J Biol Chem, Vol. 275, Issue 8, 5997-6006, February 25, 2000
Expression of Distinct ERG Proteins in Rat, Mouse, and Human
Heart
RELATION TO FUNCTIONAL IKr CHANNELS*
Amber L.
Pond
,
Bridget K.
Scheve,
Andrew T.
Benedict,
Kevin
Petrecca§,
David R.
Van Wagoner¶,
Alvin
Shrier§, and
Jeanne M.
Nerbonne
From the Department of Molecular Biology and
Pharmacology, Washington University School of Medicine, St. Louis,
Missouri 63110, the § Department of Physiology, McGill
University, Montreal, Quebec PQH3 G1Y6, Canada, and the
¶ Cleveland Clinic Foundation, Cleveland, Ohio 44195
 |
ABSTRACT |
One form of inherited long QT syndrome, LQT2,
results from mutations in HERG1, the human
ether-a-go-go-related gene, which encodes a voltage-gated K+ channel 
subunit.
Heterologous expression of HERG1 gives rise to K+ currents
that are similar (but not identical) to the rapid component of delayed
rectification, IKr, in cardiac myocytes. In
addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1)
referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional IKr
channels. In the experiments here, antibodies generated against HERG1
were used to examine ERG1 protein expression in heart and in brain. In
Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or
RERG1 (rat ERG1) probed with antibodies targeted against the C terminus
of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band
is evident in blots of extracts from cells expressing MERG1b or HERG1b.
In immunoblots of fractionated rat (and mouse) brain and heart membrane
proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F,
the 165- and 205-kDa proteins were replaced by two new bands at 175 and
130 kDa, suggesting that ERG1 is differentially glycosylated in
rat/mouse brain and heart. In human heart, a single HERG1 protein with
an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein
(and IKr) expression is higher in atria than
ventricles, whereas in humans, HERG1 expression is higher in
ventricular, than atrial, tissue. Taken together, these results suggest
that the N-terminal alternatively spliced variants of ERG1
(i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play
roles in the generation of functional cardiac
IKr channels.
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INTRODUCTION |
Long QT syndrome is an acquired or an inherited disorder that can
cause syncope and sudden death resulting from episodic ventricular arrhythmias and ventricular fibrillation (1, 2). The characteristic feature identified in surface electrocardiograms of affected
individuals is prolongation of the QT interval, consistent with the
underlying cause of long QT syndrome being a defect in ventricular
repolarization (3, 4). One form of inherited long QT syndrome, LQT2,
was localized to chromosome 7 (5), and shown to result from mutations (6) in the human
ether-a-go-go-related gene,
originally referred to as HERG (7). With the identification of
additional members of the ether-a-go-go-related
gene (ERG) family (8), the terminology HERG1 seems more
appropriate (9).
HERG1 encodes a polypeptide with a predicted molecular mass of 127 kDa
and a predicted sequence and membrane topology similar to that of other
voltage-gated K+ channel subunits (10-12).
Heterologous expression of HERG1 in Xenopus oocytes (10-12)
and in HEK-293 cells (14-16) reveals voltage-gated K+
currents that are similar to the rapid component of delayed
rectification, IKr, in myocardial cells
(17-22). Like IKr, for example, the currents produced on heterologous expression of HERG are rapidly activating and
rapidly inactivating, are K+-selective, and display marked
inward rectification at potentials positive to 0 mV (10-14). The
detailed properties of the HERG-induced currents (10-16), however, are
variable and are not identical to those of (endogenous)
IKr characterized in myocardial cells from several species (17-22), including human (20-22). Although additional members of the ERG family, ERG2 and ERG3, have been cloned (8), these
subunits appear to be nervous system-specific and therefore likely do
not contribute to cardiac IKr. Alternatively
spliced variants of HERG1 and MERG1 (the mouse homologue of HERG 1)
with unique N termini (referred to as HERG1b and MERB1b), however, have
been cloned from heart and postulated to play roles in the generation
of functional cardiac IKr channels (9, 23, 24). Indeed, coexpression of MERG1b (or HERG1b) with full-length MERG1 (or
HERG1) in Xenopus oocytes reveals voltage-gated
K+ currents that more closely resemble cardiac
IKr than the currents produced on expression of
MERG1 (or HERG1) alone (9, 23). C-terminal splice variants of HERG1
have also been identified in heart, although the functional
significance of these subunits (which do not produce functional
channels) is unclear (24, 25). HERG1 expressed in HEK-293 cells is
differentially glycosylated (14), and it has been demonstrated that
some LQT2 mutations result in changes in N-linked
glycosylation and that these underlie changes in subunit protein
processing and cell surface expression (15). Nevertheless, the
functional significance of N-linked glycosylation in the
regulation of cell surface expression of HERG1 in cardiac cells and/or
in the generation of functional cardiac IKr
channels is unclear. In fact, although ERG1 protein has been documented
in ferret heart (26), neither the expression levels nor the
distributions of alternatively spliced and/or differentially processed
ERG1 proteins have been examined directly to date in the mammalian myocardium.
The experiments here were undertaken to examine ERG1 protein expression
in mouse, rat, and human heart. Using antibodies targeted against
HERG1, two ERG1 proteins with molecular masses 165 and 205 kDa are
detected in rat and mouse atria, ventricles, and brain, and these
appear to reflect differentially (N-linked) glycosylated forms of ERG1. In contrast, a single 145-kDa HERG1 protein is identified in immunoblots of human cardiac membrane proteins. In rat,
mouse, and human heart, however, no low molecular mass proteins
corresponding to the expression of the ERG1b are detected. In rat, ERG1
protein expression and functional IKr expression are higher in atria than ventricles, whereas in mouse and human, ERG1
expression is higher in the ventricles. The results presented demonstrate that the predominant form of ERG1 expressed in myocardial tissues is the full-length ERG1 protein, suggesting that N-terminal ERG1b splice variants do not play a role in the generation of functional IKr channels in cardiac myocytes.
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MATERIALS AND METHODS |
Polyclonal Antibodies against HERG--
Peptides corresponding
to unique sequences in HERG: (i) residues 174-188, TARESSVRSGGAGGA, in
the N terminus and (ii) residues 1145-1159, LTSQPLHRHGSDPGS, in the C
terminus, were generated by the Protein Chemistry Laboratory
(Washington University Medical Center). A cysteine residue was added to
the N terminus of each peptide to allow coupling to the keyhole limpet
hemocyanin carrier protein, and the coupled peptides were sent to
Caltag (San Francisco, CA) for injection into rabbits. Sera were
screened using enzyme-linked immunosorbent assays, and antibodies were
subsequently affinity purified using the ImmunoPure Antigen/Antibody
Immobilization Kit #2 (Pierce). These antibodies are referred to as
N-anti-HERG and C-anti-HERG to denote the location of the amino acid
sequence in HERG against which the antibody was generated.
Enzyme-linked immunosorbent assays on the affinity purified antibodies
revealed that each antibody detected only the peptide against which it was generated; no cross-reactivity was evident.
In addition, a polyclonal antiserum generated against a fusion protein
corresponding to the C-terminal 181 amino acids in HERG1 coupled to
(histidine-tagged) thioredoxin was obtained from Z. Zhou and C. T. January (University of Wisconsin, Madison, WI). Details of the
preparation, purification, and characterization of the antiserum have
been published (15). An additional affinity purified anti-HERG
anti-peptide antibody generated against residues 1118-1133 in the C
terminus of HERG1 (with a tyrosine and a cysteine residue added at the
N terminus of the synthetic peptide) was purchased from Alomone Labs
(Jerusalem, Israel).
cDNA Constructs--
For cloning of the rat homologue of
HERG1, RERG1 (27), four PCR1
primers were designed to amplify the coding region of RERG1
(GenBankTM accession number RNZ96106) from a rat brain
cDNA library (CLONTECH). Two primers, RERG1
forward (5'-GGAATTCATGCCGGTGCGGAGGGGCCACGTCGCGCCGCAGAACA-3') and RERG1
reverse (5'-GGAATTCCTAACTGCCTGGATCTGAGCCATGTCTGTGCAG-3') flank the
entire coding region, whereas RERG1 17 (5'-GATGACCAGCAGCAGAATGA-3') and
RERG1 3 (5'-TCGAGCTCAGAGC-CTTAACC-3') are located within the coding
sequence. PCR amplification of the rat brain library using RERG1
forward and RERG1 17 yielded a 1.2-kilobase product, corresponding to
the 5' region of the clone. Amplification with RERG1 3 and RERG1
reverse yielded a 2.7-kilobase product corresponding to the 3' end of
the clone and included an overlap region of 
500 base pairs with the
first PCR product. The two PCR products were eluted from an agarose gel
and joined in all PCR reactions in which the RERG1 forward and reverse
primers were combined with the eluted bands. The resulting 3.5-kilobase
product was ligated into the pEF6/V5-His TOPO vector (Invitrogen) and
amplified by transformation into TOP10 (Invitrogen) competent cells.
The orientation and the fidelity of the resulting RERG1 plasmid DNA was
confirmed by sequencing both the 5' and 3' ends of the clone.
HERG1 in pBK/CMV was provided by Dr. Gail Robertson (University of
Wisconsin). The MERG1 and MERG1b in pBK/CMV cDNAs were obtained
from Dr. Barry London (University of Pittsburgh), and HERG1b in pBK/CMV
was provided by Dr. Hank Duff (University of Calgary). The
Kv4.2-pBK/CMV, Kv1.2-pBK/CMV (27), Kv1.4-pBK/CMV, and Kv2.1-pBK/CMV
(28) were all obtained from Dr. Dianne M. Barry (Washington University
Medical School). A plasmid encoding the green fluorescent protein (GFP)
driven by CMV was obtained from CLONTECH.
Cell Culture and Transfections--
QT-6 cells (29), a quail
fibroblast cell line, were cultured in medium 199 (Earle's Salts)
supplemented with 10% tryptose phosphate broth, 5% fetal calf serum,
1% dimethyl sulfoxide, 0.15% mycostatin and antibiotics (penicillin
and gentamicin) at 37 °C in 95% air/5% CO2. All media
and constituents were obtained from Life Technologies, Inc. QT-6 cells
plated on glass coverslips in 12-well (18 mm each) culture plates or in
100-mm culture dishes were transiently transfected using the calcium
phosphate precipitation method (30) with cDNA constructs encoding
HERG1, HERG1b, MERG1, MERG1b, RERG1, Kv1.2, Kv1.4, Kv2.1, or Kv4.2 (1.2 µg of cDNA/18-mm well or 20 µg of cDNA/100-mm dish). In
each case, a reporter gene construct was also used: either
lacZ or GFP in pBK-CMV (0.5 µg/18-mm well or 8 µg/100-mm
dish). For mock transfections, the same amount of the GFP-pBK-CMV or
lacZ-pBK-CMV construct was used, and carrier DNA (pSK+)
(1.2 µg of cDNA/18-mm well or 20 µg of cDNA/100-mm dish)
was added in place of the ERG1 and the Kv subunit cDNAs.
Transfection efficiencies (generally 25-35%) were determined either
by counting GFP-positive cells, which were visualized directly using an
inverted epifluorescence microscope, or by subsequent staining for
-galactosidase activity using the chromogenic substrate
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal; Sigma).
Immunohistochemistry--
The usefulness of the anti-HERG
antibodies for immunohistochemistry was assessed in experiments on
ERG1-transfected cells and on cryostat sections of adult rat
ventricles. All manipulations were performed at 22-23 °C.
Approximately 48 h after transfections, cells were washed with 0.1 M phosphate-buffered saline (PBS; pH 7.2) and fixed with
4% paraformaldehyde (in PBS). Fixed cells were washed with PBS and
incubated for 1 h in blocking buffer I (PBS containing 5% normal
goat serum, 0.2% Triton X-100, and 0.1% NaN3). Cells were
then incubated overnight at 4 °C with anti-HERG antibodies in
blocking buffer I. Following washing with PBS, cells were incubated
first with a biotinylated goat anti-rabbit secondary antibody (Sigma)
diluted 1:200, followed by avidin coupled to biotinylated alkaline
phosphatase H (Vector, Burlingame, CA) diluted 1:100 for 1 h each.
After washing, cells were exposed to the alkaline phosphatase substrate
5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium (Bio-Rad);
the reaction was monitored under brightfield illumination and stopped
by addition of PBS. Cells on coverslips were dehydrated in ethanol,
cleared in xylene, and mounted with Krystalon for viewing and photography.
For the preparation of cryostat sections, adult (Harlan Sprague-Dawley)
rats (~ 0.5 kg) were anesthetized with CO2; hearts were
rapidly removed, placed in a tissue bath perfused with Tyrode's solution at 37 °C containing 121 mM NaCl, 5 mM KCl, 15 mM NaHCO3, 1 mM Na2HPO4, 2.8 mM
sodium acetate, 1 mM MgCl2, 2.2 mM
CaCl2, and 5.5 mM glucose equilibrated with
95% O2/5% CO2 (pH 7.4), and subsequently
frozen in isopentane at 
40 °C. Small pieces of the ventricles were
removed, trimmed, attached to a tissue holder using Histo Prep
(Fisher), and placed on the rapid freeze stage of a Microm cryostat
(Zeiss). Cryostat sections were cut at 20 µm, collected, and
air-dried. Dried sections were incubated in blocking buffer II (PBS
containing 0.2% Triton X-100 and 0.5% bovine serum albumin (BSA;
Sigma)) for 1 h, followed by a 2-h incubation in one of the
anti-HERG antibodies in blocking buffer II. After washing with PBS,
sections were incubated with a Texas Red-conjugated goat anti-rabbit
IgG secondary antibody (Jackson, West Grove, PA) diluted 1:2000 in
blocking buffer II. After rinsing with PBS, sections were mounted using
ImmunoFluore (ICN, Costa Mesa, CA). For photography, sections were
placed on the stage of a Zeiss LSM 410 inverted confocal microscope and
viewed using a 63×, 1.4 numerical aperture objective (optical
thickness, 1.0 µm); sections were viewed through the entire 20 µm in 1-µm steps. Texas Red was excited with a helium/neon (543 nm)
laser and imaged on a photomultiplier; images were printed on a Kodak
XLS8300 high resolution (300 DPI) printer.
Membrane Preparations--
To harvest tissue for biochemistry,
adult Long Evans rats and adult C57BL6 mice were anesthetized (5%
halothane/95% oxygen); brains, atria, and ventricles were rapidly
removed and frozen at 70 °C. Rat and mouse brain membrane proteins
were prepared essentially as described previously (28). All procedures
were performed at 4 °C, and all solutions contained a mixture of
protease inhibitors (1 mmol/liter iodoacetamide, 1 mmol/liter
1,10-phenanthroline, 0.5 mmol/liter Pefabloc, and 1.4 µmol/liter
pepstatin). After thawing, brains were homogenized in 10 ml of Tris-HCl
buffer (5 mmol/liter Tris, pH 7.4) with 0.32 mol/liter sucrose. After
nuclei and debris were pelleted by centrifugation (1000 × g, 10 min), the supernatant was centrifuged at 100,000 × g for 1 h. The pellet was resuspended in 20 mmol/liter Tris-HCl containing 1 mmol/liter EDTA (pH 7.4), centrifuged
again (40,000 × g, 20 min), and resuspended in
solubilization buffer (20 mmol/liter HEPES, 1 mmol/liter EDTA, 10%
glycerol, 120 mmol/liter KCl, and 2% Triton X-100, pH 7.4). After
incubation on ice for 1 h, the final suspension was centrifuged at
78,000 × g for 2.5 h to pellet insoluble
material. Protein assays (Bio-Rad) were completed, and the samples were
aliquoted and frozen at 20 °C until used.
Human, rat, and mouse heart membrane proteins were isolated using a
protocol similar to that previously described for rat heart (28). Adult
human ventricular and atrial tissue samples were from normal donor
hearts that were not accepted for transplantation. These hearts were
procured by LifeBanc (Cleveland, OH) with consent for research use; one
was from a 20-year-old caucasian male, and the other was from a
60-year-old caucasian male. Macroscopic and microscopic examination
revealed no evidence of pathology or underlying cardiovascular disease.
All procedures were performed at 4 °C, and all solutions contained
the mixture of protease inhibitors described above (for the brain
membrane preparation) as well as 1 mmol/liter benzamidine, 7.9 µmol/liter aprotinin, and 0.15 µmol/liter leupeptin). For the
preparation of human cardiac membrane proteins, tissue samples (1-2
g) from individual donors were processed. For rat and mouse, ventricles
and atria from several (2-12) animals were combined. Seven separate
rat and three different mouse atrial and ventricular membrane
preparations were analyzed. Tissue samples were homogenized at 4 °C
in 10 ml of TE buffer (containing 10 mmol/liter Tris-HCl and 1 mmol/liter EDTA, pH 7.4) using a TissuemizerTM. After
centrifugation (1000 × g, 10 min), the supernatants
were retained, and the pellets were resuspended to original volume in
TE buffer, homogenized, and centrifuged. These supernatants were
collected, pooled with the original supernatants, and centrifuged (40,000 × g, 10 min). The resulting pellets were
resuspended in TE buffer containing 0.6 mol/liter KI, incubated on ice
(30 min), centrifuged (40,000 × g, 10 min), and then
washed twice with TE buffer. The final pellets were resuspended in TE
buffer containing 2% Triton X-100 and incubated on ice (1 h) to
solubilize membrane proteins. A final centrifugation (17,400 × g, 30 min) precipitated the insoluble material; supernatants
were concentrated using a Centricon 30 (Amicon, Beverly, MA). After
completing protein assays, samples were aliquoted and frozen at
20 °C until used.
Western Blots--
QT-6 cell extracts (40 µg of protein),
rat and mouse brain membrane proteins (35 µg), and rat and human
atrial and ventricular membrane proteins (60 µg) were fractionated
by SDS-PAGE and then probed by Western blot analysis with the anti-HERG
antibodies. QT-6 cells plated on 100-mm tissue culture dishes were
placed on ice and rinsed with PBS. Cold TE buffer (0.4 ml) containing 2% Triton X-100 and the same mixture of protease inhibitors used in
the heart membrane preparations was then added. Cells were scraped from
the dishes, triturated with a 1-ml syringe and an 18-gauge needle, and
transferred to an eppendorf tube on ice; after 30 min, the samples were
triturated again. Insoluble material was pelleted (17,500 × g, 3 min, 4 °C), and the supernatants were again
incubated on ice (30 min). Following a second centrifugation, the
supernatants were collected and combined. Standard protein assays were
completed, and the samples were frozen at 20 °C until used.
QT-6 cell extracts and heart and brain membrane proteins were
fractionated on 10% polyacrylamide gels and transferred to Hybond-PVDF membrane (Amersham Pharmacia Biotech). The membranes were washed (3 min) in PBS and incubated (1 h) in blocking buffer III (0.2% I-Block
(Tropix, Bedford, MA) in PBS with 0.1% TweenTM 20),
followed by an overnight incubation at 4 °C with one of the
anti-HERG antibodies in blocking buffer I. The next day, membranes were
washed (10 min) and then incubated for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-rabbit IgG (Tropix) diluted 1:10,000 in blocking buffer III. After incubation, membranes were washed in blocking buffer III for 15 min and then for 2 min in
assay buffer (0.1 mol/liter diethanolamine with 1 mmol/liter MgCl2, pH 9.8; Tropix). Bound antibodies were detected
using the chemiluminescent substrate CSPDTM (Tropix).
To determine whether MERG1 and/or RERG1 is glycosylated, mouse and rat
brain membrane proteins were incubated in the presence of PNGase F
(glycopeptidase F; Sigma), an enzyme that cleaves N-linked
sugars. Prior to exposure to the enzyme, rat/mouse brain membrane
proteins (126 µg), prepared as described above, were denatured by
boiling for 10 min. After cooling, 6.3 µl (1.26 units) of PNGase F
was added, and the samples were incubated at 37 °C for 24 h.
Following the (24 h) incubation, membrane proteins were fractionated,
and Western blots were performed as described above with the
C-anti-HERG antibody.
Isolation of Adult Rat Cardiac Myocytes--
Adult rat cardiac
myocytes were isolated using a modified version of a protocol described
previously (31). For each experiment, an adult male Harlan
Sprague-Dawley rat (250-275 g) was anesthetized with pentobarbital
(0.8 ml, 50 mg/ml). Each heart was rapidly excised and placed in cold
incubation buffer containing 118 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2, 1.2 mM KH2PO4, 0.68 mM
glutamine, 11 mM dextrose, 25 mM HEPES, 5 mM pyruvate, and 1 µM insulin (pH 7.3).
Following cannulation and flushing with 2 ml of cold incubation buffer,
the heart was mounted on a Langendorff apparatus and perfused retrogradely through the aorta with 50 ml of warmed (37 °C)
perfusion buffer containing 118 mM NaCl, 37.5 mM NaHCO3, 4.8 mM KCl, 1.2 mM MgCl2, 1.2 mM
KH2PO4, 1.0 mM CaCl2,
0.68 mM glutamine, 16.5 mM dextrose, and 7.5 mM pyruvate, equilibrated with humidified 95%
O2/5% CO2 (pH 7.2). The heart was then
perfused with 50 ml of a nominally Ca2+-free perfusion
buffer, followed by perfusion buffer (100 ml) containing Type II
collagenase (140 units/ml; Worthington, Freehold, NJ) and 0.20 mg/ml
BSA (Fraction V; Sigma). After 16 min, Ca2+ was added
gradually until the free Ca2+ concentration was 1 mM. The flow rate was then increased to 10 ml/min; when the
back pressure stabilized (30 min), the heart was removed. Atria and
ventricles were separated, minced, and transferred to fresh
collagenase-containing perfusion buffer in a shaking water bath at
37 °C, aerated with humidified 95% O2/5% CO2; for the ventricular tissue, the collagenase
concentration was increased to 400 units/ml. The secondary incubations
were 5 min for atria and 10 min for ventricles, and the tissue pieces were mechanically dispersed by gentle trituration. The resulting suspensions were filtered through gauze, and cells were pelleted by
centrifugation at 400 rpm for 1 min. Myocytes were resuspended in
incubation buffer containing 0.5% BSA and rinsed with 2 ml of
incubation buffer containing 4% BSA. After settling, myocytes were
resuspended in incubation buffer with 1 mM Ca2+
at room temperature under 100% O2. The yields of
rod-shaped atrial and ventricular myocytes were 60-80%, and cells
were used within 6 h.
Electrophysiological Recordings--
The nystatin perforated
patch, whole cell recording technique (32) was employed to record
K+ currents from isolated, Ca2+-tolerant,
rod-shaped adult rat atrial and ventricular myocytes (33). The bath
solution contained 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM NaHEPES, 3 mM sodium acetate, 5 mM glucose, and 0.002 mM nifedipine at pH 7.4. Recording pipettes, fabricated from (Corning 8161) borosilicate glass
(World Precision Instruments, Sarasota, FL), had tip diameters of 2-3
µm and resistances of 2-4 M
when filled with recording solution.
The pipette solution contained 100 mM potassium
methanesulfonate, 40 mM KCl, 5 mM
K2EGTA, 2 mM MgCl2, and 10 mM HEPES at pH 7.4. Nystatin (Sigma), dissolved in dimethyl
sulfoxide (6 mg/100 µl), was diluted in pipette solution to 100 µg/ml; the nystatin-containing pipette solution was sonicated and
used within 3 h. Nystatin-free pipette solution was added to the
tip of a recording pipette prior to adding the nystatin-containing solution. Seal resistances were 
5 G. After establishing the whole
cell configuration, ±10 mV steps were applied to allow measurements of
cell membrane capacitances and input resistances. Whole cell membrane
capacitances and series resistance were compensated ( 60-80%)
electronically; voltage errors resulting from the uncompensated series
resistance were always 
4 mV and were not corrected. Experimental data
were acquired using an Axopatch 200A amplifier (Axon, Burlingame, CA),
interfaced (Digidata 1200; Axon) to an IBM compatible 486/33 microcomputer, and pClamp 6.03 (Axon). Data were acquired at 10 kHz,
and current signals were filtered at 1 kHz prior to digitization and
storage. For experiments, myocytes in 35-mm culture dishes were placed
in a thermal stage controller (Bioptech 
T system, Butler, PA),
maintained at 35 °C, and gassed with 100% O2. E-4031 was kindly donated by Esai Co., Ltd (Tsukuba, Japan). Analysis of
digitized data was completed using pClamp and Origin (Microcal, Northhampton, MA). Averaged and normalized data are presented as
means ± S.E. Statistical significance was assessed using the Student's t test; p values are presented in the text.
| |
RESULTS |
Specific C- and N-Anti-HERG Antibodies--
The affinity purified
C- and N- anti-HERG antibodies were initially tested for specificity
and for potential cross reactivity with other voltage-gated
K+ channel subunits by immunohistochemistry and Western
blot analysis on QT-6 cells transfected with cDNA constructs
encoding HERG1, Kv1.2, Kv2.1, or Kv4.2. The immunohistochemical
experiments revealed that QT-6 cells transfected with HERG1 cDNA
stained when probed with either the C-anti-HERG (Fig.
1A, panel a) or the
N-anti-HERG (Fig. 1A, panel b) antibody, whereas
no staining was detected in either mock transfected (data not shown) or
Kv1.2-transfected (Fig. 1A, panels c and
d) cells. Similar negative results were obtained when QT-6
cells transfected with cDNA constructs encoding Kv2.1 or Kv4.2 were
probed with the anti-HERG antibodies (data not shown). As demonstrated
previously (28), expression of Kv1.2, Kv2.1, or Kv4.2 is readily
detected in QT-6 cells expressing these proteins with Kv subunit-specific antibodies.
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Fig. 1.
HERG expression in transiently transfected
QT-6 cells identified with the C- and N-anti-HERG1 antibodies.
A, in QT-6 cells transfected with the HERG1 cDNA, HERG1
expression is detected using the affinity-purified C-anti-HERG
(panel a) and N-anti-HERG (panel b) antibodies
diluted 1:500 and 1:100, respectively. No staining was evident in
Kv1.2-transfected cells probed with the C-anti-HERG (panel
c) or N-anti-HERG (panel d) antibodies. B,
the anti-HERG antibodies detect a 155-kDa protein in extracts of QT-6
cells transfected with the HERG1 cDNA. Extracts of
HERG1-transfected and mock-transfected QT-6 cells (40 µg of protein)
were fractionated by SDS-PAGE, transferred to PVDF membranes, and
blotted with the affinity-purified C-anti-HERG (lanes a and
b) and N-anti-HERG (lanes c and d)
antibodies (diluted 1:500 and 1:250, respectively). The C-anti-HERG
(lane a) and the N-anti-HERG (lane c) antibodies
detect a 155-kDa protein (arrows) in extracts from
HERG1-transfected cells; this protein is not evident in blots of
extracts of mock-transfected QT-6 cells (lanes b and
d).
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In parallel experiments, extracts from QT-6 cells transfected with
cDNA constructs encoding HERG1, Kv1.2, Kv2.1, or Kv4.2 and from
mock transfected QT-6 cells were fractionated by SDS-PAGE, transferred
to PVDF membranes, and blotted with the anti-HERG antibodies.
Immunoblots with the C-anti-HERG antibody revealed a single protein
band at approximately 155 kDa in extracts of QT-6 cells transfected
with HERG cDNA (Fig. 1B, lane a). A protein of the same molecular mass was identified in HERG1-transfected HEK-293
cells with this (and the N-anti-HERG) antibody (14, 16), although in
HEK-293 cells an additional lower molecular mass (135 kDa) protein
is also expressed (see "Discussion"). Nothing was detected with the
C-anti-HERG antibody in extracts from either mock transfected QT-6
cells (Fig. 1B, lane b) or QT-6 cells transfected
with the Kv1.2, Kv2.1, or Kv4.2 cDNA. The 155-kDa protein is also
identified in extracts of HERG1-transfected QT-6 cells probed with the
N-anti-HERG antibody (Fig. 1B, lane c) and, as
with the C-anti-HERG antibody (Fig. 1B, lane a),
this band is not detected in extracts from either mock transfected QT-6 cells (Fig. 1B, lane d) or QT-6 cells transfected
with the Kv1.2, Kv2.1, or Kv4.2 cDNA. The 155-kDa protein was also
not seen in Western blots when the antibodies were preincubated with
the peptides against which each was generated (data not shown).
Expression of ERG1 and ERG1b Revealed with C-terminal Anti-HERG
Antibodies--
Subsequent experiments were aimed at determining
whether the anti-HERG antibodies could also be used to examine the
expression of the alternatively spliced HERG1 variant, HERG1b (Fig.
2A), which has been postulated
to play a role in the generation of functional
IKr channels (9, 23), as well as to detect ERG1 protein expression in other species. Preliminary immunohistochemical experiments, similar to those described above (Fig. 1A),
revealed that expression of HERG1, MERG1, and RERG1 is reliably
detected with the C-anti-HERG and N-anti-HERG antibodies, whereas only the C-anti-HERG antibody can be used to document HERG1b or MERG1b expression (data not shown). To identify the molecular masses of
proteins produced on expression of these constructs, Western blot
experiments, similar to those described above (Fig. 1B), were completed on extracts from QT-6 cells transfected with cDNA constructs encoding HERG1, HERG1b, MERG1, MERG1b, RERG1, or Kv1.4 and
from mock transfected QT-6 cells. Immunoblots with the C-anti-HERG antibody revealed a single 155-kDa protein in extracts of QT-6 cells
transfected with the full-length HERG1, MERG1, or RERG1 cDNA
constructs (Fig. 2B, lanes a under
Herg1, Merg1, and Rerg1), whereas
nothing is detected in extracts of cells expressing Kv1.4 (Fig.
2B) or in mock transfected cells (not shown) with this
antibody. Expression of the full-length 155-kDa HERG1, MERG1, and RERG1 proteins is also identified in Western blots probed with the anti-HERG C-terminal fusion protein antiserum (15) (Fig. 2C) and with the Alomone C-terminal anti-HERG antibody (Fig. 2D). All
three C-terminal anti-HERG antibodies, therefore, identify the same full-length 155-kDa (human, mouse, and rat) ERG1 protein (Fig. 2 and
Table I).
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Fig. 2.
A, schematic of the ERG1 sequence with
the region in HERG1 selected for antibody generation, the corresponding
sequences in RERG1 and MERG1, the N-terminal splice site, and the
putative glycosylation sites indicated. B D, Western blot
analysis of HERG1, HERG1b, MERG1, MERG1b, and RERG1 expression in
transiently transfected QT-6 cells using three different antibodies
directed against sequences in C terminus of HERG1 (see "Materials and
Methods"). Extracts of HERG1- HERG1b-, MERG1, MERG1b-, RERG1-, and
Kv1.4-transfected QT-6 cells (40 µg of protein) were fractionated by
SDS-PAGE, transferred to PVDF membranes, and blotted with the
affinity-purified C-anti HERG antibody (B), the anti-HERG1
C-terminal fusion protein antiserum (15) (C), and the
anti-C-HERG antibody from Alomone Labs (D) (diluted 1:350,
1:10,000, and 1:250, respectively). Lanes a and
b under Herg1 and Merg1 in each panel
refer to the HERG1 and MERG1 constructs used in the transfections;
lane a shows extracts from cells transfected with
full-length HERG1 or MERG1, and lane b shows extracts from
cells transfected with the truncated HERB1b or MERG1b constructs (see
"Materials and Methods"). The molecular masses of the proteins
produced by the truncated HERG1b and MERG1B constructs (95 kDa) are
much lower than the molecular mass (155 kDa) of the full-length HERG1,
MERG1, and RERG1 proteins. In addition, all three anti-HERG antibodies
detect a 155-kDa protein (arrows) in extracts from cells
expressing the full-length HERG1-, MERG1- and RERG1-proteins
(solid arrow), whereas a 95- kDa protein is detected in extracts from HERG1b- and
MERG1b-transfected cells (open arrow).
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In cells transfected with constructs encoding the truncated HERG1b or
MERG1b proteins, the C-anti-HERG antibody also identifies a single
protein band, although in this case the molecular mass of the protein
is 95 kDa (Fig. 2B, lanes b under
Herg1 and Merg1). Expression of the 95-kDa HERG1b
and MERG1b proteins is also identified in Western blots probed with the
anti-HERG C-terminal fusion protein antiserum (15) (Fig. 2C,
lanes b) and with the Alomone C-terminal anti-HERG antibody
(Fig. 2D, lanes b). As with the full-length 155-kDa ERG1 proteins, all three C-terminal anti-HERG antibodies identify the same 95-kDa protein (Fig. 2, lanes b),
suggesting that all of these antibodies recognize the same ERG1b
protein(s) and that they can be used to detect ERG1b expression in
tissue. As expected, nothing is detected in Western blots of extracts of QT-6 cells expressing HERG1b or MERG1b probed with the N-anti-HERG antibody (data not shown). No specific bands were detected with the
C-terminal anti-HERG antibodies in extracts from either mock transfected QT-6 cells (data not shown) or QT-6 cells transfected with
a cDNA construct encoding Kv1.4 (Fig. 2, B-D). In
addition, neither the 155-kDa nor the 95-kDa proteins was seen in
Western blots when the antibodies were preincubated with the peptides (B, D) or the fusion protein (C)
against which each antibody was generated (data not shown).
Two ERG1 Proteins in Rat and Mouse Brain--
In Western blots of
fractionated rat brain membrane proteins, both the C- and the
N-anti-HERG antibodies identify a protein at approximately 165 kDa
(Fig. 3, A and C),
a molecular mass similar to that of the protein (155 kDa) detected with
these antibodies in immunoblots of extracts of ERG1-transfected QT-6
(Fig. 1) and HEK-293 (14-16) cells. The 165-kDa protein was not
evident when the antibodies were preincubated with the peptides against
which each was generated (Fig. 3, A and C,
+ lanes). In contrast to the findings in QT-6 (Fig. 1) and
HEK-293 (14-16) cells, however, an additional high molecular mass (205 kDa) protein, was also detected in blots of fractionated rat brain
membrane proteins probed with either the C- or the N-anti-HERG
antibodies (Fig. 3, A and C). The 205-kDa band
was also not seen when the antibodies were preincubated with the
peptides against which each (antibody) was generated (Fig. 3,
A and C, + lanes). With the
N-anti-HERG (Fig. 3C), however, the intensity of the 205-kDa
band is low relative to the 165-kDa band. This intensity difference is
not seen with the C-anti-HERG antibody (Fig. 3A), suggesting
that the affinity of the N-anti-HERG antibody is greater for the
165-kDa than the 205-kDa ERG1 protein (see "Discussion").
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Fig. 3.
Western blots reveal the presence of two
distinct ERG1 proteins in rat and mouse brain. Adult rat
(A and C) and mouse (B) brain membrane
proteins (35 µg) were fractionated by SDS-PAGE, transferred to PVDF
membranes, and immunoblotted with the C-anti-HERG (A and
B) or the N-anti-HERG (C) antibody (diluted
1:500). The antibodies were applied either directly () or after
preincubation (+) with 10 µg/ml of the peptide against
which the antibody was generated. Two bands at approximately 165 and
205 kDa (arrows) are detected with the C-anti-HERG
(A and B) and N-anti-HERG (C)
antibodies in brain (see text).
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Importantly, no protein bands at 95 kDa, as might be expected
to result from expression of the N-terminal alternatively spliced ERG1b
variant (Fig. 2), are seen in Western blots of fractionated rat brain
membrane proteins probed with the C-anti-HERG antibody (Fig.
3A), suggesting that ERG1b is not expressed (at the protein level) in rat brain (see "Discussion"). In Western blots of
fractionated mouse brain membrane proteins (Fig. 3B),
virtually identical results were obtained, i.e. two proteins
at 165 and 205 kDa were routinely identified (Table I), and there were
no prominent low molecular mass bands in these blots (Fig.
3B), as would be expected if MERG1b were expressed. The ERG1
proteins expressed in mouse and rat brain, therefore, are
indistinguishable; results similar to those presented in Fig. 3 were
obtained in Western blot analyses of seven rat and three mouse brain
membrane protein preparations.
The Two MERG1 and RERG1 Proteins Reflect N-Linked
Glycosylation--
As noted previously, the predicted molecular mass
of full-length HERG is 127 kDa (6, 7) and the molecular mass of the protein recognized in HERG1-, MERG1-, and RERG1-transfected QT-6 (Figs.
1 and 2) and in HERG1-transfected HEK-293 (14-16) cells by the C- and
the N- anti-HERG antibodies is 155 kDa. The finding of two proteins
and, particularly, the 205-kDa protein in rat and mouse brain membrane
protein preparations, therefore, was unexpected. It is theoretically
possible that the 165- and 205-kDa proteins reflect the expression of
distinct ERG subfamily genes, splice variants of the same ERG gene or,
alternatively, post-translational modifications of the same ERG
holoprotein. Although two additional members, ERG2 and ERG3, of the ERG
subfamily have recently been cloned from brain (8), the predicted amino
acid sequences of the ERG2 and ERG3 proteins are distinct from ERG1,
and neither protein would be detected by the N- and C-anti-HERG
antibodies. Although the MERG1 and HERG1 transcripts are alternatively
spliced in mouse and human heart (9, 23-25), and this could certainly also occur in (rat and mouse) brain, splicing reduces the size of the
ERG1 message and results in the production of lower (rather than
higher) molecular mass ERG1 proteins (see Fig. 2). It seemed more
likely, therefore, that the finding of two high molecular mass ERG1
proteins in rat and mouse brain reflects post-translational processing.
Examination of the sequence of HERG1 (RERG1 or MERG1) reveals multiple
potential sites for post-translational modifications, including several
sites for serine-threonine kinase phosphorylation, N-myristoylation, and N-linked glycosylation; no
O-linked glycosylation sites are found. The large difference
between the 205-kDa (rat and mouse brain) ERG1 proteins detected in the
Western blots (Fig. 3) and the predicted molecular mass (127 kDa) (6,
7) of the ERG1 proteins suggested that N-linked
glycosylation might be involved. In addition, it has been reported that
HERG1 expressed in HEK-293 cells is glycosylated (14-16). To determine
whether ERG1 is (N-linked) glycosylated in vivo,
rat and mouse brain membrane proteins were denatured (by boiling) and
incubated at 37 °C in the presence of PNGase F (see "Materials and
Methods"), which cleaves N-linked sugars. Western blot
analyses of samples incubated with PNGase F for 24 h revealed
marked reductions in the intensities of both the 165-kDa and the
205-kDa bands, as well as the appearance of two new bands at 175 and
130 kDa (Fig. 4). The results obtained on
the rat (Fig. 4A) and the mouse (Fig. 4B) brain
samples are indistinguishable, consistent with the suggestion above
that the rat and mouse ERG1 proteins are the same. Importantly, the
175-kDa and the 130-kDa proteins do not reflect breakdown of the ERG1 protein, as evidenced by the fact that the 165- and 205-kDa bands are
readily detected, whereas the 175- and 130-kDa proteins are not, in
immunoblots of rat and mouse membrane proteins incubated for 24 h
at 37 °C in the absence of PNGase F (Fig. 4). Similar results were
obtained in Western blots of four rat brain and two mouse brain
membrane protein preparations treated with PNGase F. In addition, the
results are similar to previous findings on HERG1-transfected HEK-293
(14, 15) cells and suggest that the 165- and 205-kDa bands detected in
rat and mouse brain reflect differences in N-linked
glycosylation of the same (full-length) ERG1 protein (see
"Discussion").
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Fig. 4.
The two high molecular mass ERG1 proteins in
rat and mouse brain reflect (N-linked)
glycosylation. Following boiling, adult rat (A) and
mouse (B) brain membrane proteins (30 µg) were incubated
at 37 °C for approximately 24 h in the presence and absence of
1.3 units of PNGase F. After the incubation, membrane proteins were
fractionated by SDS-PAGE, transferred to PVDF membranes, and
immunoblotted with the C-anti-HERG antibody (diluted 1:500). The two
rat (A) and mouse (B) ERG1 protein bands at 165 and 205 kDa (filled arrows) typically seen in control,
untreated membrane protein preparations (, ) are also readily
detected in samples that were boiled and stored in the absence of
PNGase F for 24 h at 37 °C (+, ). After 24 h of
incubation with PNGase F at 37 °C (+, +), the 165- and 205-kDa bands
are absent or barely detectable, and two, new prominent bands at 130 and 175 kDa are evident (open arrows).
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RERG1 Expression in Rat Heart--
Western blots of fractionated
rat ventricular and atrial membrane proteins with the C-anti-HERG
antibody also revealed proteins of 165 and 205 kDa (Fig.
5A); both bands were
eliminated when the antibody was preincubated with the C-terminal
peptide against which it was generated. The 165- and 205-kDa bands are
indistinguishable from those detected in rat brain membrane
preparations (Fig. 3A), as evidenced by Western blots of
mixed brain and heart samples in which (only) the same two (165- and
205-kDa) proteins are detected (data not shown). Immunoblots of rat
heart membrane proteins with the N-anti-HERG antibody revealed only a
prominent band at 165 kDa; the 205-kDa ERG1 protein was not evident.
Similar results were obtained with the N-anti-HERG antiserum, which
reveals intense labeling of the 165-kDa (but not the 205-kDa) protein,
as well as several other unidentified proteins (Fig. 5A).
The difficulties encountered with detecting the 205-kDa ERG1 protein in
brain (see above and Fig. 3C) and heart (Fig. 5A)
with the N-terminal (but not the C-terminal) antibody suggests that the
N-terminal amino acid sequence against which the antibody was generated
is either modified or inaccessible in the denatured (205-kDa) protein
(see below and "Discussion"). The Western blots with the
C-anti-HERG and N-anti-HERG antibodies also revealed that ERG1
expression is substantially higher in rat atrial than in ventricular
membrane protein preparations; similar results were obtained in seven
separate experiments.
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Fig. 5.
ERG1 protein expression in rat heart.
A, rat ventricular (lanes V) and atrial
(lanes A) membrane proteins (60 µg) were fractionated by
SDS-PAGE, transferred to PVDF membranes, and immunoblotted with either
the C-anti-HERG antibody (diluted 1:100) or the N-anti-HERG antiserum
(diluted 1:500). Similar to the results in brain (Fig. 3), two ERG1
proteins at 165 and 205 kDa were identified with the C-anti-HERG
antibody in immunoblots of rat atrial and ventricular membrane
proteins; both bands were eliminated when the antibody was preincubated
(+) with 10 µg/ml of the peptide against which it was
generated. In addition, ERG1 protein expression is higher in rat atrial
than in rat ventricular membrane protein preparations. In contrast to
the results in brain (Fig. 3), only the 165-kDa ERG1 is detected in rat
heart membranes with the N anti-HERG antibody. B,
immunofluorescence images of cryostat sections of rat ventricular
myocardium exposed to either the C-anti-HERG (panels a-c)
or the N-anti-HERG (panels d-f) antibody followed by Texas
Red-conjugated goat anti-rabbit IgG. Sections photographed in
panels c and f were incubated with the anti-HERG
antibodies and 10 µg/ml of the peptide against which each antibody
was generated. With the C-anti-HERG (panels a and
b) antibody, labeling is evident in the lateral membranes of
ventricular myocytes and in the T-tubules; the arrows
(panel b) indicate lateral membrane labeling. With the
N-anti-HERG antibody, no lateral membrane labeling is evident
(panels d and e); rather, this antibody appears
to label T-tubular membranes exclusively; the arrows
(panel e) indicate the absence of detectable plasmalemmal
membrane labeling (see text). Scale bars are 25 µm in
panels a, c, d, and f and
5.0 µm in panels b and e.
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Immunohistochemical analysis revealed that ERG1 is also readily
detected in the membranes of adult rat ventricular and atrial cells. As
illustrated in Fig. 5B, robust membrane (with little or no
detectable cytosolic) labeling is evident on cryostat sections of adult
rat ventricles probed with both of the anti-HERG antibodies. The
subcellular labeling patterns seen with the C- and N-anti-HERG antibodies, however, are distinct (Fig. 5B). In low and high
magnification confocal images of sections immunolabeled with the
C-anti-HERG antibody (Fig. 5B, panels a and
b), for example, staining is evident in the lateral
(i.e. the sarcolemmal) membranes and in the T-tubules; the
arrows in Fig. 5B, panel b indicate
lateral membrane labeling. With the N-anti-HERG antibody, in contrast,
only labeling of the T-tubules was revealed (Fig. 5B,
panel e). Similar results were obtained on cryostat sections
prepared from ventricles isolated from three different animals. The
difference in the labeling patterns with the N- and C-anti-HERG
antibodies is intriguing, particularly in light of the Western blot
data (Fig. 5A), demonstrating a difference between the N-
and C-anti-HERG antibodies in the detection of the 205-kDa ERG1 protein
(see "Discussion").
IKr in Rat Atrial and Ventricular Myocytes--
The
finding of robust expression of the ERG1 protein(s) in adult rat heart
was initially surprising for two reasons. First, to our knowledge,
there have been no published reports documenting the presence of
IKr in adult rat atrial myocytes. In addition, although IKr has been reported in adult rat
ventricular myocytes (34, 35), the densities of the currents are low,
particularly when compared with the densities of other voltage-gated
K+ currents in these cells (36-38). Electrophysiological
experiments were undertaken, therefore, to determine
IKr densities in these cells. Voltage-gated
K+ current tails at 30 mV were recorded (see "Materials
and Methods") under control conditions and following exposure to the
class III antiarrhythmic, E-4031, a specific blocker of
IKr (17-22). To evoke IKr, cells were depolarized to +20 mV from a
holding potential of 50 mV for times ranging from 25 to 575 ms in
50-ms increments (Fig. 6A).
These experiments revealed slowly decaying tail currents in adult rat
atrial myocytes (Fig. 6, B and C) that were
suppressed in the presence of 5 µM E-4031 (Fig.
6D), consistent with the expression of
IKr (17-22). Results similar to those presented
in Fig. 6 (B-D) were obtained on 21 other adult rat atrial
and on 22 adult rat ventricular myocytes.
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Fig. 6.
E-4031-sensitive K+ currents,
IKr, in adult rat atrial and ventricular
myocytes. B, whole cell K+ currents
recorded at 35 °C from an adult rat atrial myocyte using the
nystatin perforated patch technique; the voltage-clamp protocol is
displayed in A, and the interpulse interval was 5 s.
The holding potential was 50 mV to inactivate voltage-gated
Na+ channels, and 2 µM nifedipine was added
to the bath to suppress voltage-gated Ca2+ currents. The
tail currents at 30 mV in B are replotted in C
at a higher gain. In the presence of 5 µM E-4031, the
slowly decaying (IKr) tail currents were
suppressed completely (D). IKr tails
were recorded on repolarization to 30 mV following 200-ms
depolarizations from a holding potential of 50 mV to varying test
potentials between 30 and +50 mV. Currents in the presence of E-4031
were subtracted from the controls, and the peak amplitudes of the
E-4031-sensitive tails were determined from single exponential fits to
the tail current decays fitted from the end of the test pulse until the
current reached a steady-state level (0.5-1.0 s). Peak tail currents
determined in individual cells were then normalized to whole cell
membrane capacitance, and mean (± S.E.) peak
(IKr) tail current densities in adult rat atrial
(n = 22) and ventricular (n = 22)
myocytes are plotted versus test voltage in E
(see text).
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The time and voltage-dependent properties of
IKr in adult rat atrial and ventricular cells
are indistinguishable. Boltzman fits to the mean normalized peak tail
current amplitudes as a function of voltage, for example, yielded
V1/2 values of 19.4 mV (k = 6.4) and 18.2
mV (k = 11.1) for IKr in rat atrial and
ventricular myocytes, respectively. To compare IKr densities in adult rat atrial and
ventricular myocytes, the "E-4031-sensitive" tail currents were
obtained by subtraction of the currents in the presence of E-4031 (Fig.
6D) from the control records (Fig. 6C). Peak tail
current amplitudes at 30 mV in individual cells were then determined
from single exponential fits to the tail current decays in these
subtracted records and normalized to the whole cell capacitance. These
analyses confirmed that IKr density is
significantly higher in adult rat atrial than in adult rat ventricular
myocytes (Fig. 6E). Mean (± S.E.) peak tail current densities at 30 mV, for example, were 0.62 ± 0.03 pA/pF
(n = 22) and 0.36 ± 0.01 pA/pF (n = 22) in adult rat atrial and ventricular myocytes, respectively.
Interestingly, these differences in IKr density
parallel the observed differences in ERG1 protein expression revealed
in Western blots (Fig. 5A).
A Distinct Isoform of HERG Is Expressed in Human Heart--
In
contrast to the findings in rat, Western blot analysis of human atrial
and ventricular membrane proteins using the C-anti-HERG antibody
revealed a single 145-kDa ERG1 protein (Fig.
7A). The 145-kDa band was
eliminated when the C-anti-HERG antibody was preincubated with the
peptide against which it was generated (Fig. 7A). In
addition, the same band was identified in Western blots of human
ventricular and atrial membrane proteins probed with the N-anti-HERG
antibody, suggesting that the antibodies are detecting ERG1 protein
expression. In Western blots of fractionated mouse heart membrane
proteins (Fig. 7B), however, results identical to those in
rat heart (Fig. 5A), as well as in mouse (and rat) brain
(Fig. 3) were obtained, i.e. two proteins at 165 and 205 kDa
were routinely identified. Results similar to those presented in Fig. 7
were obtained in Western blot analyses of five human and three mouse
heart membrane protein preparations.
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Fig. 7.
Different molecular mass ERG1 proteins
expressed in human and mouse heart. A, human
ventricular (lanes V) and atrial (lanes A) (60 µg) membrane proteins were fractionated by SDS-PAGE, transferred to
PVDF membranes, and immunoblotted with the C-anti-HERG antibody (1:100)
either directly () or after preincubation (+) with 10 µg/ml of the peptide against which the antibody was generated. A
single HERG1 protein with an apparent molecular mass of 145 kDa
(arrow) is detected in atria and ventricles. B,
mouse ventricular (lanes V) and atrial (lanes A)
membrane proteins (30 µg) were fractionated by SDS-PAGE, transferred
to PVDF membranes, and immunoblotted with the C-anti-HERG antibody
(1:100) either directly () or after preincubation (+) with
10 µg/ml of the peptide against which the antibody was generated.
Similar to the results in rat heart (Fig. 5) and in rat and mouse brain
(Fig. 3), two ERG1 proteins at 165 and 205 kDa were identified in mouse
heart. In contrast to the results in rat heart (Fig. 4), ERG1 protein
expression appears to be higher in ventricular (lanes V)
than in atrial (lanes A) tissue in human (A) and
in mouse (B) heart.
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The results presented in Fig. 7A reveal that the molecular
mass of the single ERG1 protein (145 kDa) identified in human heart, therefore, is substantially lower than the molecular mass of either of
the proteins (165 and 205 kDa) identified with this antibody in rat or
mouse heart and brain (compare Figs. 3-5 and 7B),
suggesting marked species-specific differences in post-translational
processing of ERG1 proteins. In addition, and in contrast to the
results in rat (Fig. 5A) heart, the Western data (Fig.
7A) suggest that ERG1 protein expression is higher in human
ventricular than in atrial tissue. The expression of the ERG1 proteins
is also higher in mouse ventricles than atria (Fig. 7B) (see
"Discussion").
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DISCUSSION |
Generation and Characterization of Specific Anti-HERG
Antibodies--
The C- and N-anti-HERG antibodies developed here were
generated against unique peptide sequences in the C (residues
1145-1159) and N (residues 174-188) termini, respectively, of HERG1
(Fig. 2A). Experiments completed on HERG1-transfected QT-6
cells revealed that both antibodies are specific for HERG1 and reliably
detect HERG1 protein expression using immunohistochemistry or Western blot analysis. Although the sequences of mouse ERG1 (MERG1) and rat
ERG1 (RERG1) were not available at the time the peptides were selected
for antibody generation, we assumed that the sequences of MERG1 and
RERG1 would be very similar to HERG1. Subsequent sequence alignment has
indeed revealed that both RERG1 (27) and MERG1 (9, 23) are highly
homologous to HERG1; the sequence identity is 97% at the amino acid
level. In addition, the C termini of RERG1, MERG1, and HERG1, targeted
by the C-anti-HERG antibody (corresponding to residues 1145-1159 in
HERG1), are (100%) identical (Fig. 2A). There are several
amino acid substitutions in the N-terminal region (corresponding to
residues 174-188 in HERG1) against which the N-anti-HERG antibody was
generated (6, 7, 9, 23, 25). Nevertheless, 8 of the 15 amino acids
(60%) in this region are identical in RERG1 and HERG1, and 10 of the
15 (67%) are identical in HERG1 and MERG1 (Fig. 2A). More
importantly, the results presented here reveal that despite these
sequence differences, the N-anti-HERG antibody can be used to detect
RERG1 expression (Figs. 2-5). Because of the sequence differences
(between MERG1/RERG1 and HERG1) and the variable labeling intensity of
the 205-kDa RERG1 protein (relative to the 165-kDa protein), however,
the C-anti-HERG antibody is preferred for studying ERG1 protein expression.
Distinct ERG1 Proteins Expressed in Rat (Mouse) and Human
Heart--
In Western blots of fractionated rat (and mouse) brain and
heart membrane proteins probed with the C-anti-HERG antibody, two ERG1
proteins at 165 and 205 kDa were identified (Table I). Because the
predicted molecular mass of HERG1 is 127 kDa (6), and the proteins
recognized in HERG1-, MERG1-, and RERG1-transfected QT-6 cells by the
C-anti-HERG antibody is 155 kDa, the finding of two proteins and,
specifically, the 205-kDa protein, in rat and mouse (brains and hearts)
was initially unexpected. Although it was theoretically possible that
these could be the products of distinct ERG subfamily genes (8) or
splice variants of the same ERG1 gene (9, 23-25), the experiments here
reveal that the 165- and 205-kDa proteins reflect differentially
(N-linked) glycosylated forms of ERG1. It has been reported
that HERG1 expressed in HEK-293 cells is also (N-linked)
glycosylated to produce proteins of approximately 135 and 155 kDa
(14-16). The extent of N-linked glycosylation of RERG1 and
MERG1 in rat and mouse brain (and heart), therefore, is considerably
greater than for HERG1 in HEK-293 cells. The fact that the molecular
mass (145 kDa) of human heart HERG1 is much lower than that of RERG1 or
MERG1 (Table I) suggests that the extent of HERG1 glycosylation
in vivo is also substantially less than for RERG1 or MERG1.
In HERG1 (MERG1/RERG1), there are multiple (5) potential
N-linked glycosylation sites, although only two (2) of these
(asparagine 598 and 629) are extracellular (Fig. 2A).
Recently, it was demonstrated that substitution of glutamine for
asparagine at these residues leads to perinuclear localization of the
HERG1 protein stably expressed in HEK-293 cells (16). In addition, no
ionic currents are recorded from HERG1(N598Q)- or
HERG1(N629Q)-expressing HEK-293 cells (16), suggesting that N-linked glycosylation is required for cell surface
expression of functional IKr channels (16).
In Western blots of fractionated rat brain membrane proteins with the
N-anti-HERG antibody, proteins at 165 and 205 kDa were also detected,
although the intensity of the 205-kDa band was lower than the 165-kDa
band. In immunoblots of rat heart membrane proteins probed with this
antibody, however, the 205-kDa protein was not detected. Importantly,
the 165- and 205-kDa bands reflect differentially (N-linked)
glycosylated forms of ERG1, and both bands are intensely labeled with
the C-anti-HERG antibody. For these reasons, the difficulties
encountered with using the N-anti-HERG antibody to detect the 205-kDa
protein cannot reflect differences in the amino acid sequences of the
165- and 205-kDa ERG1 proteins. Rather, the experimental observations
suggest that there are additional differences in post-translational
processing of the 205-kDa, relative to the 165-kDa, ERG1 protein.
Although further experiments will be necessary to test this hypothesis
directly, it is of interest to note that there are two potential
N-myristoylation and one protein kinase C phosphorylation sites in
RERG1 between amino acids 174-188. Post-translational modifications at
nearby sites that limit access to the sequence against which the
antibody is directed could also be involved.
Subcellular Localization of ERG1 in Rat Myocardial
Cells--
Confocal images of cryostat sections of adult rat heart
revealed that both the C- and N-anti-HERG antibodies label the
membranes of ventricular myocytes; little or no cytosolic labeling was
evident. The observed subcellular staining patterns, however, are
distinct. Specifically, the C-anti-HERG antibody labels both the
T-tubules and the plasma membranes of adult rat ventricular myocytes,
and the intensity of the staining in the plasmalemmal and T-tubular membranes is similar. With the N-anti-HERG antibody, in contrast, only
the T-tubules are labeled. Taken together with the Western blot data,
these results suggest that the 165-kDa ERG1 protein is localized in the
T-tubules of adult rat ventricular myocytes. It is tempting to
speculate further that the 205-kDa ERG1 protein might be specifically
localized in the plasma membranes of rat ventricular myocytes. Further
experiments will be necessary to test this hypothesis directly.
The results of the immunohistochemical experiments also suggest that
ERG1 is distributed uniformly (or nearly so) in the sarcolemmal and
T-tubular membranes of rat ventricular myocytes, i.e. no
regions of high staining density or "hot spots" were evident. These
results are in marked contrast to previous reports demonstrating that other voltage-gated K+ channel subunits are
concentrated in the regions of the intercalated discs (28, 39). In
human atria, for example, Kv1.5 is highly localized at intercalated
disc regions, colocalized with connexin 43 and N-cadherin
(39). Other Kv subunits, including Kv4.2, are also nonuniformly
distributed in the plasma membranes of rat ventricular myocytes, with
the highest densities again being in the regions of the intercalated
discs (28). Although the number of studies examining voltage-gated
K+ channel distribution in cardiac cells to date is
admittedly small, the differences in the distributions of ERG1 seen in
the present study and of the Kv subunits reported previously (25,
39) suggest that distinct mechanisms are in place for targeting
different K+ channels to the sarcolemmal membranes of
myocardial cells.
Relationship between ERG1 and Functional IKr
Channels--
The finding of robust expression of ERG1 proteins in the
rat heart was initially surprising because IKr
had not previously been reported in rat atrial cells, and although
evident in ventricular cells, the densities of the currents are low
(34, 35), particularly compared with other voltage-gated K+
channel currents in these cells (36-38). The electrophysiological experiments here have revealed the presence of
IKr, defined as the E-4031-sensitive tail
currents (17, 18), in isolated adult rat atrial (and ventricular)
myocytes. The time- and voltage-dependent properties of the
IKr currents in the two cell types are similar, although IKr density in atrial cells is
significantly higher than in ventricular cells, consistent with the
biochemical data showing greater expression of ERG1 proteins in rat
atrial, than ventricular, membranes.
Although the results of the experiments presented here are all
consistent with a role for ERG1 in the generation of
IKr, they do not directly address questions
regarding the molecular composition of functional
IKr channels. It is possible, for example, that there are additional members of the ERG subfamily of K+
channel genes in the heart and that these coassemble with ERG1 to
produce functional IKr channels. Two additional
ERG subfamily members, ERG2 and ERG3, have been cloned from brain (8),
although these appear to be nervous system-specific. Nevertheless,
there could certainly be additional ERG subfamilies or additional
members of the ERG1, ERG2, and ERG3 subfamilies in the heart that
contribute to IKr. MERG1 and HERG1 are also
reportedly alternatively spliced in mouse and human heart (9, 23-25),
and it has been suggested that the N-terminal splice variants play a
role in the generation of functional IKr
channels (9, 23). For both HERG1 and MERG1, splicing reduces the sizes
of the transcripts resulting in HERG1b and MERG1b proteins of lower
molecular mass than full-length MERG1 and HERG1. The Western blot
analysis completed here reveals only full-length HERG1 in atria and
ventricles; there is no evidence for the expression of HERG1b (Table
I). In both rat and mouse heart, two ERG1 proteins are expressed that
reflect differences in N-linked glycosylation of full-length
ERG1 (rather than coexpression of ERG1b). The results presented here,
therefore, suggest that the ERG1b proteins do not contribute to the
generation of functional IKr channels in human,
mouse, or rat heart. Because the experiments here were performed on
adult tissues, it certainly remains a possibility that ERG1b proteins
are expressed and contribute to the formation of
IKr channels at other developmental stages.
It has also been suggested that accessory K+ channel
subunits that are present in heart contribute (with ERG1) to
IKr (40-42). In AT-1 cells (an atrial tumor
cell line), for example, IKr is attenuated on
exposure to antisense oligodeoxynucleotides targeted against the small
(130 amino acids), single transmembrane spanning domain accessory
K+ channel subunit, minK (40). In addition, heterologously
expressed minK and HERG1 reportedly coimmunoprecipitate (41). The
functional significance of this finding in terms of
IKr is not clear, however, because coexpression
of minK with HERG1 results in voltage-gated K+ currents
that are indistinguishable from those produced on expression of HERG1
alone (41). More recently, a homologue of minK, referred to as MiRP1
(minK-related peptide
1), has been identified that also coassembles with HERG1 in
Xenopus oocytes (42). In contrast to the results with minK
(41), however, coexpression of MiRP1 with HERG1 results in
voltage-gated K+ currents that are very similar to cardiac
IKr, suggesting that IKr
channels are composed of full-length HERG1 and MiRP1 proteins (42).
These observations, together with the results presented here
demonstrating only the expression of full-length ERG1 proteins in rat,
mouse, and human heart, suggest that it is quite unlikely that ERG1b
proteins play a role in the generation of functional cardiac
IKr channels. Further experiments will be
necessary to demonstrate directly that ERG1 and MiRP1 coassemble in
heart in vivo and to determine the stoichiometry of these
subunits in functional IKr channels.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Gail Robertson of the University
of Wisconsin for the HERG1 cDNA, Dr. Barry London of the University
of Pittsburgh for the MERG1 and MERG1b cDNAs, Dr. Hank Duff of the
University of Calgary for the HERG1B cDNA, and Dr. Dianne M. Barry
for the Kv1.2, Kv1.4, Kv2.1, and Kv4.2 clones. In addition, we thank
Michelle Lamorgese and Margaret Kirian for expert technical assistance and Drs. Dianne M. Barry and Haodong Xu for many thoughtful comments and discussions throughout the course of this work.
| |
FOOTNOTES |
*
This work was supported by grants from the Washington
University/Monsanto/Searle Biomedical Research Program (to J. M. N.), the NHLBI, National Institutes of Health (to J. M. N.), and the Medical Research Council of Canada (to A. S.).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.
To whom correspondence should be addressed: Washington
University School of Medicine, Dept. of Molecular Biology and
Pharmacology, Box 8103, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.:
314-362-2564; Fax: 314-362-7058; E-mail:
jnerbonn@pharmsun.wustl.edu.
| |
ABBREVIATIONS |
The abbreviations used are:
PCR, polymerase
chain reaction;
GFP, green fluorescent protein;
CMV, cytomegalovirus;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
PAGE, polyacrylamide gel electrophoresis;
PVDF, polyvinylidene
difluoride.
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REFERENCES |
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