From the Department of Physiology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104
Received for publication, August 20, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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The human ether-a-go-go-related gene (HERG)
product forms the pore-forming subunit of the delayed rectifier
K+ channel in the heart. Unlike the cardiac isoform,
the erg K+ channels in native smooth
muscle demonstrate gating properties consistent with a role in
maintaining resting potential. We have cloned the smooth muscle isoform
of HERG, denoted as erg1-sm, from human and rabbit colon. erg1-sm is
truncated by 101 amino acids in the C terminus due to a single
nucleotide deletion in the 14th exon. Sequence alignment against HERG
showed a substitution of alanine for valine in the S4 domain. When
expressed in Xenopus oocytes, erg1-sm currents had much
faster activation and deactivation kinetics compared with HERG. Step
depolarization positive to The human ether-a-go-go related gene
(HERG)1 encodes for a
K+ channel that is essential for normal repolarization of
the cardiac action potential. HERG was originally cloned from the human
hippocampal cDNA library by homology to the Drosophila
K+ channel gene, eag (1), and according
to the latest International Union of Pharmacology (IUPHAR) nomenclature
has been termed as Kv 11.1. It is strongly expressed in the mammalian
heart, and inherited mutations in this gene cause one form of long Q-T
syndrome, LQT2 (2-4). HERG forms the pore-forming subunit of the
rapidly activating delayed rectifier K+ current,
IKr, in native cardiac myocytes (5, 6), and heterologous expression of the cardiac HERG channel in Xenopus oocytes
and mammalian cells (7, 8) has demonstrated the inwardly rectifying properties of this current. HERG channels slowly activate on
depolarization and demonstrate fast inactivation at positive
potentials, resulting in small outward currents at potentials positive
to 0 mV; hence, inward rectification (9, 10). On repolarization close
to resting potentials, large outward tail currents occur that slowly deactivate. These features of HERG channel dictate its role in repolarization of the cardiac action potential and
frequency-dependent modulation of the action potential
duration (11).
Recent studies also suggest that an inwardly rectifying K+
conductance that is active near the resting membrane potential of gastrointestinal smooth muscle cells (12, 13), pituitary cells (14-16), carotid body (15-17), and microglia (18) has properties similar to that of HERG channels. In these cells HERG conductance has
been directly demonstrated in single cells, and in esophageal and
stomach smooth muscle the presence of a "window" current within the
range of the resting potential and depolarization and contraction by
HERG channel blockers of whole tissue segments strongly suggest a role
for ether-a-go-go-related gene K+ conductance in
maintaining resting membrane potential (12). Moreover, transcripts for
erg1 and immunohistochemical labeling have been shown in
gastrointestinal smooth muscle cells (13). However, it is not clear how
the kinetics of the cardiac isoform of the HERG channels can maintain
the resting membrane potential or repolarization of spike-like action
potentials of gastrointestinal smooth muscle cells. For instance, 1)
the threshold for activation of the heterologously expressed cardiac
isoform of the HERG current and its native form, IKr, is
positive to In this report, we describe the cloning and characterization of the
smooth muscle isoform of erg channels, which we have denoted as erg1-sm, from rabbit and human colon. These studies demonstrate that
erg1-sm is a truncated isoform that lacks 101 amino acids in the
C-terminal region. We also show that there is a unique substitution of
a conserved amino acid in the S4 voltage sensor region between the
cardiac and smooth muscle isoforms that confers significant
hyperpolarizing shift in the voltage dependence of activation for
erg1-sm and results in steady-state conductance within the potential
range for maintaining the resting membrane potential and demonstrates
kinetics that are consistent with this isoform being able to generate
repolarizing current during smooth muscle action potentials.
RNA Extraction and RT-PCR--
Adult male rabbits (New Zealand
White) were anesthetized with sodium pentobarbital, and the colons were
excised. Rabbits were then sacrificed by pentobarbital overdose in
accordance with the Institutional Animal Care and Use Committee of the
University of Oklahoma Health Science Center. Circular muscle strips
were carefully dissected from the underlying mucosa and quick-frozen in
liquid nitrogen followed by homogenization. Total RNA was isolated using the S.N.A.P. total RNA isolation kit (Invitrogen) as per the manufacturer's instructions. In separate experiments, rabbit colon
was embedded in OCT, and cross-sections were prepared in a
cryostat. Cross-sections of the colon were placed on glass slides, fixed in alcohol, dehydrated, dipped in xylene, and placed on the
microscope of the Arcturus PixCell II system (Arcturus Engineering Inc.). Single smooth muscle cells were selected by laser capture with a 7.5-µm spot size. Approximately 40-80 cells were collected for RNA isolation. The caps were placed in a 0.5-ml RNase-free Eppendorf tube and solubilized in lysis buffer (S.N.A.P. kit). RNA was
isolated according to the manufacturer's instructions. Human colon,
mouse small intestine, and mouse heart total RNA was purchased from
Clontech (Palo Alto, CA). All RNA samples were quantified by spectrophotometer and run on denaturing RNA gels to check
the quality and integrity of total RNA. First-strand cDNA was
synthesized using 50 units of Superscript 11 RT (Invitrogen) at
42 °C for 50 min in the presence of 3 µg of total RNA, 1 µg of
oligo-dT primers, and 10 mM dNTPs in a total 20-µl
reaction. Three 5-µl aliquots of reverse-transcribed reactions were
used for performing PCR. Appropriate controls were carried out using water or omitting the enzyme.
Primer Design--
The following PCR primers were used. Numbers
in parenthesis are GenBankTM accession numbers. For
erg K+ channels, the primers were designed to
amplify the entire coding region from the published sequence for Merg1
(NM-013569). The sense primer was (nt 317-336)
5'-CGGATCCATGGGCTCAGGATGCCGGTG-3'). The antisense primer (nt
3862-3883) (5'-CGATATCCCAAGGAGAGCGGTCAGGTAAT-3') was
designed 45 nucleotides downstream of the stop codon in the 3'-untranslated region. BamH1 and EcoRV
restriction sites were added to the forward and reverse primers,
respectively (shown in italics), to facilitate subcloning between the
BamH1 and SmaI site of pSP64 vector.
For detection of erg transcript in single smooth muscle
cells selected by laser capture, the sense and antisense primers were designed to amplify a 894-bp amplicon. The sense primer corresponds to
nt 1817-1838, and the antisense primer corresponds to nt 2711-2729 (NM-013569). Neuronal-specific primers were designed from the sequence
for PGP 9.5 (D10699) with sense (nt 34-53)
(5'-GGAGATTAACCCCGAGATGC-3') and antisense (nt 344-363)
(5'-TTGTCGTCTACCCGGCACTG-3') to reveal an amplicon of 329 bp. To detect
interstitial cells of Cajal, primers were designed against the c-Kit
receptor (X06182), sense (nt 2259-2283)
(5'-ATACATAGAAAGAGATGTGACTCC-3') and antisense (nt 2873-2897)
(5'-GAATTGATCCGCACAGAATG-3'). To detect the transcript for
ergB, the following primers were used based on the sequence for Merg1b (22) (AF034762): sense (nt 366-387)
(5'-CAGAAAGGCCGGGTGAGGCGG-3') and antisense (nt 865-885)
(5'-GCCGCAGCAGCCTCGCAGTTT-3'), resulting in an amplicon of 519 bp.
PCR and Cloning of erg Gene--
1 µg of cDNA made from
total RNA of rabbit circular muscle strips and human colon, 2.5 units
of Expand Long Template Taq DNA polymerase (Roche Molecular
Biochemicals), 10 mM dNTPs, 10 µM BamH1 forward, and EcoRV reverse primers in a
total reaction volume of 50 µl were used for PCR amplification of the
erg gene. Controls were run in the absence of template DNA.
PCR was performed in Robocycler (Stratagene, La Jolla, CA) under the
following conditions: 1 cycle at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C
for 30 s, and extension at 68 °C for 3.5 min for the first 10 cycles. Thereafter, the extension time was increased by 20s/cycle,
followed by final extension at 68 °C for 7 min. 10 µl of RT-PCR
products were separated on 1% agarose/1× Tris-acetate-EDTA
gel, and the DNA bands were visualized by ethidium bromide staining.
The full length of the erg PCR product was gel-purified
using Wizard PCR prep purification system (Promega, Madison, WI),
digested with BamH1 and EcoRV, and ligated
between the BamH1 and SmaI site of pSP64 poly(A)
vector (Promega) using T4 DNA ligase (Promega). The ligated product was
transformed in DH5 Mutagenesis--
To introduce single amino acid substitutions in
the S4 region of erg, the QuikChange site-directed
mutagensis kit (Stratagene) was used according to the manufacturer's
protocol. Primers were designed such that the nucleotide substituted
was located in the middle of the forward and reverse primers.
For creating S4 HERG mutant, the forward primer was
5'-GCGGCTGGTGCGCGCGGCGCGGAAGCT-3', and the
reverse primer was
5'-AGCTTCCGCGCCGCGCGCACCAGCCGC-3' (NM_000238).
For creating S4 erg1-sm mutant, the forward primer was
5'-CTGCGGCTGGTGCGCGTGGCTCGGAAGCTGG-3', and
reverse primer was
5'-CCAGCTTCCGAGCCACGCGCACCAGCCGCAG-3'
(AF439342). These primers were used to amplify the mutant from wt
parental erg. Parental DNA was digested after PCR with
DpnI and transformed into XL Blue supercompetent cells.
Colonies were screened for the mutant plasmid and confirmed by DNA
sequencing. HERG and erg1-sm S4 mutant plasmid DNA were linearized with
EcoR1, and cRNA was synthesized in vitro as
described above.
Protein Analysis--
In vitro translation of smooth
muscle erg and HERG plasmid DNA was performed using TNT®
coupled reticulocyte Lysate system (Promega) in combination with the
Transcend TM nonradioactive translation detection system for
incorporation of biotinylated lysine. 2 µg of smooth muscle
erg and HERG plasmid DNA was translated following the
manufacturer's instructions. The SP6 luciferase plasmid DNA was used
as a control. The in vitro translated protein products were
subjected to electrophoresis on 7.5% SDS-PAGE, transferred on to
nitrocellulose membranes, and blocked by incubation in 15 ml of TBS-T
(TBS with 0.5% Tween 20) for 1 h with gentle shaking. The
membrane was incubated in streptavidin-HRP conjugate (1:5000) for
1 h, and washed in 15 ml of TBS-T 3 times followed by several washes with deionized water. Streptavidin-HRP that binds incorporated biotinylated lysine in translated proteins was detected by
chemiluminescence according to the manufacturer's instructions.
Immunoblots were performed to detect the presence of the HERG C
terminus using HERG polyclonal antibody directed against the C-terminal
peptide sequence corresponding to amino acids 1106-1159 of HERG
(Alomone Laboratories, Jerusalem, Israel). Proteins from plasmid
DNA were translated in a similar fashion as above, except for the
omission of biotinylated lysine. These proteins were subjected to
electrophoresis on 7.5% SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes and blocked in 10 ml of 5% milk in TBS-T
(TBS with 0.1% Tween 20) for 1 h and subsequently probed with the
primary antibody directed against HERG C-terminal at a concentration of
1:200 in TBS for 1.5 h at room temperature. The membranes were
washed twice with TBS-T and incubated for 1 h with HRP-linked
secondary antibody (Santa Cruz Biotechnology). After the final washing
steps, the blots were visualized by enhanced chemiluminescence. A
similar procedure was used for detection with the anti-N-terminal HERG
antibody (1:1000) (kindly provided by Dr. Jeanne Nerbonne).
Isolation of Membrane Protein from Mice Colonic
Tissues--
Colons were excised from four mice, and muscle strips
were dissected under a microscope. Muscle strips were flash-frozen in liquid N2 and homogenized in 5 ml of TE buffer at pH 7.4. All buffer solutions contained the following protease inhibitors: 1 mM iodoacetamide, 1 mM phenanthroline, 7.9 µM aprotinin, 1 mM benzamidine, 1.4 µM pepstatin, 0.5 mM Pefabloc, 0.1 µM leupeptine, and a protease inhibitor mixture tablet
(Roche Molecular Biochemicals). The homogenized samples were
centrifuged at 1000 × g for 10 min. This procedure was
repeated twice to remove all nucleic acid, and debris and supernatant
from both spins were collected and centrifuged at 40,000 × g for 10 min. The pellet was resuspended in TE buffer
containing 0.6 M KI, incubated on ice for 30 min, and
centrifuged at 40,000 × g for 10 min. The final pellet
was solubilized in TE (2% Triton X-100) on ice for 1 h and
centrifuged at 17,400 × g for 30 min. Membrane protein
concentrations were measured and subjected to electrophoresis on
SDS-PAGE. The membrane proteins were probed with anti-N terminus HERG
antibody (1:1000) and anti-C terminus HERG antibody (1:200) (Alomone).
Electrophysiological Recordings--
Female Xenopus
laevis (Nasco, Fort Atkinson, WI) were anesthetized by a
20-30-min exposure to 3-aminobenzoic acid ethyl ester (tricaine 1.5 g/liter). All protocols were approved by the Institutional Animal Care
and Use Committee of the University of Oklahoma Health Science Center.
Ovarian lobes were removed through a small incision in the abdominal
wall and washed in a Ca2+-free OR-2 solution containing
82.5 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 5 mM HEPES (pH adjusted with NaOH).
Stage IV and V Xenopus oocytes were defolliculated by
treatment with 1 mg/ml collagenase (Type 1A; Sigma) in the OR-2
solution for 1.5 h. Oocytes were incubated at 18 °C in a
modified Barth's solution with antibiotics containing 96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 2.5 mM HEPES (free acid), 2.5 mM HEPES (sodium
salt), 0.1 g/liter streptomycin sulfate, 0.27 g/liter pyruvic acid, and
0.5 g/liter gentamycin sulfate. On the following day, oocytes were
injected with the appropriate cRNA (1.1-1.6 µg/µl) using a
Drummond Nanoject II automatic nanoliter injector (Drummond Scientific
Co., Broomall, PA). Currents were recorded by two-electrode voltage
clamp using the Geneclamp 500B amplifier (Axon Instrument, Foster City,
CA) 2-5 days after cRNA injection. All recordings were carried out at
room temperature (20-23 °C) in Barth's solution without antibiotics.
The pClamp software (Axon Instruments) was used for generation of
voltage-clamp protocols and data acquisition. Currents were evoked by
2-s depolarizing voltage steps from
The voltage dependence of steady-state inactivation (rectification
factor) was obtained using protocol as previously described (5, 23).
Channels were fully activated by voltage steps to +30 mV (5 s) followed
by test potentials from
To obtain the time constants of activation, long duration depolarizing
pulses (15 s) were applied. Selected raw traces from
To assess the rate of deactivation, a two-pulse protocol was used. A
prepulse of +30 mV (5s) was applied to fully activate and inactivate
currents following by a range of pulses from To determine the physiological properties of the smooth muscle
isoform of erg, we cloned and characterized the full-length cDNA from circular smooth muscle of the rabbit colon using RT-PCR. Fig. 1A shows the presence of
the full-length transcript for erg1 (~3.5 kilobases) from
the rabbit colon, human colon, and mouse heart. A similar size band was
also identified in mouse small intestine (data not shown). In addition
to the presence of erg1, the alternatively spliced isoform,
ergB, was also detected in the human colon (Fig.
1B). To further confirm that erg1 transcript is
present in smooth muscle cells, about 40-80 cells were selected by
laser capture microdissection. Multicell PCR of captured cells showed
the presence of erg1 without contamination from either neurons or interstitial cells of Cajal (Fig. 1C). Although
no bands were seen for neuronal or interstitial cells of Cajal in selected smooth muscle or in circular muscle strips, the fidelity of
these primers was confirmed using whole colon tissue (data not
shown).
20 mV consistently produced a transient
outward component. The threshold for activation of erg1-sm was
60 mV
and steady-state conductance was ~10-fold greater than HERG near the
resting potential of smooth muscle. Site-directed mutagenesis of
alanine to valine in the S4 region of erg1-sm converted many of the
properties to that of the cardiac HERG, including shifts in the voltage
dependence of activation and slowing of deactivation. These studies
define the functional role of a novel isoform of the
ether-a-go-go-related gene K+ channel in smooth muscle.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
50 mV (5, 19), whereas the resting potential of
gastrointestinal smooth muscle generally lies around
60 mV, 2) unlike
the native cardiac myocytes, where large tail currents occur upon
repolarization (20, 21), smooth muscle cells do not demonstrate large
tail currents upon repolarization, and 3) it is not clear how recovery
from inactivation of the cardiac HERG can generate sufficient outward
current during repolarization of shorter duration of action potential
trains that occur from a plateau potential of
30 mV in
gastrointestinal smooth muscle.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Actin
(V01217) sense (2383-2402) and antisense (3071-3091) primers were
designed to span an intron in addition to two exons. Thus contamination
by genomic DNA would be detected as a band of 708 bp rather than the
expected size of ~500 bp.
Escherichia coli cells. Colonies were
screened for positive clones by plasmid DNA isolation using Qiagen
plasmid miniprep kit (Qiagen, Valencia, CA). The orientation and
fidelity of all the resulting plasmid DNA was confirmed by running it
on 1% agarose gel after restriction digestion (EcoR1 and
XhoI). Three independent clones were sequenced in their
entirety using the dye-termination method on an ABI Prism (Foster City,
CA) automated sequencer. Possible sequence changes due to expand long
template PCR enzyme polymix-catalyzed replication errors were
examined by comparison of sequences from a minimum of three independent
PCR reactions. Nucleotide sequence common to at least two clones were
considered correct due to the low probability of proofreader-introduced
identical alterations at the same nucleotide position. For expression
studies, the plasmid DNA in pSP64 was linearized with EcoR1
and transcribed into cRNA by mMessage mMachine kit (Ambion, Austin, TX)
following the manufacturer's instructions. All cRNA samples were
quantified by UV spectroscopy, and size and integrity was checked on
denaturing RNA gels using appropriate RNA markers. HERG in pSP64 was
kindly provided by Dr. Peter Spector (University of Oklahoma).
70 mV to +50 mV in 10-mV
increments followed by a repolarizing step to
70 mV. The amplitude of
HERG and erg1-sm currents was measured at the end of depolarizing steps
then normalized and plotted against the voltage to obtain the I-V
relationship. Steady-state activation curve was determined by
normalizing the peak of tail current (I) at
70 mV, plotted against
the test potential (Vt), and fitted to a Boltzmann
function I = Imax/(1 + exp[(V0.5
Vt)/t]), where
Imax is the maximum amplitude of tail current.
140 mV to +40 mV to obtain the
current-voltage relationship of activated channels. Slope conductance
was determined from the negative arm of the I-V curve, and the
rectification factor was calculated using the formula R = I/G (Vm
EK),
where R is the rectification factor, and
EK was
95 mV. Data were fit by the Boltzmann relationship.
40 mV to
10 mV
for HERG current and from
50 mV to
20 mV for Smerg currents were
fitted with double-exponential function to obtain slow and fast time
constants. Time constants for inactivation were determined by using
three-pulse protocol similar to that previously described (24).
140 mV to +40 mV with
20-mV increments. Deactivation time constants were obtained by a
double-exponential fit.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of ether-a-go-go-related gene
(ERG) channels in smooth muscle. A,
RT-PCR detection of full-length cDNA transcript from rabbit colon
(lane 2), human colon (lane 3), and mouse heart
(lane 4). Lane 1 represents 1-kilocase markers.
B, RT-PCR product for the HERGB isoform from human colon
(lane 1). Gene-specific primers were designed to generate a
fragment of ~519 bp. The viability of samples was assessed by the
presence of cDNA for -actin (lane 2) whereby the
primers were designed to span an intron in addition to two exons. The
amplification product of 500 bp indicates no genomic DNA contamination,
which would have resulted in a 708-bp product. C,
erg1 transcript in single smooth muscle cells selected by
laser capture microdissection (lane 2). The cDNA was
checked for the presence of interstitial cells of Cajal using primers
specific for cKit (lane 3) and for neurons with
gene-specific primers for PGP 9.5 (lane 4). Lane
5 shows
-actin transcript. Lanes 1 and 6 are 100-bp markers.
The full-length transcripts for erg1 from the rabbit and
human colon were sequenced using overlapping primers. In an initial analysis, these sequences were aligned against the HERG (NM_000238) and
found to be ~87% identical at the nucleotide level and ~95% at
the protein level (deposited in GenBankTM, rabbit colon
accession number AF439342; human colon accession number AY130462).
Within the transmembrane spanning region (S1 to S6), the sequences were
identical except for the presence of a conserved amino acid
substitution, alanine for valine at position 537, in the S4 region of
the smooth muscle isoforms from human and rabbit erg (Fig.
2). This position corresponds to 535 for
HERG due to two additional amino acids in the N terminus of the smooth
muscle isoforms. The single amino acid substitution within the S4
region was also identified in the rabbit stomach smooth muscle and, as
shown below, confers important kinetic properties that define its role
in maintaining smooth muscle-resting potential and repolarization of
the action potential. We have denoted the smooth muscle isoforms as
erg1-sm.
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The open reading frame of the smooth muscle isoforms (erg1-sm)
consisted of 1058 amino acids as compared with 1159 amino acids for
HERG, resulting in a truncated C terminus of 101 amino acids. This
truncation was due to a frameshift resulting from a single-base deletion of G, corresponding to the position 3159 in HERG producing a
premature stop codon in the 14 exon (Fig.
3) (25, 26) in both human and rabbit
colon. However, a similar deletion was not observed in cDNA from
the rabbit atrial tissue, which was sequenced by RT-PCR using the same
primers as for the smooth muscle.
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To confirm that the smooth muscle isoform results in a truncated
protein, the plasmid DNA for erg1-sm and HERG were translated in
vitro and probed with streptavidin-HRP for detection of the proteins. As shown in Fig. 4A,
the protein bands for HERG were obtained around the expected size of
127 kDa. The erg1-sm band was about 10 kDa smaller in size as would be
expected for a 101-amino acid truncated protein. However, a faint band
around 127 kDa was also observed in both erg1-sm as well as in control
(luciferase DNA) lanes, indicating an additional nonspecific protein
band may be present around this molecular weight. The presence of HERG protein was, therefore, further examined by immunoblots carried out
using anti-HERG antibody that was raised against the C terminus. Fig.
4B shows that this antibody recognized the presence of HERG but did not show bands from erg1-sm or control proteins. The
anti-N-terminal HERG antibody was raised against residues 174-188 of
HERG, which is almost identical to that in erg1-sm. Fig. 4C
shows that immunoblot with anti-N-terminal antibody revealed a band of
127 kDa for HERG and a slightly lower band for erg1-sm (~120 kDa),
consistent with the hypothesis that erg1-sm is a truncated isoform of
HERG. To determine the expression of erg1-sm in native colonic tissues, immunoblots of isolated membrane proteins were carried out using the
anti-C-terminal and anti-N-terminal HERG antibodies. Fig. 4,
D and E, show that the major band revealed by
anti-C-terminal antibody was around 95 kDa, which is expected for
erg1b isoform. Larger molecular weight bands corresponding
to glycosylated forms of erg1 (27) were not detected in
colonic smooth muscle. The anti-N terminus antibody revealed a band
~120 kDa that most likely represents erg1-sm. Taken together these
data suggest that erg1-sm is truncated and that previous findings of
immunohistochemical localization of erg1 in single smooth
muscle cells using the anti-C terminus antibody (12) represent
erg1b expression.
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Properties of erg1-sm Channels-- To compare the functional properties of the smooth muscle isoform to the cardiac isoform of HERG, currents were measured from Xenopus oocytes expressing erg1-sm or HERG. Preliminary studies suggested that expression of erg1-sm currents were significantly lower than that for HERG and required at least 3-4 days for full development of currents when approximately equal amounts of RNA were injected. To obtain currents of comparable amplitude, the volume of RNA injection was doubled for erg1-sm.
Upon depolarization from a holding potential of 70 mV, erg1-sm
K+ channels activated at potentials positive to
60 mV. As shown in Fig. 5, the magnitude of
this current increased up to
20 mV, and thereafter, larger
depolarizations resulted in a progressive decrease in the amplitude, as
expected for the inwardly rectifying properties of erg
channels. At potentials positive to
20 mV, transient currents
were elicited whose rate of activation and the amplitude increased with
increasing step depolarization (inset, Fig. 5A).
Upon repolarization to the holding potential of
70 mV, tail currents
with fast deactivation kinetics were obtained. The currents from
erg1-sm were abolished by the class III anti-arrhythmic blocker, E-4031
(10 µM) (Fig. 5B). In comparison, the currents from HERG-injected oocytes demonstrated much slower activation at
negative potentials, with peak currents obtained at 0 mV. Unlike erg1-sm, currents from HERG showed no fast transient components at
positive potentials (inset, Fig. 5A, bottom
panel). Upon repolarization to
70 mV, tail currents from HERG
had much larger amplitudes and deactivated slowly compared with
erg1-sm. The current-voltage relationship measured at the end of the
test pulse showed a bell-shaped response with erg1-sm demonstrating a
leftward shift of 20 mV compared with HERG (Fig.
5C).
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The voltage dependence of activation was measured as the relative
amplitude of tail currents and plotted as a function of test potential.
This isochronal activation curve had a V1/2 of 36 ± 0.7 mV with a slope factor 10 for erg1-sm and a more
positive V1/2 potential of
16 ± 0.4 mV
(slope factor 9.5) for HERG (Fig. 6A). The voltage dependence of
the steady state inactivation (the rectification factor) was measured
using protocols as described under "Materials and Methods." The
V1/2 for erg1-sm was
26 ± 2.5 mV with a
slope factor of 17 mV, whereas that for HERG was
48 ± 1 mV with
a slope factor of 24 (Fig. 6B). The rightward shift meant
that erg1-sm channels had less steady-state inactivation, particularly
around the resting membrane potential of smooth muscle (between
70 mV
and
50 mV). To determine the voltage dependence of the steady-state
conductance, the fraction of channels activated at each potential was
multiplied by the fraction of channels inactivated at that potential.
The resulting window current showed a significant amount of
steady-state current for erg1-sm channels between
60 and
50 mV
(Fig. 6C). Within this potential range, steady-state current
was almost 10-fold greater for erg1-sm than HERG, consistent with the
findings in native smooth muscle cells of erg-like currents maintaining the resting membrane potential (12, 13).
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Both activation and inactivation rates were much faster for erg1-sm
than HERG (Fig. 7). Activation was fit by
a double-exponential, and at almost all voltages (between 50 mV and
20 mV) time constants for activation for erg1-sm were almost 3 times
smaller than for HERG currents (Fig. 7B). The time constant
for inactivation was measured using a three-pulse protocol. The oocytes
were depolarized to +40 mV for 5 s followed by a short pulse for
15 ms to
140 mV to remove inactivation, and re-inactivation was
induced at various test potentials from
60 mV to +40 mV. The
resulting currents were fit by a single exponential and demonstrated
voltage dependence. As shown in Fig. 7C, the time constant
for inactivation of erg1-sm was significantly smaller than that for
HERG at all potentials. The rate of activation was measured by
"envelope" of tail currents as shown in the voltage protocol
of Fig. 7D. The rate of activation was also faster for
erg1-sm than HERG.
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Upon repolarization to 70 mV, tail currents of much smaller amplitude
were recorded consistently in erg1-sm-injected oocytes compared with
HERG. HERG channels transition into an open state on repolarization
before slowly closing, leading to large amplitude of tail currents.
Although similar transition was evident in erg1-sm at potentials
positive to
20 mV where inactivation occurs, the amplitude of
the tail currents was similar to that of the amplitude of the transient
outward current at that potential. The significantly smaller amplitudes
of the tail currents are also consistent with findings from native
cells, where tail currents due to K+ channel activation are
quite small and do not show the typical hook observed for ventricular
myocytes (19). The most likely explanation for the smaller amplitude of
tail currents is the faster rate of deactivation in erg1-sm. Fig.
8A shows tail currents obtained upon repolarization to
60 mV from a test potential of +30
mV. Deactivation was significantly faster for erg1-sm than HERG between
80 mV and
40 mV (Fig. 8B).
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To assess whether the biophysical characteristics of erg1-sm currents
are consistent with a physiological role in the repolarization of
smooth muscle action potential, a voltage protocol was employed that
mimicked the electrical activity of gastrointestinal smooth muscle
cells. Contractions of gastrointestinal smooth muscle are elicited by
multiple action potentials that are superimposed on an underlying
rhythmic slow wave activity (28). The upstroke of the action potentials
is mediated by activation of L-type Ca2+ channels that
occur during the plateau phase of the slow wave. The duration of the
action potentials can also vary during the train. Activation of outward
currents from erg1-sm and HERG-injected oocytes were examined using
ramp depolarizations that varied in the inter-spike interval. Ramp
depolarization was applied from 70 mV to
30 mV (100 ms) to mimic
the upstroke of the slow wave that was then followed by the first spike
by ramping from
30 mV to +20 mV in 100 ms and repolarizing back to
40 mV (50 ms). The voltage was then slowly ramped back to
30 mV in
150 ms to mimic the inter-spike interval, which was followed by a
second spike as shown in the upper traces of Fig. 9
(left-hand panel). The pattern
of currents obtained from erg1-sm and HERG-injected oocytes with this
protocol is illustrated in Fig. 9 (left-hand panel). In
both, depolarization from
70 mV to
30 mV produced little outward
currents; however, in the erg1-sm-injected oocytes (middle
panel) a transient outward current was evoked during the first
spike that partly inactivated at the peak of depolarization. Upon
repolarization to
40 mV, currents slowly activated and maintained a
plateau during the inter-spike interval; thereafter, further depolarization to +20 mV (the second spike) resulted in relaxation of
the outward currents as the channels reverted to an inactivated state.
Repolarization of the second spike and return to the holding potential
resulted in significant outward currents similar to the tail currents
obtained during step repolarization.
|
The same voltage protocol evoked a different current profile from HERG-injected oocytes (Fig. 9, bottom panel). During the first spike, there was no transient component, and the currents activated slowly until the end of the inter-spike interval. Thereafter, currents decreased during the upstroke of the second spike due to inactivation, and large tail currents were observed upon repolarization similar to the erg1-sm-injected oocytes.
The differences between erg1-sm and HERG became more apparent when the duration of the spikes was decreased (Fig. 9; right-hand panel). With shorter durations, the outward currents from erg1-sm were evident during the first spike and regenerated with the second spike, whereas they were absent in HERG. These data illustrate the contribution of the erg1-sm channels to multi-spike potentials, particularly with respect to shorter action potential durations that can be frequently obtained in native gastrointestinal smooth muscle tissues.
Role of Amino Acid Substitution in the S4 Region--
Sequence
alignment of the rabbit colonic smooth muscle transcript showed a
substitution in the S4 region of an alanine (Ala-537) in erg1-sm to
valine (Val-537) in HERG (Fig. 2). Sequence by RT-PCR of transcripts
from other smooth muscle tissues of the S4 region identified the same
substitution of alanine for valine in rabbit stomach and human colon.
In comparison across species, the mouse heart, canine heart, and rabbit
heart all contain valine at this position (Fig.
10A). Fig. 10B
shows the sequence chromatogram, demonstrating that this substitution
occurred as a result of a single nucleotide change of C to T at
position 1610 in smooth muscle. To determine whether this uncharged
conserved amino acid substitution in smooth muscle results in the
changes in the biophysical properties of the channel, the alanine in
erg1-sm was mutated to valine by site-directed mutagenesis. Similarly,
valine (535) was also mutated to alanine in HERG. All mutations were
confirmed by DNA sequencing, transcribed to cRNA, and expressed in
oocytes.
|
Mutation of alanine to valine in the S4 mutant of erg1-sm (A537V)
converted many of the biophysical properties similar to that for HERG
(Fig. 10C). The A537V conversion resulted in loss of the
transient component and produced large outward tails upon repolarization, similar to those obtained with HERG. The
current-voltage relationship was also shifted to more positive
potentials with a peak at 10 mV. In contrast, mutation of V535A in
HERG resulted in transient currents at potentials positive to
20 mV, with peak currents shifted to
20 mV compared with 0 mV in
wt-HERG (Fig. 10D). Moreover, tail currents were also
smaller to peak currents, comparable with that of the wt-erg1-sm
with fast deactivation kinetics. Fig.
11A shows the changes in the
voltage dependence of activation, which was shifted from a
V1/2 of
36 ± 0.7 mV in wt-erg1-sm to
23 ± 0.8 mV in the erg1-sm S4 mutant. In contrast, the S4
mutant of HERG had a mid-point of steady-state activation of
66 ± 12 mV compared with
16 mV for the wild type. The rate of
activation was also shifted to the left for the V535A mutant of HERG
and rightward for A537V mutant for erg1-sm from its corresponding wild
type (Fig. 11B). The activation time constants were slower in the S4 V537A mutant of erg1-sm and enhanced in the S4 mutant of HERG
(not shown). Interestingly, the time constant for inactivation was not
significantly changed by the S4 mutations. Thus, inactivation in V535A
in HERG remained slower than that in A537V in erg1-sm (Fig.
11C) similar to those obtained with the corresponding wild types. However, the rate of deactivation was markedly slower in the
erg1-sm S4 mutant compared with the HERG S4 mutant (Fig.
11D).
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DISCUSSION |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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The findings of the present study demonstrate the functional
properties of the smooth muscle isoform of the ether-a-go-go-related gene K+ channel and show the differences compared with the
cardiac isoform. At least three members of the ether-a-go-go-related
gene family, namely erg1, erg2, and
erg3, have been previously identified. erg1 is
present in several tissues including strong expression in the heart (2,
29) and brain (1). erg2 and erg3 are neuronal-specific isoforms (23). A weak signal for the presence of
erg1 in smooth muscle was also previously reported in the
small intestine (30) and stomach (22). Subsequently, patch-clamp studies in single smooth muscle cells from the opossum esophagus (12),
rat stomach (13), and murine portal vein (31) have demonstrated the
presence of HERG-like K+ channels in native cells. HERG
channel blockers, including the gastrointestinal prokinetic agent,
cisapride, were found to depolarize smooth muscle cells, and the
presence of a window current around the typical resting potential of
50 mV strongly indicates that these channels were responsible for
maintaining the resting membrane potential. In the present study, we
show that the cloned erg K+ channel from
gastrointestinal smooth muscle shares almost 95% homology at the amino
acid level with the cardiac isoform up to the truncation at the
C-terminal end. However, a critical substitution of an alanine for
valine within the S4 region is present in several gastrointestinal
smooth muscle cells, and this substitution confers a hyperpolarizing
shift in the activation threshold and a significantly larger
steady-state conductance around the resting membrane potential. Moreover, differences in the rates of activation, recovery from inactivation, and deactivation compared with the cardiac HERG define
the mechanisms for the smooth muscle isoform in the repolarization of
multiple spike-like action potentials that are typically observed in
native gastrointestinal smooth muscle cells.
The smooth muscle isoforms contained a premature stop codon that occurs in the 14th exon, resulting in a truncated C terminus. erg1 consists of 15 exons and at least 2 alternatively spliced variants of HERG1 have been identified in the heart, HERGB and HERGuso. HERGB and its mouse counterpart, MergB, appear to contain a unique N terminus (22, 32) that on co-injection with HERG1 results in a current that approximates native cardiac IKr. However, recent studies by Pond et al. (29) suggest that HERGB protein may not be expressed in the heart. HERGuso is alternatively spliced at exon 9 and leads to a truncated isoform (33). However, expression of HERGuso in mammalian cells does not appear to generate a functional channel. erg1-sm on the other hand appears to encode for a functional protein that is truncated at the C terminus. The in vitro translation of erg1-sm resulted in a protein band that was ~10 kDa smaller compared with HERG, as would be expected for a 101-amino acid truncation. Furthermore, immunoblots using an anti-HERG antibody that is raised against the C-terminal amino acids 1106-1159 of HERG while recognizing the HERG protein failed to detect erg1-sm. This is in contrast to the immunohistochemical localization of HERG in single smooth muscle cells using a similar antibody (12, 13). A likely explanation for this is that smooth muscle cells also appear to express the ergB isoform that would, thus, result in immunolocalization of the C terminus (Fig. 1) (22, 32). Further confirmation that erg1-sm is truncated in native tissues was obtained by using the anti-N-terminal HERG antibody. This antibody recognized the in vitro translated protein as well as a 120-kDa protein in colonic membrane. Further studies are, however, necessary to define whether the ergB isoform from smooth muscle makes functional channels alone or in combination with erg1-sm.
Although erg1-sm currents showed similar inward rectification as the
HERG channel, notable differences were the presence of a large
transient component at potentials positive to 20 mV, a lower
threshold for activation, a larger steady-state conductance around the
resting membrane potential, and faster activation, inactivation, and
deactivation kinetics. These differences are consistent with the
ability of the smooth muscle isoform to provide significant outward
currents during smooth muscle action potential, particularly from a
plateau phase of the gastrointestinal slow wave. The transient
component is only occasionally seen in cardiac HERG (34, 35) and mainly
at potentials positive to +20 mV. In single channel recordings, Kiehn
et al. (34) show that HERG channels undergo direct
closed to inactivated states, and the absence of a transient component
allows for the delayed repolarization of the cardiac action potential.
The upstroke of the action potential in smooth muscle cells is
principally mediated by L-type Ca2+ channels, whose
threshold lies around
30 mV, at the plateau of the slow wave. The
action potentials are also of shorter duration, and as demonstrated by
using voltage protocols that mimic the gastrointestinal smooth muscle
action potential, erg1-sm currents can activate during the first action
potential spike. HERG on the other hand requires significant longer
duration to fully activate and contribute to this type of action
potential, as would be expected with slower activation kinetics.
There are several similarities between the current profile of erg1-sm
and the nervous system specific isoform, erg3. Shi et al. (23) show that erg3 produces a transient component
at potentials of +20 mV and has a more negative threshold for
activation and a larger steady-state conductance around 50 mV than
erg1. These properties allow for substantial contribution of
these channels in the shorter duration action potentials in neurons.
The transient component of erg3 occurs as a result of much
slower rate of inactivation compared with erg1. However, in
erg1-sm we found that the rate of inactivation was considerably faster
than HERG. Moreover, the activation of the transient outward component
appears to depend on the presence of an alanine in the S4 voltage
sensor region, whereas erg3, like HERG, contains a valine in
this position. erg3 is also only 57% identical to
erg1, whereas erg1-sm is ~95% identical at the amino acid
level up to the truncated C-terminal portion.
It is noteworthy that in several gastrointestinal smooth muscle tissues there is a substitution of an alanine for valine in the S4 region. The S4 segment of voltage-gated ion channels is highly conserved, consisting of repeating basic residues. The S4 segment is proposed to function as a voltage sensor with positively charged residues critical in voltage-dependent transitions for activation (36). Recent studies by Smith-Maxwell et al. (37, 38) suggest that single conserved uncharged amino acid substitution within the S4 region may be important in cooperativity and voltage dependence of Shaker K+ channels. These authors demonstrated that substitution of leucine for isoleucine (that differ in the attachment of a methyl group) resulted in changes in the gating charge movement. This could be attributed to steric interactions within the S4 region. Mutation of an alanine to valine in erg1-sm resulted in loss of the transient component and shifted the voltage dependence of activation to more positive potentials. Moreover, there was an obvious increase in the amplitude of the tail currents as well as a decrease in the rate of deactivation. In contrast, mutation of valine in HERG to alanine resulted in a transient component, leftward shift in the threshold and voltage dependence of activation and a faster rate of deactivation. These findings are consistent with interactions between the amino and carboxyl termini with the S4 region. Deactivation of the HERG channels is strongly dependent on the initial N-terminal residues that constitute the PAS domain (39, 40). Previous studies from several laboratories have confirmed that the interaction of the PAS domain with the channel core, including the S4 region (41) and/or the S4-S5 linker (42), determines the slow deactivation kinetics of the HERG channel. The PAS domain is identical in erg1-sm and HERG and suggests that the faster deactivation process may be related to the interaction with the S4 domain. Thus, mutation of the S4 alanine to valine resulted in significant slowing of the deactivation process, similar to that for wt-HERG. At present, it is not clear how single nucleotide substitution occurs within the S4 region of smooth muscle isoform. However, it is interesting to note that the C to T substitution is reminiscent of RNA editing. Further experiments to determine whether this may be due to specific RNA editing in smooth muscle cells will be required.
In addition to the initial PAS domain, the stretch of amino acids from 138 to 373 (just before the S1 region) can also affect channel activation. The deletion of this segment appears to accelerate channel activation and shifts voltage dependence to hyperpolarized potentials (43 (44). There are several differences in the amino acid sequence between erg1-sm and HERG within this region including the addition of two amino acids in the smooth muscle isoform that could account for both faster rates of activation and hyperpolarized shift in the voltage dependence. However, further studies with specific mutations in this domain are needed to confirm the role of the N-terminal domain.
Interestingly, inactivation was significantly faster in erg1-sm than HERG and, unlike activation or deactivation, was not altered by S4 mutation. This could be related to the specific interaction of the C and N termini in maintaining the inactivation gate. Aydar and Palmer (45) demonstrate that truncation of the C terminus of HERG resulted in faster deactivation kinetics and that double deletion mutants of N and C termini increased channel inactivation time constants. It is possible that in erg1-sm a truncated C terminus specifically interacts with the N terminus, resulting in faster inactivation. The C terminus is also known to be responsible for expression. Kupershmidt et al. (46) recently demonstrated that removal of the C-terminal 147 amino acids resulted in endoplasmic reticulum retention of the HERG channel. However in mutants in which the RXR motif is absent, there is robust expression of functional channels. Our findings that expression levels were reduced in erg1-sm-injected oocytes could be related to the presence of the RGR sequence. The fact that erg1-sm is expressed, albeit to a lesser extent, suggest that part of the forward trafficking domain and/or protein folding is intact. These findings are also consistent with lower expression levels in smooth muscle, where high K+ concentrations are required to identify HERG-like currents (12).
In conclusion, our studies demonstrate several features of the smooth
muscle isoform of the ether-a-go-go-related gene K channel that may
explain its role in the native tissues. (a) The negative threshold for activation and steady conductance around the resting potential is consistent with these channels maintaining the resting membrane potential, (b) the faster rate of activation and
recovery from inactivation define the role of erg1-sm in repolarization of the action potential and of gastrointestinal slow waves,
(c) the lower levels of expression of erg message
in smooth muscle may be due to a truncated C terminus, and
(d) substitution of a conserved amino acid within the S4
voltage-sensing region defines some of the biophysical differences
between smooth muscle and cardiac ether-a-go-go-related gene
K+ channels. Further studies to determine the association
of erg1-sm with accessory K+ channel subunits, such as
MiRP1, that co-assemble with HERG (47) will be necessary to demonstrate
the functional basis of erg currents in native smooth muscle tissues.
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ACKNOWLEDGEMENTS |
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We thank Drs. Wayne Giles, Leo Tsiokas, Kennon Garrett, Mark Coggeshall, and Liz Howard for helpful discussion during the course of this work and Nemat Morsy for technical assistance. We also thank Dr. Jeanne Nerbonne for providing the anti-N terminus HERG antibody.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK59777 and DK46367.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF439342 and AY130462.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Physiology, University of Oklahoma Health Science Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73194. Tel.: 405-271-2226; Fax: 405-271-3181; E-mail: hamid-akbarali@ouhsc.edu.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M208525200
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ABBREVIATIONS |
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The abbreviations used are: HERG, human ether-a-go-go-related gene; RT, reverse transcription; nt, nucleotides; wt, wild type; TBS, Tris-buffered saline; HRP, horseradish peroxidase.
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