© 1999 American Heart Association, Inc.
Molecular Medicine |
Oxygen Sensitivity of Cloned Voltage-Gated K+ Channels Expressed in the Pulmonary Vasculature
From the Departments of Physiology (J.T.H., E.A.C., J.R.M, M.M.T.) and Biochemistry and Molecular Biology (M.M.T.), Colorado State University, Ft. Collins, Colo, and the Departament de Bioquimica i Biologia Molecular (A.F.), Universitat de Barcelona, Barcelona, Spain. Joanne T. Hulme is currently affiliated with the Department of Pharmacology, University of Washington, Seattle.
Correspondence to Michael M. Tamkun, Department of Physiology, Colorado State University, Ft. Collins, CO 80523. E-mail tamkunmm{at}lamar.colostate.edu
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Abstract—Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O2-sensitive K+ current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3
subunits expressed in mouse L
cells using the whole-cell patch-clamp technique. Hypoxia
(PO2=
30 mm Hg) reversibly inhibited
Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV.
In contrast, hypoxia did not alter Kv1.5 current. Currents
generated by coexpression of Kv2.1 with Kv9.3 subunits were
reversibly inhibited by hypoxia in the voltage range of the
resting membrane potential (EM) of PA VSMCs (28% at
-40 mV). Coexpression of Kv1.2 and Kv1.5 subunits produced
currents that displayed kinetic and pharmacological properties distinct
from Kv1.2 and Kv1.5 channels expressed alone. Moreover,
hypoxia reversibly inhibited Kv1.2/Kv1.5 current
activated at physiologically relevant
membrane potentials (65% at -40 mV). These results indicate that
(1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5
homomeric channels, (2) Kv1.2 and 1.5 subunits can assemble to form
an O2-sensitive heteromeric channel, and (3) only
Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric channels are inhibited by
hypoxia in the voltage range of the PA VSMC EM.
Thus, these heteromeric channels are strong candidates for the
K+ channel isoforms initiating hypoxic pulmonary
vasoconstriction.
Key Words: Kv channel • hypoxia • pulmonary artery • heteromeric
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Hypoxia induces constriction of the small pulmonary resistance arteries, a process known as hypoxic pulmonary vasoconstriction (HPV).1 This constrictor response is the opposite of that which occurs in resistance vessels of the systemic circulation. In these vessels, hypoxia results in vasodilation.2 In the fetus, HPV contributes to high pulmonary arterial resistance by diverting blood flow through the ductus arteriosus.3 In the adult, HPV reduces blood flow through atelectatic or underventilated areas of the lung where ventilation is not adequate for oxygenation.3 In this way, short-term HPV is an essential mechanism that helps match ventilation to perfusion, diverting blood flow away from poorly ventilated regions of the lung to maximize arterial saturation.4 However, when hypoxia becomes more generalized, as seen in patients suffering from either long-term obstructive lung diseases or high altitude exposure,4 5 the subsequent pulmonary vasoconstriction causes an increase in pulmonary arterial pressure that can lead to the development of pulmonary hypertension.
In pulmonary arterial (PA) vascular smooth muscle
cells (VSMCs), voltage-gated potassium (Kv) channels play an important
role in setting the resting membrane potential
(EM=-55 mV) and, consequently, vascular
tone.6 7 8 It is thought that hypoxia reversibly
inhibits Kv channels and, thereby, regulates
vasoconstriction.7 9 10 11 12 This hypothesis is supported by
the observation that hypoxia inhibits whole-cell
K+ currents and causes membrane depolarization in
both acutely isolated and cultured PA VSMCs.7 9 11 12 13 14
Membrane depolarization is followed by an influx of calcium through
voltage-gated calcium channels, PA VSMC contraction, and
vasoconstriction.
Although electrophysiological and pharmacological studies have attempted to identify which Kv channel proteins contribute to the hypoxic-sensitive Kv current in PA VSMCs,11 14 15 16 the literature remains filled with contradictions. Yuan et al16 used both reverse transcription–polymerase chain reaction (RT-PCR) amplification and Western blotting to determine which of the cloned cardiovascular channels is expressed in rat PA VSMCs in short-term cultures. This group documented the expression of Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv9.3, Kvß1.1, Kvß1.2, and Kvß2.1 by PCR and confirmed expression of Kv1.2, Kv1.4, Kv1.5, and Kv2.1 channel proteins by Western blot analysis. In addition, Wang et al17 demonstrated that long-term hypoxia down-regulates Kv1.2 and Kv1.5 mRNA and protein expression in cultured PA VSMCs. However, a potential problem with studies of VSMCs in short-term cultures is that, with time, myocytes show alterations in the expression of outward currents.15 Archer et al14 used Western blotting of whole lung and acutely isolated VSMCs to argue in favor of the expression of Kv2.1 and Kv1.5 in rat pulmonary arteries. Furthermore, this group reported that the addition of Kv1.5 and Kv2.1 antibodies to the patch-clamp pipet solution inhibits the whole-cell hypoxic-sensitive Kv current. Similarly, Gelband and Gelband18 demonstrated that anti-Kv1.5 antibodies inhibit the hypoxia-induced membrane depolarization in rat PA VSMCs. Thus, Kv2.1 and Kv1.5 channel subunits may be important molecular components of the native O2-sensitive K+ current.
More recently, Patel et al15 used both defined and degenerate RT-PCR primers to suggest that, of the known Kv channels, only Kv1.2, Kv1.3, and Kv2.1 channels are expressed in rat pulmonary arteries. They failed to detect Kv1.5, even when using defined primers specific for this isoform, and disregarded Kv1.2 and Kv1.3 as important O2-sensitive Kv channels in PA VSMCs on the basis of their sensitivity to the K+ channel toxins charybdotoxin (CTX) and dendrotoxin (DTX). Moreover, this group cloned a novel K+ channel (Kv9.3) from rat pulmonary arteries that does not functionally express as a homomeric channel, but rather assembles with Kv2.1 to form a functional heteromeric channel.15
Although the Kv2.1/Kv9.3 channel complex seems to be the best candidate
to date for the O2-sensitive Kv channel in PA
VSMCs, the O2 sensitivity of other potentially
important Kv channels (ie, Kv1.2 and Kv1.5) has not been directly
studied. In the present study, we examined the effects of
hypoxia on Kv1.2, Kv1.5, Kv2.1, and Kv9.3 subunits
expressed in mouse L cells in an attempt to clarify which Kv channels
may contribute to the O2-sensitive
K+ current in PA VSMCs. Here, we present data
indicating that Kv1.2 and Kv2.1, but not Kv1.5, homomeric channels are
reversibly inhibited by hypoxia, and we provide
electrophysiological and pharmacological
evidence that Kv1.2 and Kv1.5 subunits can form an
O2-sensitive heteromeric channel. Especially
noteworthy is the finding that heteromeric channels show the greatest
O2 sensitivity at voltages near the
EM of PA VSMCs. Thus, the potential for
heteromeric channel formation may provide one explanation for the
pharmacological and electrophysiological
discrepancies between cloned Kv channels and native
K+ currents.
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DNA Constructs
A 1600 bp PvuII-EcoRI fragment encoding the open reading frame of the rat Kv1.2 cDNA was blunted with Klenow and subcloned into the blunted KpnI site of the pCMV4 expression vector using standard techniques.19 The 2100-bp NotI-EcoRI fragment of the rKv2.1 cDNA20 was blunted and subcloned into the EcoRV site of the pMSVNeo expression vector.21 Kv9.3 cDNA was obtained using the Stratagene RT-PCR kit and rat lung mRNA. Primers were designed from the rat Kv9.3 sequence reported in GenBank.15 The upper primer began at -2 (the adenine of the initiating codon ATG was at +1), whereas the lower primer started at nucleotide 1623. The amino acid sequence of Kv9.3 was 98.3% homologous to that previously reported by Patel et al,15 with one amino acid substitution at position 209 (E to G). PCR yielded a 1625 bp fragment that was gel-purified and subcloned into the pCR2.1 TA cloning vector (Invitrogen). The fragment was then subcloned into the XbaI and SmaI sites of a modified pBKCMV expression vector that had the ß-galactosidase ATG deleted. This vector was used to increase expression efficiency and remove the possibility that the expressed channels contained a ß-galactosidase protein sequence on the N-terminus.
The Kv1.5/Kv1.2 tandem was constructed by using overlap PCR to link the C-terminus of Kv1.5 to the N-terminus of Kv1.2. The stop codon in Kv1.5 was removed, and 4 amino acids (GPEE) were inserted before the Kv1.2 starting methionine. The 3' end of Kv1.2 was as described above, whereas the 5' end of Kv1.5 started 22 bp upstream from the initiating ATG. The tandem was blunt-end ligated into the SmaI site of the modified pBK vector.
All DNA constructs were confirmed by restriction analysis and automated sequencing.
Transfection and Tissue Culture
The mouse L-cell line expressing human Kv1.5 was used as
described previously.21 The rKv2.1-containing
expression vector was transfected into mouse L cells, and stable cell
lines were produced and cultured as previously
described.21 rKv1.2 and rKv9.3 were studied using
transiently transfected L cells. Mouse L cells (40% to 60% confluence
in 60-mm dishes) were transiently transfected with either rKv1.2 or
rKv9.3 and green fluorescent protein (GFP) to assess
transfection efficiency and to identify cells for voltage-clamp
analysis, as previously described.23 The transient
transfection procedure used either 1 µg of
rKv1.2/pCMV4 or 0.6 µg of rKv9.3 and 0.4 µg
of GFP/pCI; each was mixed with 15 µL of the lipofectamine reagent,
which was 0.5 mL of serum-free Dulbecco's modified Eagle medium
incubated at 37°C for 30 minutes. Cells were then incubated
with the lipofectamine mixture for 6 to 8 hours; after this time, the
mixture was removed and replaced with standard culture medium for 36 to
48 hours. Cells were removed from the dish using brief trypsinization;
they were then briefly centrifuged and resuspended in fresh
standard culture medium at room temperature for recording
within the next 12 hours.
The mouse L cells were used specifically because they endogenously express the Kvß2.1 subunit, which is needed for efficient expression of Kv1.2.22 Therefore, the Kv1.2/Kv1.5 experiments were performed in the presence of the Kvß2.1 subunit. Comparison of the hypoxia sensitivity of Kv1.2 and Kv1.5 ± this ß was attempted by using the ß-free HEK293 cells. However, Kv1.2 was difficult to express in these cells, perhaps due to the lack of the Kvß2.1 subunit.22
Heteromeric Formation of Kv1.2/Kv1.5 and Kv2.1/Kv9.3
Channels
Mouse L cells stably expressing rKv2.1 or hKv1.5 were
transiently transfected with either 0.6 or 1 µg
of rKv9.3 and rKv1.2, respectively (see above for
details). These DNA amounts gave saturating effects in terms of
heteromeric channel formation. After transfection, cells were incubated
for 36 hours; then, the transfected cells were incubated with 2
µmol/L dexamethasone for 18 to 24 hours to induce the
expression of Kv1.5 or Kv2.1 channels and, thereby, allow heteromeric
channel formation of these subunits.
Kv9.3 is electrically silent when expressed as a homomeric channel, but
when expressed with Kv2.1, it modifies the activation and deactivation
kinetics of the Kv2.1 channel and shifts the activation curve in the
hyperpolarizing direction.15 However, because confirmation
of heteromeric assembly between Kv1.2 and Kv1.5 subunits was more
difficult to ascertain, the following criteria were used as a guide to
assess heteromeric channel formation. The current kinetics and
activation curves generated from Kv1.2 and Kv1.5 homomeric channels and
Kv1.2/Kv1.5 heteromeric channels allowed one level of characterization
because the activation midpoints were approximately 22 and -12 mV for
the Kv1.2 and Kv1.5 homomeric channels, respectively. When these
channels were coexpressed, activation curve data best fit by the sum of
2 Boltzmann equations were disregarded based on the assumption
that these data reflected the summation of distinct populations of
Kv1.2 and Kv1.5 homomeric channels. Only data best fit with a single
Boltzmann equation were analyzed. The DTX sensitivity of
expressed currents was also used to assess heteromeric channel
formation because DTX specifically blocks Kv1.2 homomeric channels
(Kd=20 nmol/L),23
whereas Kv1.5 homomeric channels are virtually insensitive
(Kd>300 nmol/L). Recent studies suggest
that the DTX sensitivity of a Kv1.2/Kv1.5 heteromeric channel is
dominated by the presence of the Kv1.5 toxin-insensitive
subunit.24 Therefore, in the present study,
Kv1.2/Kv1.5 heteromeric channels containing 
1 Kv1.5 DTX-insensitive
subunit should be insensitive to 50 nmol/L DTX. Only those currents
that were DTX-insensitive were considered to represent
heteromeric Kv1.2/Kv1.5 channel complexes. Currents showing any DTX
sensitivity were not analyzed further.
Electrophysiology
Experiments were performed in a small volume (180 µL) bath
mounted on the stage of an inverted microscope (Nikon) continuously
superfused at a flow rate of 0.5 to 1.0 mL/min. Recordings
were made with an Axopatch 200A amplifier (Axon Instruments)
using the whole-cell configuration of the patch-clamp
technique.25 Patch pipettes (1 to 2 megohms) were pulled
from borosilicate glass capillaries. Junction potentials were corrected
after placing the tip of the electrode in the bath solution and before
gigohmn seal formation, which was achieved via gentle suction. After
establishing the whole-cell configuration, capacitive transients
elicited by symmetrical 10 mV voltage clamp steps from -80 mV were
recorded to calculate cell capacitance and access resistance. Cells
expressing currents>5 nA were discarded. Currents were sampled at 1 to
5 kHz after anti-alias filtering at 0.5 to 2 kHz. Data acquisition and
command potentials were controlled by pClamp software (v6.04, Axon
Instruments) and stored for later off-line analysis. All
experiments were performed at room temperature (21°C to
23°C).
Solutions
The intracellular pipette solution contained (in mmol/L):
KCl 110, HEPES 10, K2BAPTA 5,
K2ATP 5, and MgCl2 1; it
was adjusted to pH 7.2 with KOH. The bath solution contained (in
mmol/L): NaCl 110, KCl 4, MgCl2 1,
CaCl2 1.8, HEPES 10, and glucose 1.8; it was
adjusted to pH 7.35 with NaOH. The effects of hypoxia were
studied by switching between normoxic and hypoxic perfusate
reservoirs. Normoxic solutions were equilibrated with 100%
O2, whereas hypoxic solutions were achieved by
bubbling with 100% N2 for at least 20 minutes
before cell perfusion and by passing a stream of
N2 over the surface of the bath. These procedures
produced PO2 values of 140 to 160
mm Hg and 30 to 40 mm Hg, respectively.
PO2 was continuously monitored in the
bath chamber using an O2-sensitive microelectrode
(ISO2/OXEL-1, World Precision
Instruments). The pH and temperature of these solutions were regularly
monitored and maintained at pH 7.35 and 21°C to 23°C to guard
against pH- and temperature-induced changes in channel function. DTX-I
was kindly provided by Dr R. Hartshorne (Oregon Health Sciences,
Portland, Ore) and was added to the bath solution where
indicated.
Pulse Protocols and Analysis
The holding potential was -80 mV, and the cycle time within
each protocol was 10 s to allow full recovery from inactivation
between pulses. The standard protocol used to obtain current-voltage
relationships and activation curves consisted of 250-ms voltage-clamp
pulses applied in 10 mV steps between -70 and 60 mV, unless indicated
otherwise. Steady-state current-voltage relationships were obtained by
measuring the current at the end of the voltage-clamp pulse and plotted
against test potential. Currents were normalized relative to each
cell's control (normoxic) current at 60 mV. Deactivating tail currents
were recorded at -40 mV, and activation curves were obtained from
the maximum value of the tail current amplitude immediately after the
capacitive transients.
Activation curves were fitted with a Boltzmann equation:
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2 criterion.
Statistical Analysis
All data are expressed as the mean±SEM of n cells. Comparisons
between groups were made using a paired Student's t test.
Values of P<0.05 were considered significant.
Immunohistochemistry
The production and use of the Kv1.5 N-terminal antibody
and the immunolocalization of Kv1.2 and Kv1.5 in rat pulmonary
resistance arteries were performed as previously
described.27 The Kv1.2 antibody was produced against a
glutathione transferase (GST) fusion protein containing
C-terminal sequence amino acids 436 to 495 using previously described
protocols.27 A second, affinity-purified Kv1.2 antibody
directed against a synthetic peptide, CNEDFREENLKTANCTLANT, was
provided by Dr Jeanne Nerbonne (Washington University, St. Louis, Mo).
Immunofluorescence staining of transfected tissue
culture cells confirmed that both Kv1.2 antibodies detected Kv1.2
protein (data not shown). Rat pulmonary resistance vessels
(third division, 250 to 300 µm in diameter) were placed in
cryomolds, embedded in Tissue Tek (Baxter), and slowly frozen at
-80°C. Cryosections measuring 10 µm were collected on
gelatin-coated coverslips and then incubated with primary antibody
followed by biotin-conjugated goat anti-rabbit IgG (Jackson
Immunoresearch Laboratories) and CY3-conjugated streptavidin (Jackson
Immunoresearch Laboratories), as previously described.27
Samples were analyzed using a Nikon E800 microscope equipped
with standard epifluorescence and a Princeton
Instruments CCD camera.
Immunogen block of tissue staining was performed to demonstrate antibody specificity. Staining was performed as described above, except that cryosections were stained with antiserum that had been preincubated overnight at 4°C with 40 nmol/L of either glutathione transferase (GSH-T) or the appropriate GSH-T fusion protein construct, as previously described.27 The 112 N-terminal amino acids of Kv1.5 coupled to GSH-T were used to block Kv1.5 antiserum binding, while C-terminal residues 429 to 496 of Kv1.2 coupled to GSH-T blocked Kv1.2 antiserum binding.
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O2 Sensitivity of rKv2.1 Stably Expressed in Mouse L Cells
Because previous work implicated the Kv2.1 channel as an important molecular component of the PA VSMC O2-sensitive K+ current, we first investigated the O2 sensitivity of this channel as stably expressed in mouse L cells. Representative outward currents recorded from a holding potential of -80 mV to a test potential of 60 mV, before and after 10 minutes of exposure to the hypoxic solution, are shown in Figure 1A
.
Hypoxia reversibly inhibited Kv2.1 current; in the example
shown, hypoxia significantly (P<0.05) inhibited the
current recorded at 60 mV by 23%. The onset of Kv2.1 current
inhibition occurred within 1 minute after exposure to the hypoxic
solution; inhibition continued to decrease to a new steady state after
7 minutes of exposure to hypoxia. This decrease in current
paralleled the decrease in
PO2 levels. The mean
current-voltage (I-V) relationship of Kv2.1 (Figure 1B) shows that Kv2.1 currents activated at potentials
more positive than 30 mV were significantly inhibited by
hypoxia (P<0.05; n=10). In contrast, little Kv2.1
current was detected at potentials more negative than -20 mV (Figure 1C), and this current was not significantly inhibited by
hypoxia. These data suggest that although Kv2.1 seems to be
O2 sensitive, this sensitivity does not
occur in the voltage range of the PA VSMC EM.
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The effect of hypoxia on the voltage dependence of activation
was also examined; mean data are shown in Figure 1C
.
Hypoxia had no effect on the voltage dependence of activation
(control, V0.5=11.37±1.35 mV, n=15;
hypoxia, V0.5=10.73±1.27 mV, n=10),
which suggests that the observed decrease in Kv2.1 current was not due
to a shift in the voltage dependence of activation.
Functional Expression and O2 Sensitivity of Kv2.1/Kv9.3
Expressed in Mouse L Cells
Patel et al15 reported the cloning of a novel Kv
channel (Kv9.3) from a rat pulmonary artery; this channel does
not functionally express as a homomeric channel when expressed alone,
but it assembles with Kv2.1 to form a functional heteromeric channel.
Although this group reported that the heteromeric channel was
reversibly inhibited by hypoxia, the hypoxic sensitivity of
this channel was not fully characterized, and it was detected only in a
subset (56%) of transfected COS-7 cells.15 In the
present study, 99% of the cells expressing the Kv2.1 channel
exhibited a hypoxic response; therefore, the O2
sensitivity of the Kv2.1/Kv9.3 heteromeric channel was examined using
the L-cell expression system.
Representative current records from L cells
expressing Kv2.1, Kv9.3, or Kv2.1/Kv9.3 are shown in Figure 2A. Kv9.3-transfected cells failed to
express any Kv channel activity (Figure 2A; n=3); however,
coexpression of Kv9.3 with Kv2.1 produced several important
modifications in the biophysical properties of the Kv2.1 channel. Kv9.3
increased activation kinetics (Kv2.1: 
=11.25±1.67 ms; Kv2.1/Kv9.3:
=7.27±0.56 ms; Figure 2A) and slowed deactivation kinetics
(Kv2.1: fast=12.66±0.39 ms,
slow=62.1±3.16 ms; Kv2.1/Kv9.3:
fast=20.13±1.08 ms,
slow=142.12±6.55 ms; Figure 2A)
relative to Kv2.1 alone. In addition, Kv9.3 caused a hyperpolarizing
shift in the voltage dependence of activation (Kv2.1:
V0.5=11.37±1.35, k=13.34±1.23, n=15;
Kv2.1/Kv9.3: V0.5=-8.73±1.2 mV,
k=12.67±1.13; n=6); therefore, the Kv2.1/Kv9.3 heteromeric
channel activated at physiologically
relevant membrane potentials (data not shown). These data are
consistent with those of Patel et al,15 who
reported similar Kv9.3-induced changes in Kv2.1 current expressed in
both COS-7 cells and Xenopus oocytes.
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Figure 2B shows that hypoxia reversibly inhibited
Kv2.1/Kv9.3 current at 60 mV. Moreover, hypoxic inhibition of
Kv2.1/Kv9.3 current was detected in all cells studied. Data summarized
in Figure 2C compare the effect of hypoxia on the mean
I-V relationship for Kv2.1/Kv9.3 current. Mean hypoxic
inhibition of Kv2.1/Kv9.3 current at 60 mV was 21±0.9% (n=6), which
was not significantly different from Kv2.1 alone (compare values at 60
mV; Figure 1B). However, in contrast to Kv2.1 alone, a greater
amount of Kv2.1/Kv9.3 current was activated at more
physiologically important potentials, and this
current was reversibly inhibited by hypoxia. The observation
that hypoxia inhibited the Kv2.1/Kv9.3 heteromeric current at
negative potentials (see inset in Figure 2B for currents at -20
mV), where homomeric Kv2.1 current does not activate, further
supports the existence of a Kv2.1/Kv9.3 heteromeric complex. Note that
at -20 mV and 300 ms, the currents have not reached steady state.
However, O2 sensitivity calculated during
activation was similar to that observed at steady state. For example,
at 60 mV, the steady-state Kv2.1/Kv9.3 current was inhibited 20% with
hypoxia, whereas the inhibition was 25% at 50% activation. As
illustrated in Figure 2D, hypoxia caused a reduction of
48% in current activated at -30 mV (P<0.05).
These data suggest, therefore, that the Kv2.1/Kv9.3 heteromeric channel
may indeed be an important molecular component of the native
O2-sensitive K+
current.
O2 Sensitivity of Kv1.5
Some authors suggested that Kv1.5 was an important
O2-sensing Kv channel present in PA
VSMCs14 ; therefore, the O2
sensitivity of this channel was investigated next. Figure 3 shows the effect of hypoxia on
hKv1.5 currents stably expressed in mouse L cells. In contrast to Kv2.1
and Kv2.1/Kv9.3, hKv1.5 current at 60 mV was not inhibited by
hypoxia (Figure 3A). Moreover, the mean I-V
relationship for hKv1.5 current under control and hypoxic conditions
shows that although Kv1.5 current activated at
physiologically relevant potentials, this
current was not O2 sensitive at any potential
(Figure 3B; n=9). Hypoxia also had no significant effect
on the rat Kv1.5 channel expressed in the same L cells (data not
shown).
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Functional Expression and O2 Sensitivity of rKv1.2
Expressed in Mouse L Cells
The O2 sensitivity of rKv1.2 transiently
transfected into L cells was examined next.
Representative traces of rKv1.2 current elicited in
response to 1-s voltage-clamp pulses to 80 mV are shown in Figure 4A. Under normoxic conditions, these
currents were noninactivating, delayed-rectifying,
outward K+ currents that activated much
more slowly than the Kv channels described above. For example, a 1-s
voltage-clamp pulse to 80 mV was required to fully activate
Kv1.2; a 250-ms pulse to 60 mV was needed to fully activate
Kv1.5, Kv2.1, and Kv2.1/Kv9.3 (Figures 1 through 3). Figure 4A also shows that hypoxia reversibly
inhibited rKv1.2 current. At 80 mV, hypoxic inhibition of rKv1.2
averaged 19±0.2% (n=7; Figure 4B); this inhibition was not
associated with a shift in the voltage dependence of activation (data
not shown). Although Kv1.2 current at depolarized potentials (>40 mV)
was significantly inhibited by hypoxia, no Kv1.2 current was
detected at more physiologically relevant
potentials (ie, -50 to -20 mV), suggesting that this homomeric
channel is unlikely to contribute to the PA VSMC
O2-sensitive K+
current.
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Kv1.2 and Kv1.5 Subunits Coassemble to Form an
O2-Sensitive Heteromeric Channel
To determine whether Kv1.2 and Kv1.5 subunits can assemble to
form an O2-sensitive heteromeric channel, L cells
stably expressing Kv1.5 were transiently transfected with Kv1.2 cDNA.
Only those cells meeting the criteria described in Materials and
Methods were analyzed.
The electrophysiological and
pharmacological characteristics of K+ currents
generated by coexpression of Kv1.2 and Kv1.5 subunits in L cells
were examined first. As illustrated in Figure 5A, coexpression of Kv1.2 with Kv1.5
produced currents that exhibited faster activation kinetics and partial
inactivation compared with Kv1.2 alone. Nonetheless, it was possible
that this K+ current reflected the summation of
distinct populations of Kv1.2 and Kv1.5 homomeric channels rather than
heteromeric channels composed of Kv1.2 and Kv1.5 subunits.
Analysis of the voltage dependence of activation, however,
revealed that these data were best fit with a single Boltzmann equation
with an activation midpoint of -4.55±1.09 mV and
k=16.13±0.65 (n=5), compared with 21.73±0.65 mV and
k=11.11 (n=7) for Kv1.2 alone and -12.88±0.68 mV and
k=8.57±0.54 (n=8) for Kv1.5, suggesting that this
K+ current represented a single
population of Kv channels.
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To provide pharmacological evidence for the heteromeric formation of
Kv1.2 and Kv1.5 subunits, the blocking effects of the
K+ channel toxin DTX-I on Kv1.2, Kv1.5, and
Kv1.2/Kv1.5 currents were investigated. Figure 5C shows that 50
nmol/L DTX-I markedly inhibited the Kv1.2 current elicited during a
voltage-clamp pulse to 80 mV. Mean data summarized in Figure 5D
show that this concentration of DTX-I inhibited Kv1.2 current by
80% (n=6). In contrast, Kv1.5 current was insensitive to 50 nmol/L
DTX-I (n=4; data not shown). Moreover, Figures 5E and 5F
show that 50 nmol/L DTX-I also had no effect on Kv1.2/Kv1.5 current
(n=5); this is consistent with the idea that the presence of a
toxin-insensitive Kv1.5 subunit dominates the pharmacology of the
heteromeric channel.24 28
The electrophysiological and
pharmacological data described thus far suggest that heteromeric
assembly of Kv1.2 and Kv1.5 subunits was occurring (Figure 5). To determine whether this DTX-insensitive
K+ current was O2
sensitive, the effects of hypoxia were examined. Figure 6A shows that hypoxia
significantly inhibited the Kv1.2/Kv1.5 current re- corded at 80 mV;
in the example shown, hypoxia reversibly inhibited current by
18%. In addition, the inset in Figure 6A shows that
hypoxia also inhibited this current at -20 mV. Figures 6B and 6C summarize the meaned data confirming
that hypoxia significantly inhibited current at
physiologically important potentials. For
example, hypoxia inhibited current at -40 mV by 65%
(P<0.05; n=5). The observation that hypoxia
significantly inhibited current at potentials more negative than -20
mV provides further evidence supporting a heteromeric assembly of Kv1.2
and Kv1.5 subunits and suggests that this
O2-sensitive heteromeric channel may be an
important molecular component of the O2-sensitive
K+ current in PA VSMCs.
|
To control the subunit stoichiometry of the Kv1.2/Kv1.5 heteromeric
complex, a tandem construct was created in which Kv1.5 was placed
upstream of, and in frame with, Kv1.2. This tandem had similar
activation kinetics and voltage-sensitivity of the heteromeric currents
shown in Figures 5A and 5B, respectively (data not
shown). Figure 6D shows the tandem construct also had the
expected O2 sensitivity.
To confirm the expression and examine the cellular distribution of the
Kv1.2 and Kv1.5 channel subunits in vivo, cyrosections of rat
pulmonary resistance vessels were stained with Kv1.2 and Kv1.5
antibodies, as shown in Figure 7. Figures 7A and 7B illustrate that Kv1.2 staining was localized to
the smooth muscle layer. Because the anti-GST-Kv1.2 fusion protein
antibody had not been used previously to stain vascular tissue, Kv1.2
immunolocalization was repeated with a second antibody directed against
a peptide immunogen. The same staining pattern was observed with this
antipeptide, the affinity-purified Kv1.2 antibody (data not shown).
Figures 7E and 7F show the localization of the Kv1.5
channel protein; Kv1.5 was also localized to the smooth muscle layer,
as previously described by Archer et al.14 The remaining
panels of Figure 7 show controls for antibody specificity.
Figures 7C and 7D illustrate Kv1.2 antibody binding after
preincubation with either GSH-T or GSH-T/Kv1.2 fusion protein,
respectively. Although the antibody binding was unaffected by
preincubation with GSH-T, it was eliminated with the channel containing
the fusion protein. The same results were obtained with the Kv1.5
antibody after preincubation with GSH-T and GSH-T/Kv1.5 fusion protein
(Figures 7G and 7H. Taken together, these data confirm
that both the Kv1.2 and Kv1.5 channels are expressed in PA smooth
muscle cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The observation that hypoxia reversibly inhibited Kv2.1 current is consistent with previous reports suggesting that this channel is an important Kv channel contributing to the O2-sensitive K+ current in PA VSMCs.14 15 It seems unlikely, however, that the Kv2.1 channel alone could account for the native O2-sensitive K+ current because this channel, when heterologously expressed, does not activate in the voltage range of PA VSMC EM (Figure 1
).15 Indeed,
Patel et al15 reported that Kv2.1 coassembles with Kv9.3
in COS-7 cells and Xenopus oocytes to form a functional
heteromeric channel that activates near the range of the PA
VSMC EM. In support of this hypothesis, in the
present study, coexpression of Kv2.1 and Kv9.3 channel subunits
in mouse L cells generated currents that activated at
physiologically relevant potentials (Figure 2). More importantly, however, hypoxia reversibly
inhibited Kv2.1/Kv9.3 current in the voltage range of PA VSMC
EM (Figure 2). Although Patel et
al15 reported that the Kv2.1/Kv9.3 heteromeric
channel was inhibited by hypoxia, these effects were only
detected in a subset (56%) of transfected COS cells. In contrast, in
the present study, hypoxia reversibly inhibited Kv2.1/Kv9.3
current in all cells studied. Perhaps O2 sensing
and the machinery required to couple it to channel function are
endogenously expressed in mouse L cells. The finding that
hypoxia inhibited Kv2.1/Kv9.3 current activated at
physiologically relevant membrane potentials
suggests that this heteromeric channel may be an important component of
the O2-sensitive K+ current
in PA VSMCs.
Although a number of studies have reported that Kv1.5 is expressed in
PA VSMCs,14 16 the O2 sensitivity of
this channel and its role in PA VSMCs is less clear. Two independent
groups used antibodies specific for the Kv1.5 channel to argue in favor
of Kv1.5 as an important molecular component of HPV.14 18
Furthermore, Wang et al17 reported that chronic
hypoxia down-regulates Kv1.5 mRNA and protein expression in
cultured PA VSMCs. Although these data suggest that Kv1.5 channels may
contribute to the O2-sensitive
K+ current in PA VSMCs, in the present study,
hypoxia failed to inhibit Kv1.5 current expressed in mouse L
cells, despite the fact that this current activated in the
range of the PA VSMC EM (Figure 3). This
was not due to a species difference because hypoxia failed to
inhibit both human and rat Kv1.5 current. Thus, the inability of Kv1.5
to respond to hypoxia suggests that Kv1.5 homomeric channels do
not contribute to the O2-sensitive
K+ current in PA VSMCs.
Although the presence of Kv1.2 channel mRNA and protein in PA VSMCs has
been documented,15 16 17 the idea of Kv1.2 as an
O2-sensitive Kv channel has been disregarded by
several investigators on the basis of its CTX and DTX
sensitivity14 15 : Kv1.2 is blocked in the nanomolar range
(Kd<20 nmol/L),22 whereas the
PA VSMC O2-sensitive K+
current is virtually DTX- and CTX-insensitive
(Kd>300 nmol/L).8 11 15
Heterologous expression of Kv1.2 in mouse L cells, however, revealed
that hypoxia reversibly inhibited Kv1.2 current, although
hypoxic inhibition was detected only at depolarized potentials (Figure 4). Data presented in Figures 3 and 4
suggest that Kv1.2, but not Kv1.5, homomeric channels are reversibly
inhibited by hypoxia. However, comparison of the
electrophysiological and pharmacological
properties of Kv1.2 and Kv1.5 homomeric channels with those of the
native current suggests that, of these two channels, Kv1.5 encodes a
channel most resembling the O2-sensitive
K+ current in PA VSMCs. One possible explanation
for the discrepancies between our data and the literature is that Kv1.2
and Kv1.5 subunits assemble to form an
O2-sensitive heteromeric channel. Indeed, it has
been demonstrated in vitro that Kv1.2 and Kv1.5 subunits can
assemble to form a functional heteromeric
channel.23 28
Our electrophysiological and
pharmacological data suggest that Kv1.2 and Kv1.5 subunits can
assemble to generate currents with distinct kinetic and pharmacological
properties from those displayed by either Kv1.2 or Kv1.5 homomeric
channels (Figure 5). It seems unlikely that this current
reflected the summation of distinct populations of Kv1.2 and Kv1.5
homomeric channels rather than a heteromeric channel for several
reasons. The activation curve data were best fit with a single
Boltzmann function, suggesting the presence of a single population of
Kv1.2/Kv1.5 heteromeric channels. It should be noted, however, that in
65% of the voltage-clamped cells, activation curve data were best fit
with 2 Boltzmann functions, suggesting that summation of Kv1.2 and
Kv1.5 homomeric channels was occurring. However, in 35% of the cells,
activation curves were best fit with a single Boltzmann function,
suggesting that in these cells, heteromeric channel formation between
Kv1.2 and Kv1.5 subunits was most likely. This low percentage of
true heteromeric complexes most likely reflects the less efficient
transient expression of Kv1.2 relative to stable Kv1.5 expression. The
percentage of GFP-positive cells expressing Kv1.2 current, when
transfected into normal L cells, was 80%, and the current densities
were lower than those observed in the cells stably expressing Kv1.5.
Thus, the reason only 35% of the Kv1.5-expressing cells showed
complete heteromeric channel formation after transient transfection
with Kv1.2 is probably due to the fact that (1) only 80% of the cells
express any Kv1.2 current and (2) in those cells expressing Kv1.2, less
than half make enough Kv1.2 protein to ensure complete heteromeral
formation given the amount of Kv1.5 protein already expressed in the
cell line.
These heteromeric currents were also insensitive to 50 nmol/L DTX,
consistent with the idea that the presence of the Kv1.5
toxin-insensitive channel subunit dominates the pharmacology of the
heteromeric channel.24 28 More importantly, however, the
DTX-insensitive currents were reversibly inhibited by hypoxia
at physiologically relevant potentials (-40 to
-20 mV). The latter observation cannot be explained by the summation
of distinct populations of Kv1.2 and Kv1.5 homomeric channels because
at these negative potentials (reflecting current flow purely through
Kv1.5 channels), hypoxia would not be expected to inhibit
current due to the fact that Kv1.5 channels are
O2 insensitive. The finding that hypoxia
inhibited current at these negative potentials (Figure 6)
further argues in favor of heteromeric formation of Kv1.2 and Kv1.5
subunits. It seems, therefore, that Kv1.2 and Kv1.5 subunits can
assemble to form a functional heteromeric channel and that the presence
of two Kv1.2 subunits, as in the tandem, confer
O2 sensitivity on this heteromeric channel. The
Kv1.2/Kv1.5 heteromeric current is a delayed rectifier with apparent
slow inactivation (activation between -40 and -30 mV) and a lack of
DTX sensitivity. Thus, it does resemble the
O2-sensitive currents recorded from native
rat PA VSMCs.15
In summary, the present study examined the O2
sensitivity of several cloned Kv channels expressed in mouse L cells in
an attempt to identify which of these Kv channels may contribute to the
O2-sensitive K+ current in
PA VSMCs. Our results suggest that (1) Kv1.2 and Kv2.1, but not Kv1.5,
homomeric channels are reversibly inhibited by hypoxia; (2)
Kv1.2 and Kv1.5 subunits assemble to form an
O2-sensitive heteromeric channel; and (3) of
these channels, only the Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric
channels are reversibly inhibited by hypoxia in the voltage
range of the PA VSMC EM. Although beyond the
scope of the present study, an obvious question deals with the
mechanism linking hypoxic conditions to Kv channel function. Possible
mechanisms include channel internalization, channel protein
phosphorylation, or direct O2
sensing by channel sulfhydryl groups. Whatever the mechanism, it is
channel isoform–specific and influenced by subunit composition. A
major limitation of our study is that it deals with cloned channels
expressed in heterologous systems and not with native VSMCs.
Confirmation of the expression of heteromeric complexes and their
regulation by hypoxia in native PA VSMCs is the next step in
elucidating the molecular mechanisms responsible for
hypoxia-induced pulmonary vasoconstriction.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health grant RO1 HL49330 (to M.M.T.), Animal Health and Disease Funds through the College of Veterinary Medicine and Biomedical Sciences at Colorado State University, and a Grant-in-Aid from the American Heart Association (to M.M.T.). The authors thank Dr Jeanne Nerbonne for providing the second Kv1.2 antibody.
Received March 3, 1999; accepted July 19, 1999.
|
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V. Hampl, J. Bibova, Z. Stranak, X. Wu, E. D. Michelakis, K. Hashimoto, and S. L. Archer Hypoxic fetoplacental vasoconstriction in humans is mediated by potassium channel inhibition Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2440 - H2449. [Abstract] [Full Text] [PDF]
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 J. Malysz, G. Farrugia, Y. Ou, J. H. Szurszewski, A. Nehra, and S. J. Gibbons The Kv2.2 {alpha} Subunit Contributes to Delayed Rectifier K+ Currents in Myocytes From Rabbit Corpus Cavernosum J Androl, November 1, 2002; 23(6): 899 - 910. [Abstract] [Full Text] [PDF]
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A. Olschewski, Z. Hong, D. P. Nelson, and E. K. Weir Graded response of K+ current, membrane potential, and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L1143 - L1150. [Abstract] [Full Text] [PDF]
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E. D. Michelakis, I. Rebeyka, X. Wu, A. Nsair, B. Thebaud, K. Hashimoto, J. R.B. Dyck, A. Haromy, G. Harry, A. Barr, et al. O2 Sensing in the Human Ductus Arteriosus: Regulation of Voltage-Gated K+ Channels in Smooth Muscle Cells by a Mitochondrial Redox Sensor Circ. Res., September 20, 2002; 91(6): 478 - 486. [Abstract] [Full Text] [PDF]
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I. So and Y. E Earm Is Kv channel inhibition a common path to hypoxic pulmonary vasoconstriction? Cardiovasc Res, August 1, 2002; 55(2): 233 - 235. [Full Text] [PDF]
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D. S Hogg, A. R.L Davies, G. McMurray, and R. Z Kozlowski KV2.1 channels mediate hypoxic inhibition of IKV in native pulmonary arterial smooth muscle cells of the rat Cardiovasc Res, August 1, 2002; 55(2): 349 - 360. [Abstract] [Full Text] [PDF]
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 P. Escoubas, S. Diochot, M.-L. Celerier, T. Nakajima, and M. Lazdunski Novel Tarantula Toxins for Subtypes of Voltage-Dependent Potassium Channels in the Kv2 and Kv4 Subfamilies Mol. Pharmacol., July 1, 2002; 62(1): 48 - 57. [Abstract] [Full Text] [PDF]
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G. D. Thorne, L. Conforti, and R. J. Paul Hypoxic vasorelaxation inhibition by organ culture correlates with loss of Kv channels but not Ca2+ channels Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H247 - H253. [Abstract] [Full Text] [PDF]
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A. E. Belevych, R. Beck, P. Tammaro, L. Poston, and S. V. Smirnov Developmental changes in the functional characteristics and expression of voltage-gated K+ channel currents in rat aortic myocytes Cardiovasc Res, April 1, 2002; 54(1): 152 - 161. [Abstract] [Full Text] [PDF]
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S. V Smirnov, R. Beck, P. Tammaro, T. Ishii, and P. I Aaronson Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries J. Physiol., February 1, 2002; 538(3): 867 - 878. [Abstract] [Full Text] [PDF]
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J. X.-J. Yuan Oxygen-sensitive K+ channel(s): where and what? Am J Physiol Lung Cell Mol Physiol, December 1, 2001; 281(6): L1345 - L1349. [Full Text] [PDF]
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E. A. Coppock and M. M. Tamkun Differential expression of KV channel alpha - and beta -subunits in the bovine pulmonary arterial circulation Am J Physiol Lung Cell Mol Physiol, December 1, 2001; 281(6): L1350 - L1360. [Abstract] [Full Text] [PDF]
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Y. Yu, O. Platoshyn, J. Zhang, S. Krick, Y. Zhao, L. J. Rubin, A. Rothman, and J. X.-J. Yuan c-Jun Decreases Voltage-Gated K+ Channel Activity in Pulmonary Artery Smooth Muscle Cells Circulation, September 25, 2001; 104(13): 1557 - 1563. [Abstract] [Full Text] [PDF]
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A. Cheong, A. M. Dedman, S. Z. Xu, and D. J. Beech KV{alpha}1 channels in murine arterioles: differential cellular expression and regulation of diameter Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1057 - H1065. [Abstract] [Full Text] [PDF]
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A.J. Patel and E. Honore Molecular physiology of oxygen-sensitive potassium channels Eur. Respir. J., July 1, 2001; 18(1): 221 - 227. [Abstract] [Full Text] [PDF]
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E. A. Coppock, J. R. Martens, and M. M. Tamkun Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L1 - L12. [Abstract] [Full Text] [PDF]
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H. L. Reeve, E. Michelakis, D. P. Nelson, E. K. Weir, and S. L. Archer Alterations in a redox oxygen sensing mechanism in chronic hypoxia J Appl Physiol, June 1, 2001; 90(6): 2249 - 2256. [Abstract] [Full Text] [PDF]
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E. D. Michelakis, E. K. Weir, X. Wu, A. Nsair, R. Waite, K. Hashimoto, L. Puttagunta, H. G. Knaus, and S. L. Archer Potassium channels regulate tone in rat pulmonary veins Am J Physiol Lung Cell Mol Physiol, June 1, 2001; 280(6): L1138 - L1147. [Abstract] [Full Text] [PDF]
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O. Platoshyn, Y. Yu, V. A. Golovina, S. S. McDaniel, S. Krick, L. Li, J.-Y. Wang, Lewis. J. Rubin, and J. X.-J. Yuan Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes Am J Physiol Lung Cell Mol Physiol, April 1, 2001; 280(4): L801 - L812. [Abstract] [Full Text] [PDF]
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 M. T. Perez-Garcia, J. R. Lopez-Lopez, A. M. Riesco, U. C. Hoppe, E. Marban, C. Gonzalez, and D. C. Johns Viral Gene Transfer of Dominant-Negative Kv4 Construct Suppresses an O2-Sensitive K+ Current in Chemoreceptor Cells J. Neurosci., August 1, 2000; 20(15): 5689 - 5695. [Abstract] [Full Text] [PDF]
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L. Conforti, I. Bodi, J. W Nisbet, and D. E Millhorn O2-sensitive K+ channels: role of the Kv1.2 {alpha}-subunit in mediating the hypoxic response J. Physiol., May 1, 2000; 524(3): 783 - 793. [Abstract] [Full Text] [PDF]
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M. T. Perez-Garcia and J. R. Lopez-Lopez Are Kv Channels the Essence of O2 Sensing? Circ. Res., March 17, 2000; 86(5): 490 - 491. [Full Text] [PDF]
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O. N. Osipenko, R. J. Tate, and A. M. Gurney Potential Role for Kv3.1b Channels as Oxygen Sensors Circ. Res., March 17, 2000; 86(5): 534 - 540. [Abstract] [Full Text] [PDF]
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J. R. Martens, R. Navarro-Polanco, E. A. Coppock, A. Nishiyama, L. Parshley, T. D. Grobaski, and M. M. Tamkun Differential Targeting of Shaker-like Potassium Channels to Lipid Rafts J. Biol. Chem., March 10, 2000; 275(11): 7443 - 7446. [Abstract] [Full Text] [PDF]
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J. R. Martens, N. Sakamoto, S. A. Sullivan, T. D. Grobaski, and M. M. Tamkun Isoform-specific Localization of Voltage-gated K+ Channels to Distinct Lipid Raft Populations. TARGETING OF Kv1.5 TO CAVEOLAE J. Biol. Chem., March 9, 2001; 276(11): 8409 - 8414. [Abstract] [Full Text] [PDF]
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S. V Smirnov, R. Beck, P. Tammaro, T. Ishii, and P. I Aaronson Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries J. Physiol., February 1, 2002; 538(3): 867 - 878. [Abstract] [Full Text] [PDF]
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P. M. Kerr, O. Clement-Chomienne, K. S. Thorneloe, T. T. Chen, K. Ishii, D. P. Sontag, M. P. Walsh, and W. C. Cole Heteromultimeric Kv1.2-Kv1.5 Channels Underlie 4-Aminopyridine-Sensitive Delayed Rectifier K+ Current of Rabbit Vascular Myocytes Circ. Res., November 23, 2001; 89(11): 1038 - 1044. [Abstract] [Full Text] [PDF]
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