. D, differential interference contrast image of another cardiac myocyte. E, same myocyte probed with an HERG antibody shows a bright signal at nuclear poles consistent with Golgi localization. Membrane signal is out of the plane of focus.

Like the GM130 antibody, the HERG antibody exhibited a bright perinuclear signal at intracellular planes of focus (Fig. 3, D and E). HERG signal was also observed at the plasma membrane and T-tubules, which appear as out-of-focus background in Fig. 3E. These images suggest that the interaction between HERG and GM130 takes place largely in the cis-Golgi.

By using a HEK-293 cell line stably expressing FLAG-tagged HERG, we observed co-localization of HERG and native GM130 in the same cells (Fig. 4). Fig. 4A shows HERG/HEK-293 cells in culture. In Fig. 4B, the same cells are probed with an anti-FLAG monoclonal antibody (anti-mouse Texas Red secondary) to demonstrate the subcellular HERG distribution (21). Fig. 4C shows the cells imaged for the GM130 antibody (anti-rabbit FITC secondary), highlighting the Golgi apparatus. Fig. 4D melds these images, where the yellow defines the overlap of the two signals, presumably in the Golgi apparatus.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4.   Co-localization of HERG and GM130 in HEK-293 cells. A, differential interference contrast image of stably transfected HERG-FLAG/HEK-293 cells. B, same cells probed with anti-FLAG primary and Texas Red secondary show characteristic HERG distribution. C, GM130 primary antibody with FITC secondary highlights the cis-Golgi compartments. D, overlap of Texas Red and FITC signals (in yellow) is consistent with co-localization of HERG and GM130 in the Golgi apparatus.

Suppression of HERG Current by GM130 Overexpression-- To determine whether GM130 alters the functional properties of HERG channels, we measured HERG currents using two-electrode voltage clamp in Xenopus oocytes where expression levels could be carefully controlled by quantitative RNA injection and incubation times (Fig. 5). We compared currents in oocytes co-injected with HERG and GM130 cRNA to those injected with HERG cRNA and water. We estimated channel density by extrapolating the deactivating tail currents (see "Experimental Procedures"). In the experiment shown in Fig. 5, GM130 reduced HERG currents by about 35% from a mean of 16.5 ± 2.1 µA to 10.1 ± 1.28 (mean ± S.E.; n = 8 oocytes, p < 0.05). No differences in channel deactivation rates were observed. Similar results from nine other experiments are summarized in Table II. HERGDelta C channels missing the last 278 amino acids, including sequences required for GM130 interaction, exhibited no significant differences in current amplitudes when expressed alone or in combination with GM130 (Table II), indicating that the suppression by GM130 was due to a specific interaction with the HERG C terminus and not to competition with HERG for the translational machinery. The expression of HERGDelta C channels indicates that interaction with GM130 is not required for the perfunctory assembly and delivery of channels to the plasma membrane in Xenopus oocytes. This result confirms a previous report that the HERG C terminus is not required expression in oocytes, although the truncation was reported to reduce current levels (42). Suppression of HERG currents by GM130 is consistent with the slowing of transport observed for other membrane proteins as a consequence of interactions with anchorage or targeting proteins (see "Discussion").


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   Suppression of HERG currents by GM130 in Xenopus oocytes. A, typical currents and voltage clamp protocol stepping to positive voltages from a holding potential of -80 mV. Current density was estimated by fitting tail currents with a double exponential function and extrapolating back to the moment of the voltage change (arrows). B, summary of one experiment showing suppression of HERG amplitude by GM130. Experiments were controlled and matched with respect to time between injection and recording as well as quantity of HERG RNA injected.

                              
View this table:
[in this window]
[in a new window]
 
Table II
HERG expression in Xenopus oocytes
Numbers in 1st column refer to oocyte batches, each from a different frog; and n refers to number of oocytes per batch from which measurements were made. Current measurements are the means of maximum tail currents ± S.E. Student t tests were performed on data represented in consecutive rows, and p indicates whether differences were significant (at the 0.01 or 0.05 level) or not ("no").


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study demonstrates an interaction between HERG, a potassium channel target of congenital and acquired LQTS, and GM130, a Golgi-resident protein. GM130 provides an anchorage for HERG en passant, as captured by confocal images of the two proteins in the Golgi apparatus of rat cardiac myocytes. Over-expression of GM130 suppresses HERG currents in a heterologous expression system, as if by providing an excess of the anchorage or checkpoint substrate.

GM130 was originally identified as Golgin-95, the target of autoantibodies isolated from patients with systemic rheumatic illnesses (17). A marker for the Golgi apparatus (17), GM130 was characterized as a structural component localizing primarily to the cis-Golgi compartment (18). A role in maintaining Golgi structure was established when anti-GM130 antibodies were shown to interfere with the reformation of the Golgi from vesicular structures after mitosis (18). Those antibodies also led to an accumulation of vesicular structures at the cis-Golgi interface, suggesting a role in trafficking in post-mitotic cells (43).

Identification of binding partners by several laboratories shows that GM130 is part of a complex involved in the dynamic regulation of trafficking at the Golgi apparatus. GM130 is a component of a vesicle tethering complex, attached to the Golgi by its interaction with GRASP65 (27, 28) and to COPI and COPII vesicles by an interaction with p115 (39, 44, 45). Antibodies against GM130 disrupt transport of the viral protein VSV-G, suggesting that GM130 is required for delivery of cargo-containing vesicles to the mannosidase II-containing Golgi compartment (26). Other studies show a role for GM130 at the cis-Golgi entry point where vesicular-tubular clusters fuse (43). These transport events are regulated by Rab GTPases, with the GM130-GRASP65 complex serving as a Rab1 effector in delivering COPII vesicles to the cis-Golgi (45).

Our findings suggest a novel role for GM130 in cargo interactions during vesicular transport in the secretory pathway. The proximity of the GM130-binding domains for HERG and the tethering protein GRASP-65 raises the possibility that the HERG C terminus might specify its own targeting by forming a component of the tethering complex. A related mechanism was recently proposed for the infectious bronchitis virus (IBV), which targets to the Golgi apparatus where it recruits proteins required to form the viral envelope (46). It was shown that a stretch of amino acids in the IBV cytoplasmic C terminus is sufficient to target and retain a reporter protein in the Golgi. Thus, the virus directs its trafficking to the appropriate subcellular membrane compartment. The authors suggest that an interaction with a Golgi-resident protein might mediate this targeting and facilitate the budding that ensues (46). Perhaps GM130 is a candidate for such an interaction.

Much of the HERG C terminus may be involved in the interaction with GM130, given that either a deletion of the most C-terminal 100 amino acids or a conservative substitution some 337 residues upstream disrupts GM130 binding. Alternatively, upstream regions including the CNBD could bind cyclic nucleotides or undergo phosphorylation (47, 48) to allosterically modulate the binding of GM130 to the C-terminal 100 residues.

Growing evidence supports the concept of checkpoints or anchorages along the transport pathways for membrane proteins. In kidney epithelial cells, overexpression of the Golgi protein cytosolic group IV phospholipase A(2) retards the constitutive surface expression of aquaporin and the Na/K ATPase alpha  subunit, causing retention in the rough endoplasmic reticulum (49). CFTR transport is slowed by a resident Golgi protein, BAP31, such that reducing BAP31 expression by antisense knock-down enhances CFTR density at the plasma membrane (50). Regulatory checkpoints in distal subcellular compartments have been identified in neurons, where regulated interactions with proteins such as PICK1 are required for the proper AMPA receptor localization to synapses (51). As in the previous examples, overexpression of PICK1 also suppresses AMPA receptor surface expression. When present in excess, checkpoint proteins may simply out-compete the subsequent protein in the transport pathway. Alternatively, they may saturate a modification enzyme, such as a kinase, that triggers cargo protein release from a given compartment. The suppression of HERG currents by GM130 overexpression is consistent with the checkpoint model.

Future studies are required to determine the role of the GM130 checkpoint in normal HERG transport. GM130 apparently accompanies HERG during protein maturation in the Golgi, but whether it is required for glycosylation has not yet been determined. Another important question surrounds the specificity of the HERG-GM130 interaction. Additional experiments will be required to corroborate our initial finding that GM130 interacts specifically with HERG rather than KvLQT1 and other membrane proteins. These and other studies will help define the roles of GM130 and its cargo, HERG, in the transport and targeting of this important ion channel.

    ACKNOWLEDGEMENTS

We thank Jessica Stein, Hillary Anderson, and Erin McCarthy technical support; Corey Anderson for cell culture assistance; Ravi Kochhar for computer support; Dr. Timothy Kamp and Dr. Jeffery Walker for isolated rat myocytes; Dr. Jia-Chang He, Dr. Ravi Balijapali, Seth Robia, Valentin Robu, Dr. Eugenia Jones, and Dr. Diana Pitterle for technical advice; Dr. Qiuming Gong and Dr. Zhengfeng Zhou for guidance in establishing the HERG-FLAG/HEK stable cell line and for the FLAG-tagged cDNA; Dr. Ebru Aydar and Dr. Chris Palmer for constructing the HERGDelta C truncation; Lance Rodenkirk and the University of Wisconsin-Keck Confocal Facility; and Dr. Edward Ziff (New York University School of Medicine and Howard Hughes Medical Institutes), Dr. Jinling Wang, and Dr. Cynthia Czajkowski for insightful discussions. We gratefully acknowledge Dr. Mark Keating (Harvard University and Children's Hospital, Boston) for the gift of the KvLQT1 clone and Dr. Diane Papazian (UCLA) for the gift of the Shaker clone.

    FOOTNOTES

* This work was supported by a Howard Hughes Medical Institutes-University of Wisconsin-Madison Medical School Faculty Development award and National Institutes of Health Grant R01-HL68868 and in part by a National Science Foundation Career award and National Institutes of Health R01-HL55973 (to G. A. R.), a Clinical Scientist Development award from the Doris Duke Charitable Foundation (to M. J. A.), and National Institutes of Health Grant R01-HL60723 (to C. T. J.). This work was originally presented in abstract form (Roti Roti, E. C., Myers, C. D., Ayers, R. A., Boatman, D. E., January, C. T., and Robertson, G. A. (2001) Mol. Biol. Cell 12, 2577).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.

§ Present address: Infigen, Inc., 1825 River Rd., DeForest, WI 53532.

Dagger Dagger To whom correspondence should be addressed: Dept. of Physiology, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-265-3339; E-mail: robertson@physiology.wisc.edu.

Published, JBC Papers in Press, September 20, 2002, DOI 10.1074/jbc.M206638200

2 G. Robertson, Q. Gong, Z. Zhou, and C. January, unpublished data.

3 E. C. Roti Roti, C. D. Myers, and G. A. Robertson, unpublished data.

    ABBREVIATIONS

The abbreviations used are: LQTS, long QT syndrome; HERG, Human Ether-à-go-go-Related Gene; LQT2, type 2 long QT syndrome; VTC, vesicular tubular compartment; aa, amino acid; CNBD, cyclic nucleotide-binding domain; FITC, fluorescein isothiocyanate; RT, room temperature; Ab, antibody; BD, binding domain; CFTR, cystic fibrosis transmembrane regulator; AMPA, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Zhou, Z., Gong, Q., Epstein, M. L., and January, C. T. (1998) J. Biol. Chem. 273, 21061-21066[Abstract/Free Full Text]
2. Furutani, M., Trudeau, M. C., Hagiwara, N., Seki, A., Gong, Q., Zhou, Z., Imamura, S., Nagashima, H., Kasanuki, H., Takao, A., Momma, K., January, C. T., Robertson, G. A., and Matsuoka, R. (1999) Circulation 99, 2290-2294[Abstract/Free Full Text]
3. Splawski, I., Shen, J., Timothy, K. W., Lehmann, M. H., Priori, S., Robinson, J. L., Moss, A. J., Schwartz, P. J., Towbin, J. A., Vincent, G. M., and Keating, M. T. (2000) Circulation 102, 1178-1185[Abstract/Free Full Text]
4. Robertson, G. A. (2000) Circ. Res. 86, 492-493[Free Full Text]
5. Ficker, E., Thomas, D., Viswanathan, P. C., Dennis, A. T., Priori, S. G., Napolitano, C., Memmi, M., Wible, B. A., Kaufman, E. S., Iyengar, S., Schwartz, P. J., Rudy, Y., and Brown, A. M. (2000) Am. J. Physiol. 279, H1748-H1756
6. Trudeau, M. C., Warmke, J. W., Ganetzky, B., and Robertson, G. A. (1995) Science 269, 92-95[Abstract/Free Full Text]
7. Sanguinetti, M. C., Jiang, C., Curran, M. E., and Keating, M. T. (1995) Cell 81, 299-307[CrossRef][Medline] [Order article via Infotrieve]
8. Sanguinetti, M. C., and Jurkiewicz, N. K. (1991) Am. J. Physiol. 260, H393-H399[Medline] [Order article via Infotrieve]
9. Curran, M. E., Splawski, I., Timothy, K. W., Vincent, G. M., Green, E. D., and Keating, M. T. (1995) Cell 80, 795-803[CrossRef][Medline] [Order article via Infotrieve]
10. Roden, D. M., George, A. L., Jr., and Bennett, P. B. (1995) J. Cardiovasc. Electrophysiol. 6, 1023-1031[Medline] [Order article via Infotrieve]
11. Ackerman, M. J. (1998) Mayo Clin. Proc. 73, 250-269[Medline] [Order article via Infotrieve]
12. Sanguinetti, M. C., Curran, M. E., Spector, P. S., and Keating, M. T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2208-2212[Abstract/Free Full Text]
13. Keating, M. T., and Sanguinetti, M. C. (1996) Curr. Opin. Genet. & Dev. 6, 326-333[CrossRef][Medline] [Order article via Infotrieve]
14. Chen, J., Zou, A., Splawski, I., Keating, M. T., and Sanguinetti, M. C. (1999) J. Biol. Chem. 274, 10113-10118[Abstract/Free Full Text]
15. Sanguinetti, M. C., and Spector, P. S. (1997) Neuropharmacology 36, 755-762[CrossRef][Medline] [Order article via Infotrieve]
16. Lees-Miller, J. P., Duan, Y., Teng, T. Q., Thorstad, K., and Duff, H. J. (2000) Circ. Res. 86, 507-513[Abstract/Free Full Text]
17. Fritzler, M., Hamel, J., Ochs, R., and Chan, E. (1993) J. Exp. Med. 178, 49-62[Abstract/Free Full Text]
18. Nakamura, N., Rabouille, C., Watson, R., Nilsson, T., Hui, N., Slusarewicz, P., Kreis, T., and Warren, G. (1995) J. Cell Biol. 131, 1715-1726[Abstract/Free Full Text]
19. James, P., Halladay, J., and Craig, E. A. (1996) Genetics 144, 1425-1436[Abstract]
20. Seipel, K., O'Brien, S. P., Iannotti, E., Medley, Q. G., and Streuli, M. (2001) J. Cell Sci. 114, 389-399[Abstract]
21. Zhou, Z., Gong, Q., Ye, B., Fan, Z., Makielski, J. C., Robertson, G. A., and January, C. T. (1998) Biophys. J. 74, 230-241[Medline] [Order article via Infotrieve]
22. Zhou, Z., Gong, Q., and January, C. T. (1999) J. Biol. Chem. 274, 31123-31126[Abstract/Free Full Text]
23. He, J.-Q., Pi, Y., Walker, J. W., and Kamp, T. J. (2000) J. Physiol. (Lond.) 524, 807-820[Abstract/Free Full Text]
24. Wang, J., Myers, C. D., and Robertson, G. A. (2000) J. Gen. Physiol. 115, 749-758[Abstract/Free Full Text]
25. Wang, J., Trudeau, M. C., Zappia, A. M., and Robertson, G. A. (1998) J. Gen. Physiol. 112, 637-647[Abstract/Free Full Text]
26. Alvarez, C., Garcia-Mata, R., Hauri, H. P., and Sztul, E. (2001) J. Biol. Chem. 276, 2693-2700[Abstract/Free Full Text]
27. Barr, F. A., Nakamura, N., and Warren, G. (1998) EMBO J. 17, 3258-3268[CrossRef][Medline] [Order article via Infotrieve]
28. Barr, F. A., Puype, M., Vandekerckhove, J., and Warren, G. (1997) Cell 91, 253-262[CrossRef][Medline] [Order article via Infotrieve]
29. Eystathioy, T., Jakymiw, A., Fujita, D. J., Fritzler, M. J., and Chan, E. K. (2000) J. Autoimmun. 14, 179-187[CrossRef][Medline] [Order article via Infotrieve]
30. Jakymiw, A., Raharjo, E., Rattner, J. B., Eystathioy, T., Chan, E. K., and Fujita, D. J. (2000) J. Biol. Chem. 275, 4137-4144[Abstract/Free Full Text]
31. Wang, Q., Curran, M. E., Splawski, I., Burn, T. C., Millholland, J. M., VanRaay, T. J., Shen, J., Timothy, K. W., Vincent, G. M., de Jager, T., Schwartz, P. J., Toubin, J. A., Moss, A. J., Atkinson, D. L., Landes, G. M., Connors, T. D., and Keating, M. T. (1996) Nat. Genet. 12, 17-23[CrossRef][Medline] [Order article via Infotrieve]
32. Papazian, D. M., Schwarz, T. L., Tempel, B. L., Jan, Y. N., and Jan, L. Y. (1987) Science 237, 749-753[Abstract/Free Full Text]
33. Satler, C. A., Walsh, E. P., Vesely, M. R., Plummer, M. H., Ginsburg, G. S., and Jacob, H. J. (1996) Am. J. Med. Genet. 65, 27-35[CrossRef][Medline] [Order article via Infotrieve]
34. Berthet, M., Denjoy, I., Donger, C., Demay, L., Hammoude, H., Klug, D., Schulze-Bahr, E., Richard, P., Funke, H., Schwartz, K., Coumel, P., Hainque, B., and Guicheney, P. (1999) Circulation 99, 1464-1470[Abstract/Free Full Text]
35. Laitinen, P., Fodstad, H., Piippo, K., Swan, H., Toivonen, L., Viitasalo, M., Kaprio, J., and Kontula, K. (2000) Hum. Mutat. 15, 580-581[Medline] [Order article via Infotrieve]
36. Saraste, J., and Svensson, K. (1991) J. Cell Sci. 100, 415-430[Abstract/Free Full Text]
37. Hauri, H. P., and Schweizer, A. (1992) Curr. Opin. Cell Biol. 4, 600-608[CrossRef][Medline] [Order article via Infotrieve]
38. Balch, W. E., McCaffery, J. M., Plutner, H., and Farquhar, M. G. (1994) Cell 76, 841-852[CrossRef][Medline] [Order article via Infotrieve]
39. Marra, P., Maffucci, T., Daniele, T., Tullio, G. D., Ikehara, Y., Chan, E. K. L., Luini, A., Beznoussenko, G., Mironov, A., and DeMatteis, M. A. (2001) Nat. Cell Biol. 3, 1101-1114[CrossRef][Medline] [Order article via Infotrieve]
40. McNutt, N. S., and Fawcett, D. W. (1969) J. Cell Biol. 42, 46-67[Abstract/Free Full Text]
41. Fawcett, D. W., and McNutt, N. S. (1969) J. Cell Biol. 42, 1-45[Abstract/Free Full Text]
42. Aydar, E., and Palmer, C. (2001) J. Physiol. (Lond.) 534, 1-14[Abstract/Free Full Text]
43. Seemann, J., Jokitalo, E. J., and Warren, G. (2000) Mol. Biol. Cell 11, 635-645[Abstract/Free Full Text]
44. Nakamura, N., Lowe, M., Levine, T. P., Rabouille, C., and Warren, G. (1997) Cell 89, 445-455[CrossRef][Medline] [Order article via Infotrieve]
45. Moyer, B. D., Allan, B. B., and Balch, W. E. (2001) Traffic 2, 268-276[CrossRef][Medline] [Order article via Infotrieve]
46. Corse, E., and Machamer, C. E. (2002) J. Virol. 76, 1273-1284[Abstract/Free Full Text]
47. Cui, J., Melman, Y., Palma, E., Fishman, G. I., and McDonald, T. V. (2000) Curr. Biol. 10, 671-674[CrossRef][Medline] [Order article via Infotrieve]
48. Kiehn, J. (2000) Trends Cardiovasc. Med. 10, 205-209[CrossRef][Medline] [Order article via Infotrieve]
49. Choukroun, G. J., Marshansky, V., Gustafson, C. E., McKee, M., Hajjar, R. J., Rosenzweig, A., Brown, D., and Bonventre, J. V. (2000) J. Clin. Invest. 106, 983-993[Medline] [Order article via Infotrieve]
50. Lambert, G., Becker, B., Schreiber, R., Boucherot, A., Reth, M., and Kunzelmann, K. (2001) J. Biol. Chem. 276, 20340-20345[Abstract/Free Full Text]
51. Perez, J. L., Khatri, L., Chang, C., Srivastava, S., Osten, P., and Ziff, E. B. (2001) J. Neurosci. 21, 5417-5428[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Circ. Res.Home page
H. Sale, J. Wang, T. J. O'Hara, D. J. Tester, P. Phartiyal, J.-Q. He, Y. Rudy, M. J. Ackerman, and G. A. Robertson
Physiological Properties of hERG 1a/1b Heteromeric Currents and a hERG 1b-Specific Mutation Associated With Long-QT Syndrome
Circ. Res., September 26, 2008; 103(7): e81 - e95.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
S. L. Cockerill, A. B. Tobin, I. Torrecilla, G. B. Willars, N. B. Standen, and J. S. Mitcheson
Modulation of hERG potassium currents in HEK-293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits
J. Physiol., June 1, 2007; 581(2): 479 - 493.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
P. Marra, L. Salvatore, A. Mironov Jr, A. Di Campli, G. Di Tullio, A. Trucco, G. Beznoussenko, A. Mironov, and M. A. De Matteis
The Biogenesis of the Golgi Ribbon: The Roles of Membrane Input from the ER and of GM130
Mol. Biol. Cell, May 1, 2007; 18(5): 1595 - 1608.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Phartiyal, E. M. C. Jones, and G. A. Robertson
Heteromeric Assembly of Human Ether-a-go-go-related Gene (hERG) 1a/1b Channels Occurs Cotranslationally via N-terminal Interactions
J. Biol. Chem., March 30, 2007; 282(13): 9874 - 9882.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Nakajima, K. Hayashi, P. C. Viswanathan, M.-Y. Kim, M. Anghelescu, K. A. Barksdale, W. Shuai, J. R. Balser, and S. Kupershmidt
HERG Is Protected from Pharmacological Block by {alpha}-1,2-Glucosyltransferase Function
J. Biol. Chem., February 23, 2007; 282(8): 5506 - 5513.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Grishin, H. Li, E. S. Levitan, and E. Zaks-Makhina
Identification of {gamma}-Aminobutyric Acid Receptor-interacting Factor 1 (TRAK2) as a Trafficking Factor for the K+ Channel Kir2.1
J. Biol. Chem., October 6, 2006; 281(40): 30104 - 30111.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. V. Soldovieri, P. Castaldo, L. Iodice, F. Miceli, V. Barrese, G. Bellini, E. M. del Giudice, A. Pascotto, S. Bonatti, L. Annunziato, et al.
Decreased Subunit Stability as a Novel Mechanism for Potassium Current Impairment by a KCNQ2 C Terminus Mutation Causing Benign Familial Neonatal Convulsions
J. Biol. Chem., January 6, 2006; 281(1): 418 - 428.
[Abstract] [Full Text] [PDF]