870
K' Currents and K' Channel mRNA in
Cultured Atrial Cardiac Myocytes (AT-1 Cells)
Tao Yang, Mark S. Wathen, Antonio Felipe, Michael M. Tamkun,
Dirk J. Snyders, Dan M. Roden
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Abstract Atrial tumor myocytes derived from transgenic mice
(AT-1 cells) maintain a well-differentiated cardiac biochemical
and histological phenotype. In addition, they beat spontaneously
in culture and exhibit long action potentials whose repolarization
resembles that observed in native mammalian myocytes. In this
study, we identified the major depolarization-activated outward
currents in AT-1 cells; also, the presence of mRNAs that encode
outwardly conducting ion channels was determined by cloning
from an AT-1 cDNA library or by Northern hybridization.
Among K' channel isoforms, Kv2.1, minK, and Kvl.4 were
readily detected in tumors and at 1 day in culture. Their
abundance remained relatively stable (twofold or less change)
after 14 days. The major outward current in AT-1 cells is a
delayed rectifier that displays prominent inward rectification,
activates rapidly (eg, 182±27 milliseconds [mean±SEMl at +20
mV, n=12), exhibits biexponential deactivation kinetics, and is
extremely sensitive to the methanesulfonanilide dofetilide (ICa,
12 nmol/L). These characteristics identify this current as IKr, a
delayed rectifier observed only in cardiac cells. IK, in AT-1 cells
displayed slow inactivation: dofetilide-sensitive deactivating tails
were greater after 1-second than after 5-second pulses. When IK,
was blocked by .0.5 ,umol/L dofetiide, time-independent current was usually recorded (50 of 65 experiments); rapidly inactivating (6 of 65) or slowly inactivating (9 of 65) outward currents
were occasionally observed. We conclude that AT-1 cells express
mRNAs encoding cardiac K' channels and display a cardiac
electrophysiological phenotype. This system may therefore be a
useful tool in studies of regulation of cardiac ion channel gene
expression and its functional consequences. The presence of a
readily recorded 1Kr will facilitate study of the physiology and
pharmacology of this important repolarizing current. (Circ Res.
1994;75:870-878.)
Key Words * electrophysiology * ion currents * atrial
tumor myocytes * transgenic mice * ion channel genes
AT-1 cells are derived from atrial tumors in transLI genic mice carrying fusions between the tran1 k scriptional regulatory elements of atrial natriuretic factor (ANF) and those encoding the simian virus
40 (SV40) large T antigen.' As described by Field,
Claycomb, and colleagues2-4 and as described further in
"Materials and Methods," in vivo passage of the tumors
into subcutaneous tissues of syngeneic animals results in
secondary tumor growth. More than 90% of the cells in
the secondary tumors display sarcomeric banding and
other structural features typical of atrial cardiac myocytes, including transverse tubules, gap junctions, myofibrils, and atrial-specific cytoplasmic granules.23 These
cells proliferate in culture and can be passaged and
recovered from frozen stocks.3 Most important, they
retain a highly differentiated cardiac phenotype, including expression of adult cardiac-specific proteins, spontaneous beating that is reported to be modulated by
muscarinic agonists, and secretion of ANF that is modulated by a or 83 agonists, as well as by endothelin,
phorbol ester, or KCL.2-5 Thus, important intracellular
signaling mechanisms appear intact.
As described further below and by others,2,5 the cells
demonstrate spontaneous beating activity and action
potentials with slow cardiac-like repolarization.25 However, individual ion channels in AT-1 cells have not been
characterized in detail, nor has the repertoire of genes
that encode cardiac ion channels and are expressed in
this cell line been investigated. In the present study, we
identified the major K' currents controlling repolarization in AT-1 cells; we also probed these cells for the
presence of several known K' channel mRNAs. Our data
raise a number of important questions with regard to the
relation between expression of ion channel genes and the
electrophysiological properties they encode. Thus, AT-1
cells should provide an interesting new system for further
studies of the mechanisms and functional consequences
of ion channel gene expression. In addition, we find that
the rapidly activating delayed rectifier K' current ('Kr) is
a major repolarizing current in AT-1 cells. However, in
contrast to IKr in other myocytes, IKr in AT-1 cells is
readily isolated from other currents, making AT-1 cells a
particularly attractive system in which to further examine
the physiology and pharmacology of this important repolarizing current.
Received April 22, 1994; accepted July 22, 1994.
From the Departments of Pharmacology (T.Y., M.M.T., D.J.S.,
D.M.R.), Medicine (M.S.W., D.J.S., D.M.R.), and Molecular
Physiology and Biophysics (A.F., M.M.T.), Vanderbilt University
School of Medicine, Nashville, Tenn.
Correspondence to Dan M. Roden, MD, Director, Division of
Clinical Pharmacology, Department of Pharmacology, Vanderbilt
University School of Medicine, 532C Medical Research Bldg,
Nashville, TN 37232-6602.
© 1994 American Heart Association, Inc.
AT-1 cells were kindly supplied by Loren Field, Krannert
Institute, Indianapolis, Ind. Cells were injected subcutaneously into new syngeneic hosts ([C57BL/6JxDBA/2J]F, female mice, Jackson Laboratories, Bar Harbor, Me). In 6 to 8
weeks, subcutaneous tumors became palpable, and cells were
isolated 12 to 16 weeks after inoculation. With each cell
isolation, further syngeneic hosts were inoculated to propagate
the colony. To isolate cells, tumor-bearing mice were anesthetized with methoxyflurane and placed in 70% ethanol. The
tumor mass was excised, rinsed with phosphate-buffered saline
Materials and Methods
Cell Preparation
Yang et al K' Channels in AT-1 Cells
871
Passive Properties of the AT-1 Cell System
R., MQ
Diameter, pzm
Cm, pF
rc, p5
Ra, MO
RP, mV
5.6
-61
27
63
353
2.9
2.1
2
0.1
0.3
3.6
22
50
40
110
50
50
110
as
described
in
"Materials
and
DC
resistance;
cell
capacitance
calculated
indicates
microelectrode
Re
Cm,
Methods"; rc, time constant of the uncompensated capacitive transient; Ra, uncompensated access resistance
(Ra=rc/Cm); RP, resting potential; and N, number of cells.
Mean
SEM
N
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(PBS), minced finely, and placed for 1 hour at 37°C with gentle
rocking in PBS containing pen-strep (100 U/mL penicillin and
100 jtg/mL streptomycin, GIBCO) and 0.1% collagenase. The
cell suspension was spun, washed with PBS, resuspended, and
then plated at a density of 250x103 to 325X103 cells per
milliliter in 10-mm Primaria dishes (Falcon). The media (PC1
[Ventrex Laboratories], which included pen-strep, 10% fetal
bovine serum, and 10 nmol/L dexamethasone) were changed
every other day until used. Primary cultures grew to confluence and usually beat spontaneously at -1 week. Isolated cells
can also be repassaged or frozen for subsequent replating,2 but
none of the experiments reported here used these methods.
The electrophysiological findings described here were obtained from cells that had been in culture for 7 to 14 days. For
electrophysiological studies, cells were removed from the
culture dish by a 2-minute exposure to a trypsin-containing
solution (0.125% in Ca2'-Mg 2+-free Hanks' solution), decanted into sterile culture tubes, and held at room temperature for at least 4 hours before study. Isolated cells held in this
fashion could be studied up to 3 days after trypsinization.
cDNA Library Construction and Screening
AgtlO libraries were constructed from an AT-1 tumor and
from beating AT-1 cells by using a cDNA synthesis system
(Pharmacia) and methods previously described.6 Size-fractionated cDNA (.2 kb) was inserted into the AgtlO arms using
EcoRI-Not I adapters. The libraries (5 x 10 to 1.4x 106 primary plaques) were screened at low stringency by using
methods described previously6 and by using probes derived
from rat or human cDNA libraries. Positive recombinants
were isolated, amplified, and subcloned in either EcoRI or Not
I sites from pGEM7 or Bluescript KS, respectively. DNA
sequence analysis was performed by previously described
methods, and the sequences obtained were analyzed by
STRIDER and MACVECIOR DNA software.6-8
Northern Analysis
Total RNA from AT-1 tumors or cells was isolated by
centrifugation in guanidinium thiocyanate and a cesium gradient.6 8 Total RNA (10 to 20 ,ug) was fractionated by electrophoresis on 1% agarose-3% formaldehyde gels; ethidium
bromide staining was used to ensure equal loading of the lanes.
RNA was partially hydrolyzed by submerging the gel for 5
minutes in 50 mmol/L NaOH and 1.5 mol/L NaCl, followed by
neutralization for 30 minutes in 1 mol/L Tris (pH 6.8) and 1.5
mol/L NaCl. The RNA was then transferred overnight to a
Nytran filter by capillary action in 20x standard saline citrate
(SSC, which contains 3 mol/L NaCl and 300 ptmol/L sodium
citrate [pH 7.0]) and cross-linked to the Nytran by irradiation
with ultraviolet light for 3 minutes. The filter was prehybridized overnight at 65°C in 20% formamide, 10% dextran
sulfate, 4x SSPE (600 mmol/L NaCl, 40 mmol/L NaH2P04,
and 4 mmol/L EDTA [pH 7.4]), 5 x BFP (1 g/L bovine serum
albumin, 1 g/L polyvinyl pyrolidone 40, 1 g/L Ficoll, and
0.001% sodium azide), 0.1 mg/mL sonicated salmon sperm
DNA, 0.2 mg/mL yeast RNA, and 5% sodium dodecyl sulfate
(SDS) and hybridized for 24 hours at 65°C in the same solution
with 106 cpm/mL of the appropriate random primer-labeled
cDNA probe having a specific activity of =7.5xi0' cpm/mg.
The filters were then sequentially washed for 30 minutes each
at 65°C with 3 x SSC and 1% SDS, 1 x SSC and 1% SDS, and
0.2x SSC and 1% SDS. The filters were processed in a
Phosphor Imager (Molecular Technologies) to quantify radioactivity; also, filters were exposed on photographic film for
presentation here.
Three K' channel superfamilies have been described: the
Shaker (six membrane-spanning domain) superfamily, which
includes genes coding not only Shaker-derived channels (the
Kvl.x group) but also channels of the Shab, Shaw, and Shal
families6'8-13; genes that encode channels with two putative
membrane-spanning domains that appear to display inward
rectifier behavior14"15; and minK (also termed ISK), a gene that
encodes a very small protein that is thought to have one
membrane-spanning domain and whose expression appears to
result in a very slowly activating delayed rectifier current (IKS)
in native cardiac cells.16-18 The cDNA probes used in the
present study included Kv2.1 and minK, cloned from the AT-1
library as described above, and Kvl.1, Kv1.2, KvL.4, and Kvl.5,
cloned from a rat heart library.6 We have also cloned Kvl.5
from a murine genomic library, and mKvl.5 was also used in
Northern hybridizations.
Electrophysiological Recording
Recordings were performed with an Axopatch-lA patchclamp amplifier (Axon Instruments, Inc) in the whole-cell
configuration of the patch-clamp technique.19 Electrodes were
pulled from borosilicate glass (Radnoti Co) and heat-polished.
Ionic currents were recorded at room temperature (22°C to
23°C). The currents were sampled at 3 to 10 times antialias
filter setting and stored on the hard disk of an 80386-based
microcomputer for subsequent analysis. Data acquisition and
command potentials were controlled with a commercial software program (PcLAMP, Axon Instruments). To ensure voltage-clamp quality, electrode tip resistance (Re) was kept below
3.5 Mfl; the average R, was 2.9±0.1 MQ (Table).
Junction potential was zeroed with an electrode in the
standard bath solution. The microelectrodes were gently lowered onto the cell surface and a gigaohm tight seal was formed
by suction (range, 5 to 50 GfQ). After the whole-cell configuration was established, the capacitive transients elicited by
symmetrical 10-mV voltage-clamp steps from -80 mV (Fig 1)
were recorded at 50 kHz (filtered at a bandwidth of 10 kHz, -3
dB) for calculation of capacitive surface area, time constant,
and access resistance (Ra). Thereafter, capacitance and series
resistance compensation were optimized; 80% compensation
was usually obtained. The passive properties of AT-1 cells are
presented in the Table. No significant voltage errors (<5 mV)
are expected when using the electrodes described above, and
this was confirmed by the calculated R, (Table). Fig 1 shows a
recording of uncompensated capacitive transient in one AT-1
cell, which improved further after compensation. In current
clamp, action potentials in AT-1 cells were elicited by 1-Hz
current pulses from the resting potential.
Solutions and Drugs
Unless otherwise noted, the extracellular solution was normal Tyrode's (to which blockers described below were added
as required). Normal Tyrode's solution contained (mmol/L)
872
Circulation Research Vol 75, No 5 November 1994
0o
1 msec
FIG 1. Passive properties of an AT-1 cell. A capacitive transient
elicited by a 10-mV depolarization from -80 mV is shown after
linear leak subtraction. Numerical integration of the area under
the curve yielded a membrane capacitance of 67 pF. A time
constant (r) of 356 microseconds was obtained with a monoexponential fit to the declining phase of the curve. Calculated
uncompensated access resistance (Ra=7./Cm, where Cm is cell
capacitance) was 5.3 MQI. The sampling rate was 50 kHz with a
low-pass filter (-3 dB) of 10 kHz.
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NaCl 130, KCI 4, CaCl2 1.8, MgCl2 1, HEPES 10, and glucose
10, and the solution was adjusted to pH 7.35 with NaOH. The
intracellular pipette filling solution contained (mmol/L) KCI
110, KBAPTA 5, K2ATP 5, MgCl2 1, and HEPES 10, and the
solution was adjusted to pH 7.2 with KOH, yielding a final
intracellular K' concentration of -145 mmol/L. Na+ current
(INa) and Ca2, current are frequently recorded in AT-1 cells
and will be the subject of a separate report. In these experiments, the L-type Ca2, current (ICaL) was eliminated using
nisoldipine (1.0 ,mol/L) or Cd21 (100 ,umol/L). In most
experiments, INa and the T-type Ca2+ current (ICa-T) were
eliminated by holding at .-50 mV. When a more negative
holding potential was used, INa was eliminated by substituting
N-methyl-D-glucamine for extracellular Na' and 'Ca T was
blocked by 100 to 200 gmol/L Ni2+.
Salts were purchased from Sigma Chemical Co, as were
4-aminopyridine and isoproterenol. Nisoldipine was obtained
from Miles Pharmaceutical, Inc, and dofetilide was provided
by Pfizer Central Research. Stock solutions were stored at 4°C,
and the final concentrations in the bath were obtained by
diluting the stock solutions in the external solution during
experiments.
Voltage-Clamp Protocols and Data Analysis
Specific protocols are presented in "Results." In all these
experiments, cycle time between clamp pulses was >10 seconds. In general, currents following exposure to drugs were
recorded after apparent steady state was reached. To compare
current densities among cells, currents are reported as current
density per unit area (picoamperes per picofarad) after linear
leak subtraction and normalization relative to cell surface area
determined by measurement of capacitance, as described
above. The voltage dependence of channel opening and inactivation was determined by fitting a Boltzmann function:
y=1/(1+exp[-(E-Eh)/kI) to activation curves, where k represents the slope factor; E, membrane potential; and Eh, the
voltage at which 50% of the channels are activated. Activation
kinetics were fit with a monoexponential function; the time
course of tail currents and of slow inactivation were fit by using
sums of multiple-exponential terms. The IC50 for drug block of
ion current was analyzed by using a Hill function: y= 1/
{1 + (IC50/ [D])N}, where [D] is the blocker concentration and N
is the Hill coefficient. Nonlinear curve-fitting routines written
in FORTRAN and using a SIMPLEX algorithm were used to fit
functions to the data.12,20,21 All electrophysiological data presented here are from at least three separate cell isolations.
Student's t test was used for comparisons between means,
with a value of P<.05 considered significant. Results are
expressed as mean+ 1 SEM.
1 sec
FIG 2. Action potentials recorded from an AT-1 cell displaying
spontaneous beating at 104 beats per minute. Maximum diastolic potential was -61 mV.
Results
When AT-1 cells were cultured for 7 days, spontaneous synchronous beating at a rate of %0.5 to 2 Hz was
usually observed (58 of 86 experiments). Action potentials from a cell displaying such spontaneous beating
activity are shown in Fig 2: diastolic depolarization and
a takeoff potential of :-40 mV are evident. After
trypsinization, round cells with a diameter of 20 to 30
gm (mean, 27-0.3 gm; Table) were chosen for further
experiments. Resting potentials were measured only in
cells with either no leak or only a linear leak at
potentials positive to -40 mV. Under these conditions,
resting potential varied from -45 to -82 mV (mean,
-61±2 mV; n=40). In current clamp, action potential
shapes without (Fig 3A) and with (Fig 3B through 3D)
prominent plateaus were recorded. In most cells, an
apparent ICa-L, which ran down rapidly, was recorded;
because of this rundown, specific correlations between
action potential configurations and the membrane currents present in individual cells were not undertaken in
the present study.
A Prominent Inwardly Rectifying Delayed Rectifier
K' Current
When INa and ICa-T were inactivated by holding at -40
mV and IC.-L was blocked by 1 gmol/L nisoldipine, a
rapidly activating outward current was observed (Fig
4A). With 250-millisecond depolarizing pulses to +30
mV, this current did not inactivate. Moreover, as indicated in Fig 4B, the activating current displayed inward
rectification and deactivating current tails that were
voltage dependent and saturated at -+20 to +30 mV.
All of these characteristics are consistent with the
properties of the rapid component of cardiac delayed
A
B
50
c
E
01
msce
D
E
0
50 misc
50 masc
FIG 3. A through D, Action potential shapes in four different
AT-1 cells.
Yang et al K' Channels in AT-1 Cells
+30
+30
1000
msec
FIG 4. A rapidly activating delayed rectifier K+ current (I,). A, A
family of typical l,. recordings (1
j,mol/L nisoldipine was present).
-An
-v mV
MY
-d
A
500
pA
--b.. %-
B
pA/pF
5 l1
h
C
700
-
600
-
o
*
*
500
-
10 400
E
-
activation
fast deactivation
slow deactivation
(n-1 2)
T
5.O 300
-
1
200
-
'7-l
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
100 0-
-60
-40
-20
0
+20
Membrane potential CmV)
+40
873
+60
a
.
a
-80
-80
-40
-20
0
+20
+40
+80
The activation threshold for this
current was near -30 mV, and
the activating current was maximal at -+20 mV, with less activating current at more positive
potentials. Deactivating tail currents recorded at -40 mV were
saturated at z'+20 mV. B, Graph
showing IK, current-voltage relations, assessed as in panel A. The
activating current (o) shows
prominent inward rectification,
and the current tails (a) show saturation. The dashed line is the fit
to the tails using the Boltzmann
equation described in "Materials
and Methods." Eh indicates the
voltage at which 50% of the channels are activated. C, Graph
shows voltage dependence of the
time constant (Xr) of activation and
deactivation.
Membrane potential (mV)
rectifier current, termed 'Kr by Sanguinetti and Jurkiewicz.2 The IKr-like current was the most prominent
outward current in AT-1 cells and was found in 66 of 77
experiments. It did not run down even during prolonged
experiments (eg, >2 hours), which were readily
achieved in AT-1 cells. It was the dominant outward
current in cells studied as early as day 3 in culture and
as late as day 14 in culture. At room temperature (22°C
to 23°C), this current activated rapidly (eg, 182±+27
milliseconds at +20 mV, n=12) and, like 'Kr in other
myocytes,22 its deactivation kinetics were biexponential
(Fig 4C).
We compared the time course of current activated
during a depolarizing step to the envelope of deactivating tail current amplitudes after steps of varying duration. This is a widely used method (the "envelope test")
for determining whether multiple overlapping currents
are present in myocytes.20'22'23 In contrast to guinea pig
myocytes20'22 and sheep23 or dog24 Purkinje fibers, the
envelope test is clearly satisfied in AT-1 cells (Fig 5),
indicating that a single component constitutes the timedependent current shown in Figs 4 and 5. A number of
new methanesulfonanilide "class III' antiarrhythmic
drugs, including E4031 and dofetilide, are potent and
specific blockers of IKr222526; in fact, Sanguinetti and
Jurkiewicz22 defined 'Kr as the E4031-sensitive component of the delayed rectifier current in guinea pig
myocytes, where a prominent IKS is also recorded.
Therefore, we evaluated the effects of dofetilide on
outward current in AT-1 cells. As shown in Fig 6, 1
,gmol/L dofetilide inhibited time-dependent activating
current as well as all current tails; the possible nature of
the dofetilide-resistant current such as that shown in Fig
6A is discussed below. Fig 6B shows the concentration
dependence of dofetilide block at +20 mV: the IC50 was
12 nmol/L (n=8). The voltage dependence of dofetilide-sensitive currents (Fig 6C) was very similar to that
shown for time-dependent current in a different group
of cells (Fig 4B), again arguing that IK, is the predomi-
nant outward current under these conditions in AT-1
cells. La3` is another fairly selective blocker of IKr over
IJ02,27; as shown in Fig 6D, outward current in AT-1
cells was suppressed by 1 ,umol/L La3", which also
appeared to block ICa-L-
The reversal potential (Erev) of this current after
1-second prepulses to +20 mV (Fig 7) was -79.4±1.9
mV (n=11) in 4 mmol/L extracellular K+. Under these
+20
-40 mV
-40
pA
I
500|
1
sec
B
1.51
m
.E
+...
0
0
1.0
1.
m
5
1.10
0.5-
0
.0
0j
1
0
400
800
1200
At (msecl
8
1600
2000
FIG 5. Envelope tests in AT-1 cells. A, An example of the
currents recorded during this procedure. B, Graph showing
mean data in eight envelope tests. The ratio of current activated
during the pulse to deactivating tail amplitude is independent of
pulse duration; ie, the envelope test is satisfied. IKr indicates
rapidly activating delayed rectifier K+ current.
Circulation Research Vol 75, No 5 November 1994
874
B 1
A
1000
+20
msec
+20
-40 mv
-40
/ control
10 nM dofettlide
100 nM
/-4%~~c:z
500
1sec
o
Li
-40 mv
-t11
k
<|> 0,5 ._
iM
A_
p
'tNN
00
nM
0
-
10
100
Dofetilide (nM)
D
+10
-40
1000
1000 msec
-40 mV
control
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i
500
pA |
-60 -40 -20
a
FIG 6. Drug block of the rapidly
activating delayed rectifier K' current (Q4. A, Tracings showing block
by a range of concentrations of
dofetilide. At high concentrations,
time-dependent outward current
and deactivating tails were completely inhibited. B, Graph showing
concentration dependence of
dofetilide block. C, Graph showing
current-voltage relation of the current blocked by a high concentration (1 gmol/L) of dofetilide. As in
Fig 4B, the activating current (o)
displays prominent inward rectification, and the tail currents (i)
show saturation. Eh indicates the
voltage at which 50% of the channels are activated. D, Tracings
showing block by 1 gmol/L La3+. In
this experiment, nisoldipine was
omitted, and an L-type Ca2+ current was recorded.
La+ 1 yM
+20 +40 +60
Membrane potential ImV)
conditions, the Nernst equation predicts a K' equilibrium potential (EK) of -95 mV, assuming an intracellular K' concentration of 145 mmol/L, the pipette K+
concentration. Erev measured after 5-second prepulses
was -76.6±1.6 mV (n=11), a value similar to that
observed after 1-second prepulses. These data indicate
that the difference between EK and E,,, was not due to
extracellular K+ accumulation; rather, imperfect K'
selectivity (as reported for other delayed rectifier K+
currentS28) is more likely.
Other K' Channels
AT-1 tumors, AT-1 cells, and the AT-1 cDNA libraries were screened to identify K' channel isoforms
present in this preparation. Kv1.4, a member of the
Shaker family, was detected in cells in culture early as
day 1, and its abundance tended to decrease with time
in culture (Fig 8). Kvl.1, KV1.2, and KV1.5, other
members of the Shaker family, were not detected. All
screening with these members of the KvI.x family was
performed at low stringency by using probes cloned
as
from rat heart, except in the case of KvL.5, where a
genomic clone
used. Kv2.I (drkl, a member
of the Shab family) and minK (ISK), the mRNA which is
thought to encode Iy, in guinea pig myocytes,16-18 were
identified in both tumor and cultured cells; their sequences were identical to those previously reported for
the corresponding cDNAs isolated from mouse heart.18
These K' channel transcripts were readily detected in
AT-1 cells that had been in culture for only 24 hours,
and their abundance did not appear strongly time
dependent (Fig 8). Kv2.1 transcripts increased 1.6- and
2.3-fold (duplicate experiments), and minK transcripts
increased 1.5- and 2.9-fold from 1 to 14 days, whereas
those encoding Kv1.4 decreased, by 46% and 38%.
Further experiments were then undertaken to determine if the channels encoded by these genes were
expressed in AT-1 cells.
Despite the fact that the minK mRNA was present in
this preparation, the corresponding current, Iy,, was not
mouse
was
,., b, 611Y\ 15,1b..1 6Isrso
(b
+20
1
sec
Kvl.4
-40
-40 mv
-100 mv
-40
mY
500
Kv2. 1
pA
Er.,
mV-
-100 mV
FIG 7. Tracings showing reversal potential (Erev) of rapidly
activating delayed rectifier K' current in 4 mmol/L extracellular
K+. In 11 cells, the mean Er,, was -79.4±1.9 mV at 4 mmol/L
extracellular K+.
minK
FIG 8. Northern analysis of mRNA encoding outward currents in
AT-1 cells in culture 1 to 14 days. The probes used were Kv2.1
and minK (IsK), both cloned from the AT-1 cell library, and rat
Kvl.4.
Yang et al K' Channels in AT-1 Cells
5 sec
B
+400,
-40 l:
LIL
A
11
baseline
------- dofetilide
11 pMp
o-o 1 sec
r-u_ 5 sec
7-
-40
mv
My
N
<
6-
1n=10)
*
*P<0.01
To
'P
3
..
2
-30 mV
I1A1....
nA
.+10 m
................................. .........
z
z
10]
-50
-30
-10
+10
Membrane potential
-20 mV
-10 mV
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
nA
1
AL
.
. ......
......
.......
...
.0 m;
1
nA -=
1
nA
+30
+50
(mV)
+20 mY
~~~~~~~~~~~~..................................1...........
.....
;
M
nA
n1A
1;
875
+30 mV
+40
FIG 9. Effect of pulse duration on rapidly activating delayed
rectifier K+ current (1Q) tail current. A, With long (5-second)
pulses, IK was still the most prominent outward current.
Slow inactivation is evident at positive potentials, but no
slowly activating delayed rectifier K+ current was recorded.
The dotted tracings are those recorded during the administration of 1 gmol/L dofetilide. B, Graph showing that tail
currents after 5-second depolarizing pulses were smaller
than after 1-second pulses (n=10, *P<.01).
mV
==_
observed: tail currents were actually larger after 1-second than after 5-second depolarizations (Fig 9),
whereas the opposite would be expected if IKS were
present. The fact that these tail currents were >95%
decreased by dofetilide (dotted tracings, Fig 9A) also
argues against the presence of IKS since, by definition, IKS
is dofetilide resistant. In this regard, it is important that
dofetilide is thought to be a highly specific blocker of
1Kr.2930 At concentrations up to 1 gmol/L, it does not
affect Na+ current, Ca 2+ current, rat transient outward
current (ITO),2930 or Kv1.4, Kvl.5, or Kv2.1 in heterologous expression systems.
When inward currents were eliminated as described
above and lKr was eliminated by high concentrations of
dofetilide, there was usually (50 of 65 experiments) little
or no residual time-dependent current (eg, Figs 6A and
9A), even from a holding potential of -80 mV. Exposure to isoproterenol increased the amplitude of the
residual time-independent current and exaggerated its
outwardly rectifying properties; thus, it may represent a
f,-stimulated Cl- current.31 In a minority of such experiments, inactivating currents were observed. Kvl.4 and
Kv2.1 encode channels that inactivate rapidly32 and
slowly,33 respectively. However, no pharmacological
probe clearly distinguishes between these isoforms, and
in heterologous expression systems they are not blocked
by dofetilide; thus, these experiments could not identify
with certainty specific channel behaviors clearly linked
to the expression of a specific isoform. The inactivating
behaviors appeared to fall into two groups. In 6 of 65
cells, rapid inactivation (44±+8 milliseconds at +50 mV;
range, 20 to 75 mV) was observed, whereas in 9 of 65
cells, inactivation was much slower (226±90 milliseconds at +50 mV; range, 109 to 354 mV). Both of these
behaviors were suppressed by <1 mmol/L 4-aminopyridine, arguing that they are also K+ currents.
Discussion
The most important findings in the present study are
that (1) the major repolarizing current in AT-1 cells is a
rapidly activating delayed rectifier heretofore reported
only in heart cells, and (2) mRNA transcripts encoding
cardiac K' channels are expressed in AT-1 cells. The
repolarizing current displays prominent inward rectification (Figs 4B and 6C), rapid activation kinetics and
biexponential deactivation kinetics (Fig 4C), nanomolar
sensitivity to dofetilide (Fig 6A and 6B), and block by
La3` (Fig 6D). In addition, the envelope test (Fig 5)
indicates that this current comprises a single component. These characteristics therefore identify 'Kr as the
major repolarizing current in AT-1 cells.
Steinhelper et a12 described the initial characteristics
of the AT-1 cell line. Histological examination of AT-1
cells showed a muscle structure similar to that observed
in normal atria and prominent perinuclear electrodense
atrial granules containing immunoreactive ANF. Direct
immunofluorescence detected the presence of sarcomeric myosin indistinguishable from that in normal cardiac tissue, and a-cardiac actin was observed in AT-1
cells, whereas a-skeletal muscle actin was absent. One
difference between AT-1 cells and atrial cells was
greater expression of the BB isoform of creatine phosphokinase in AT-1 cells. In their study, spontaneous
beating activity was observed in vitro, and action potentials with maximum diastolic potentials in the -64- to
-79-mV range were reported. Delcarpio et a13 further
reported ultrastructural features including well-organized myofibrils, gap junctions, and atrial-specific cytoplasmic granules, as well as a well-developed transverse
T-tubule system and the presence of connexin43. Further studies from these laboratories4 have demonstrated
that ANF is stored as a prohormone and processed to
the bioactive form in a fashion similar to that observed
in normal cells. In addition, stimulation of ANF secretion by endothelin, KCI, the ,3-agonist isoproterenol, the
a,-agonist phenylephrine, and a phorbol ester (12-0tetradecanoylphorbol 13-acetate) demonstrated the
presence of cell surface receptors and their coupling to
intracellular signaling systems. The latter conclusion is
also supported by the reported carbachol sensitivity of
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Circulation Research Vol 75, No 5 November 1994
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beating behavior in AT-1 cells,5 as well as our observation that the amplitude of 1Ca L in AT-1 cells is increased
with isoproterenol (data not shown).
different K' channel proteins to faithfully reconstitute
function has been reported for Kvl.4 and Kvi.2.32,'4
IKr in AT-1 Cells
In guinea pig ventricular myocytes, the other major
outward current is a very slowly activating delayed
rectifier that is prominent even at room temperature20
and that Sanguinetti and Jurkiewicz22 termed IK, (for
slowly activating delayed rectifier). Although a cDNA
encoding lK, has not been cloned, a cDNA (minK or ISK)
encoding a slowly activating delayed rectifier has been
isolated from rat renal tubule, estrogen-primed uterus
in rat, mouse heart, and neonatal guinea pig myocytes.16- 8The protein encoded by minK is small, 129 to
130 amino acids, and is predicted to have one membrane-spanning domain. The mechanism whereby this
small protein encodes a delayed rectifier is uncertain.
Injection of minK cRNA in Xenopus oocytes does result
in expression of a very slowly activating delayed rectifier. The fact that the amplitude of IKS is increased by
second-messenger stimulation and that consensus sequences for protein kinases A and C are present in the
predicted cytoplasmic portion of the protein also argues
that the minK protein actually encodes the channel.18'4546 Moreover, mutations in or near the putative
membrane-spanning domain result in altered selectivity
of the expressed current.47 However, a recent report
suggests the minK gene encodes a protein whose function may be to regulate expression of a "silent" endogenous K' channel in Xenopus oocytes.48 We have detected minK mRNA in AT-1 tumors and cells (Fig 8),
but long pulses (Fig 9) did not elicit IK,-like behavior. A
number of possibilities, each of which require further
testing, may explain our findings. We have considered
the possibility that trypsinization digests functional
protein encoded by minK. However, two pieces of
experimental evidence argue against this hypothesis.
First, we and others49 have used trypsinization during
isolation of guinea pig myocytes, and IKS is still observed,
indicating that the channel is not markedly sensitive to
trypsin. Second, we have conducted a limited number of
experiments in which AT-1 cells have been cultured
directly on coverslips and voltage-clamped without
trypsinization. In these experiments, gigaohm seals are
more difficult to obtain; however, when experiments
have been successful, prolonged pulses produced no
suggestion of a slowly activating delayed rectifier. It is
possible that minK mRNA is present but not transcribed into protein or that the protein is present but its
activation and/or assembly into a fully functional channel requires further element(s) that are either absent or
not appropriately stimulated in these cells. Our data are
also consistent with the possibility that minK does not,
in fact, encode an ion channel but plays an important
role in promoting expression of other proteins that do.
Finally, the minK protein may be expressed only in a
subpopulation of cells that were not included in those
chosen for voltage clamp.
Other Cell Lines
These findings support the continued investigation of
this cell line as a surrogate for cardiac cells in culture.
Another system that others have proposed is H9c2 cells,
derived from embryonic rat ventricle.50'51 H9c2 cells
have some characteristics of cardiac cells, including
In guinea pig cardiac myocytes, the multicomponent
nature of the delayed rectifier has been described2022:
an envelope-of-tails test was not satisfied until blockers
(E4031 or La3`) were used. Sanguinetti and Jurkiewicz22
characterized the E4031-sensitive component in detail
by using a subtraction approach. They found that it
displayed very prominent inward rectification and activated rapidly, and they proposed the name lK, (for rapid
delayed rectifier). It is now apparent that IKr is a major
target for a new generation of antiarrhythmics that are
methanesulfonanilide analogues of N-acetylprocainamide34 and include E4031, D-sotalol, and dofetilide.2225'26 IKr has not been reported in preparations
other than heart cells, and drugs such as dofetilide do
not appear to exert significant noncardiac effects.
'Kr has now been described in other species. In rabbit,
ITO appears to be the major repolarizing current. However, after inactivation of ITO, a dofetilide-sensitive
delayed rectifier is also observed.26'35 In adult rats,
dofetilide does not alter action potentials, and IKr is
thought to be absent.36 In contrast to guinea pig and
rabbit, where IKr is but one of multiple depolarizationactivated outward currents, IKr appears to be the most
prominent outward current in cat myocytes.37 However,
in that preparation, deactivation is far slower than in
other species. Wang et a138 have identified a current
strongly resembling IKr, with rapid activation kinetics
and inward rectification, in 71% of myocytes isolated
from human right atria; 82% of cells showing the
delayed rectifier also displayed a prominent ITO.38 Humans also develop QT prolongation with IKr blockers,39-42 indicating that the current is likely present in
ventricular tissue. In mouse ventricular cells, multiple
subtypes of ITO have been identified, but no data are yet
available on K' currents in native mouse atrial cells.
Our data demonstrate that in AT-1 cells, IK, can be
observed directly, as opposed to guinea pig myocytes,
where the characteristics of the current can only be
studied by using a drug-subtraction approach. One
difference between the description of IKr provided by
Sanguinetti and Jurkiewicz22 and our findings here is the
apparent slow inactivation of the current. Outward
current such as that shown in Figs 4 and 6A could
represent not only IKr but also the other inactivating
components that we occasionally observed. However,
Fig 9A directly demonstrates inactivation of the dofetilide-sensitive current. In addition, the findings that timedependent currents (both activating and tails) were
virtually abolished by dofetilide and were significantly
larger after 1-second than after 5-second pulses (Fig 9)
further argue for slow inactivation of IK, in this preparation. Recently, 'Kr in human atrial myocytes, defined
as E4031-sensitive current, has also been reported to
display slow inactivation.43 A cDNA encoding IKr has
not yet been isolated. The abundance of other K'
channel mRNAs shown in Fig 8 raises the possibility
that the channels that give rise to IKr result from
coassembly of multiple K' channel transcript products
such as minK, Kv2.1, and Kvi.4. Such assembly of
MinK and IKS
Yang et al K' Channels in AT-1 Cells
1Ca L, which resembles that seen in the heart. However,
other studies have suggested that H9c2 cells show
characteristics more typical of skeletal muscle, including the pattern of expression of G proteins and, at least
in the hands of some workers, a response to acetylcholine suggesting skeletal muscle end plate-type nicotinic
acetylcholine receptors.5052 H9c2 cells have also been
reported to express both transient outward and delayed
outward currents, which are thought to represent both
K+-selective and nonspecific channels.5051 No Na+ currents have been recorded from this cell line, nor do they
display spontaneous beating in culture.
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Unanswered Questions
Our studies do indicate that AT-1 cells express cardiac electrophysiological properties more faithful to the
"cardiac" phenotype than other cell lines available to
date. A number of interesting questions are raised
whose answers would provide important fundamental
information on the regulation of ion channel genes and
the ion currents they encode. Examples include the
following: (1) What factors control the development of
ion channel gene expression as a function of time? (2)
Do other factors also regulate ion channel gene expression? (3) What is the relation between ion channel
mRNA and the observed ion currents? (4) What individual currents are responsible for the variable action
potentials presented in Fig 3? (5) What ion current(s)
underlies pacemaker activity in these cells? Studies to
address these questions are currently in progress; the
availability of cells expressing cardiac ion channel genes
and ion currents will now allow the use of powerful
methods such as antisense or antibody strategies to
study regulation of ion channel gene expression and its
functional consequences.
In summary, AT-1 cells, derived from transgenic
animals carrying fusions between the SV40 large T
antigen and an ANF promoter proliferate in cell culture
and retain a cardiac phenotype by biochemical and
histological criteria. Our studies now indicate that these
cells also display cardiac-type repolarization characteristics, most notably a prominent delayed rectifier, IKr.
The minK (ISK) mRNA is detected, but the corresponding ion current signature, a dofetilide-resistant slowly
activating delayed rectifier, is not; the latter observation
raises a number of possibilities, the testing of which
should provide new information on the functional importance of this channel. The ability to study lKr physiology and pharmacology in cells in which overlapping
currents are readily eliminated is one potential application of this model system. More generally, this system
may provide an opportunity to study regulation of ion
channel gene expression and its functional consequences in a cardiac cell line.
Acknowledgments
This study was supported in part by grants from the US
Public Health Service (HL-46681, HL-47599, and HL-49330).
Dr Tamkun is an Established Investigator of the American
Heart Association. Dr Felipe was a recipient of a fellowship
from the DGICYT, Spain, during the conduct of these studies.
The excellent technical assistance of Holly Waldrop and of
Charles Mashburn and the excellent secretarial assistance of
Patricia James are greatly appreciated.
877
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Circ Res. 1994;75:870-878
doi: 10.1161/01.RES.75.5.870
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