Molecular basis and function of voltage-gated
K+ channels in pulmonary arterial
smooth muscle cells
Xiao-Jian
Yuan1,2,
Jian
Wang1,
Magdalena
Juhaszova2,
Vera A.
Golovina2, and
Lewis J.
Rubin1,2
1 Division of Pulmonary and
Critical Care Medicine, Department of Medicine, and
2 Department of Physiology,
University of Maryland School of Medicine, Baltimore, Maryland 21201
 |
ABSTRACT |
K+-channel
activity-mediated alteration of the membrane potential and cytoplasmic
free Ca2+ concentration
([Ca2+]cyt)
is a pivotal mechanism in controlling pulmonary vasomotor tone. By
using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+ channels and the
role of different K+ currents in
regulating
[Ca2+]cyt
and explored the molecular identification of voltage-gated K+
(KV)- and
Ca2+-activated
K+
(KCa)-channel genes expressed in
pulmonary arterial smooth muscle cells (PASMC). Two kinetically
distinct KV currents
[IK(V)],
a rapidly inactivating (A-type) and a noninactivating delayed
rectifier, as well as a slowly activated
KCa current
[IK(Ca)]
were identified. IK(V) was
reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca) was
significantly inhibited by charybdotoxin (10-20 nM). K+ channels are composed of
pore-forming
-subunits and auxiliary
-subunits. Five
KV-channel
-subunit genes from
the Shaker subfamily (KV1.1,
KV1.2,
KV1.4,
KV1.5, and
KV1.6), a
KV-channel
-subunit gene from
the Shab subfamily
(KV2.1), a
KV-channel modulatory
-subunit
(KV9.3), and a
KCa-channel
-subunit gene
(rSlo), as well as three
KV-channel
-subunit genes
(KV
1.1,
KV
2, and
KV
3) are expressed in PASMC.
The data suggest that 1) native
K+ channels in PASMC are encoded
by multiple genes; 2) the delayed rectifier IK(V)
may be generated by the KV1.1,
KV1.2,
KV1.5,
KV1.6, KV2.1, and/or
KV2.1/KV9.3
channels; 3) the A-type
IK(V) may be generated by the KV1.4 channel
and/or the delayed rectifier
KV channels
(KV1 subfamily) associated with
-subunits; and 4) the IK(Ca) may be
generated by the rSlo gene product.
The function of the KV channels
plays an important role in the regulation of membrane potential and
[Ca2+]cyt
in PASMC.
potassium channel; polymerase chain reaction; fluorescence
microscopy; patch clamp; cytoplasmic calcium
 |
INTRODUCTION |
K+ channels play a critical role
in the regulation of pulmonary vasomotor tone by governing membrane
potential (Em)
(39). Inhibition of K+ channels
depolarizes pulmonary arterial (PA) smooth muscle cells (PASMC) to a
threshold that opens voltage-gated
Ca2+ channels and thus increases
the cytoplasmic free Ca2+
concentration
([Ca2+]cyt)
(39, 40, 67). In contrast, activation of
K+ channels hyperpolarizes PASMC
and inhibits the evoked rise in [Ca2+]cyt
(4, 72). The elevation of
[Ca2+]cyt
in PASMC is a major trigger for pulmonary vasoconstriction and an
important stimulus for vascular smooth muscle cell proliferation, which
leads to vascular remodeling. K+
channels in vascular smooth muscle cells are also potential targets of
vasoactive neurotransmitters or therapeutic agents (e.g., nitric oxide)
(4, 72). Altered K+-channel
function has been implicated in the pathogenesis of several cardiovascular diseases including primary pulmonary hypertension (69)
and systemic arterial hypertension (32).
K+ channels are ubiquitously
expressed in almost all excitable or nonexcitable cells (11, 52). With
the use of electrophysiological approaches, three types of
K+ currents have been identified
in PASMC: voltage-gated K+
(KV) current
[IK(V)]
(16, 18, 47, 67, 70),
Ca2+-activated
K+
(KCa) current
[IK(Ca)]
(2, 4, 18, 46), and ATP-sensitive K+
(KATP) current
[IK(ATP)]
(13). IK(V) is an
important regulator of resting
Em, whereas
IK(Ca) functions
as a critical negative feedback pathway in the regulation of
Em and vascular
contractility (7, 17-19, 29, 67).
KV channels are composed of
pore-forming
-subunits and associated cytoplasmic
-subunits,
which act mainly as a regulatory moiety (26). The
KV-channel
-subunit gene
encoded by the Shaker locus
(KV1) in
Drosophila was first isolated in 1987 (44); three related fly KV-channel
genes, Shab
(KV2),
Shaw
(KV3), and
Shal (KV4), were subsequently isolated
using molecular biological approaches (9). At least 18 vertebrate genes
encoding KV-channel
-subunits have been isolated from mammals and frogs with
Shaker probes (11). Each of these
KV-channel genes produced
functionally distinct KV channels
(11, 24). The slowpoke gene
(Slo) encoding the KCa channel
[high-conductance (BK) channel] was first identified in the
Drosophila
Slo
(dSlo) locus (5). The subsequent
cloning and expression of the genes encoding
KCa channels from
Drosophila (5), mouse
(mSlo) (8), and human
(hSlo) (33) demonstrate that the
protein shares extensive homology with the
KV channels of the
Shaker subfamily.
Recently, four new subfamilies of the electrically silent
KV-channel
-subunits have been
cloned: KV5 (74),
KV6 (49), KV8 (25, 54), and
KV9 (45, 58). Expression of these
modulatory
-subunits (e.g.,
KV9.3 or
KV8.1) per se does not produce
K+-channel activity; however,
coexpression of the modulatory
-subunits with other functional
KV-channel
-subunits (e.g.,
KV2.1) significantly modulates
kinetics, expression level, and voltage dependence of the functional
KV channels (45, 54, 55).
The
-subunits of KV channels
were identified as 38- to 41-kDa polypeptides associated with the
-subunits (64). Studies on cloning of the cDNAs encoding three
KV-channel
-subunits
(KV
1.1, KV
2, and
KV
3) and coexpression with
KV-channel
-subunits indicate that the
-subunits are highly conserved and play an important role
in modulating gating properties of certain
-subunits (23, 24, 26,
50). It has recently been reported that the
Shaker KV-channel
-subunits belong to
an NAD(P)H-dependent oxidoreductase superfamily (34), suggesting that
-subunits may play a critical role in sensing changes in redox
status (3, 72), oxidoreductive metabolism (72), and oxygen tension (30,
42, 45, 47, 48, 59, 66, 71).
Although the electrophysiological properties of
K+ channels have been extensively
studied (10, 40), the molecular identity of
K+-channel genes in PASMC has not
yet been elucidated. In this study, we used patch-clamp techniques,
digital-imaging fluorescence microscopy, PCR, and immunoblotting to
define the distinct K+ currents
and their associated function in regulating
[Ca2+]cyt and
to identify the corresponding K+-channel
genes expressed in PASMC. Because
KV-channel
-subunits specifically bind with Shaker-related
KV-channel
-subunits
(KV1 family) (24, 56), the
molecular study focused on the
KV1-channel
-subunits and
KV-channel
-subunits.
 |
MATERIALS AND METHODS |
Cell preparation. Primary cultures of
rat PASMC were prepared as previously described (70). Briefly, the
intrapulmonary arterial branches as well as the right and left branches
of the main PA were incubated for 20 min in Hanks' balanced salt
solution containing 1.5 mg/ml of collagenase (Worthington Biochemical, Freehold, NJ). After the incubation, a thin layer of adventitia was
carefully stripped off with fine forceps, and the endothelium was
removed by gently scratching the intimal surface with a surgical blade.
The remaining PA smooth muscle was then digested with 1.5 mg/ml of
collagenase, 0.5 mg/ml of elastase (Sigma, St. Louis, MO), and 1 mg/ml
of bovine albumin (Sigma) for 45 min at 37°C to create a
single-cell suspension of PASMC. The cells were then resuspended and
plated onto 25-mm coverslips (for electrophysiological and fluorescent
microscopy experiments) or 10-cm petri dishes (for molecular biological
experiments) and incubated in a humidified atmosphere of 5%
CO2 in air at 37°C in 10%
fetal bovine serum culture medium. Before each experiment and
extraction of total RNA and protein, the primary cultured PASMC were
incubated in 0.3% fetal bovine serum culture medium for 12-24 h
to stop cell growth. This treatment would also minimize the effects of
growth factors, DNA synthesis, and tyrosine kinase activation on
channel expression.
Immunofluorescence labeling. The
primary cultured PASMC were fixed in 95% ethanol and stained with the
membrane-permeable nucleic acid stain
4',6'-diamidino-2-phenylindole (DAPI; 5 µM; Molecular
Probes); the blue fluorescence emitted at 461 nm was used to visualize
the cell nuclei and estimate total cell numbers in the cultures. The
specific monoclonal antibody raised against
-smooth muscle actin
(Boehringer Mannheim, Indianapolis, IN) was used to evaluate cellular
purity of the cultures, and a secondary antibody conjugated with
indocarbocyanine (Cy3; Jackson ImmunoResearch, West Grove, PA) was used
to display the fluorescent image (emitted at 570 nm). The cells were
mounted in 10% 1 M Tris · HCl-90% glycerol (pH 8.5)
containing 1 mg/ml of
p-phenylenediamine. The cell images were processed by a MetaMorph Imaging System (Universal Imaging, West
Chester, PA); indocarbocyanine fluorescence was colored red and DAPI
fluorescence was colored green to display images with a red-green
overlay. All the DAPI-stained cells in the primary cultures also
cross-reacted with the smooth muscle cell
-actin antibody,
indicating that the cultures were all smooth muscle cells.
Recording of
K+ current.
Whole cell and single-channel K+
currents (IK)
were recorded with an Axopatch-1D amplifier and a TL-1 DMA digital
interface (Axon Instruments, Foster City, CA) with the patch-clamp
technique as described (67, 70). Patch pipettes (2-4 M
) were
fabricated from microhematocrit tubes (VWR Scientific, Bridgeport, NJ)
and were fire polished on a Narishige microforge. Step-pulse protocols and data acquisition were performed with pCLAMP software. Currents were
filtered at 1-2 kHz (
3 dB) and digitized at 2-4 kHz
with the Axopatch-1D amplifier. For whole cell current recording,
series resistance and capacitance were routinely compensated for
(40-70%) by adjusting the internal circuitry of the patch-clamp
amplifier. Leakage currents were subtracted with the P/4 protocol in
pCLAMP software. In experiments with cell-attached patches, the actual transpatch potential
(Epatch) was
unknown; however, it was assumed that
Epatch equals the
difference between the resting
Em and the applied pipette command potential
(Eapp); i.e.,
Epatch = Eapp
Em.
The resting Em in
the cells used in this study was approximately
40 mV (67, 70)
when the cells were bathed in a solution containing 4.7 mM
K+. Thus the single-channel
IK measured in
the cell-attached patches did not reverse at 0 mV, although the
K+ equilibrium
potential was ~0 mV (the pipette solution contained 135 mM K+). The currents actually
reversed at approximately +35 to +45 mV. To make them clear, the
voltages shown in the figures are expressed as
Eapp values. All
experiments were performed at room temperature (24°C).
Measurement of Em.
The Em in single
PASMC was measured in a current-clamp configuration with the
conventional patch-clamp technique. The extracellular and intracellular
solutions were the same as those for the whole cell current recording.
Voltage measurement was performed when the cell was held at zero
current. Data were acquired by the TL-1 DMA digital interface coupled
to a computer and the Axopatch-1D amplifier and analyzed with pCLAMP
software (67, 70).
Measurement of
[Ca2+]cyt.
Details of the digital-imaging methods employed for measuring
[Ca2+]cyt
have been previously published (20). Briefly, PASMC grown on 25-mm
coverslips were incubated in culture medium containing 3.3 µM fura
2-AM for 30-40 min at room temperature (22-24°C) under an
atmosphere of 5% CO2 in air. The
fura 2-loaded cells were then superfused with a standard bath solution
for 20-30 min at 32-34°C to wash away extracellular dye
and permit intracellular esterases to cleave cytosolic fura 2-AM into
active fura 2. Fura 2 fluorescence (510-nm emission; 380- and 360-nm
excitation) from the cells and background fluorescence were imaged with
a Nikon Diaphot microscope equipped for epifluorescence. Fluorescent
images were obtained with a microchannel plate-image intensifier
(Amperex XX1381, Opelco, Washington, DC) coupled by fiber optics to a
Pulnix charge-coupled device video camera (Stanford Photonics,
Stanford, CA).
Image acquisition and analysis were performed with a MetaMorph Imaging
System (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells as well as the corresponding background images
(fluorescence from fields devoid of cells) were digitized at a
resolution of 512 horizontal × 480 vertical pixels and 8 bits
with a Matrix LC imaging board operating in an IBM-compatible computer
(66 MHz, 486). To improve the signal-to-noise ratio, 8-32
consecutive video frames were usually averaged at a video frame rate of
30 frames/s. Images were acquired at a rate of one averaged image every
3 s when the
[Ca2+]cyt
was changing and every 60 s when the
[Ca2+]cyt
was relatively constant. The
[Ca2+]cyt
was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (20). In most experiments, multiple cells
(usually 6-10) were imaged in a single field, and one arbitrarily
chosen peripheral cytosolic area (4-6 × 4-6 pixels) from each cell was spatially averaged.
Solution and reagents. A coverslip
containing the cells was positioned in the recording chamber (
0.75
ml) and superfused (2-3 ml/min) with the standard extracellular
(bath) physiological salt solution (PSS) for either recording
IK or measuring
[Ca2+]cyt.
The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose,
buffered to pH 7.4 with 5 M NaOH. In
Ca2+-free PSS,
CaCl2 was replaced by equimolar
MgCl2 and 1 mM EGTA was added to
chelate residual Ca2+. The
internal (pipette) solution for recording whole cell
IK(V) contained
(in mM) 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP, buffered to pH 7.2 with 1 M KOH. In the experiments for
recording whole cell
IK(Ca)
[together with
IK(V)], 8.8 mM CaCl2 was added to yield
~2.65 M free Ca2+ in the pipette
solution at room temperature (24°C). In experiments with
cell-attached patches, the pipette was filled with a 135 mM
K+ solution (with 4 mM
MgCl2, 10 mM HEPES, 2 mM EGTA, and
10 mM glucose, buffered to pH 7.4 with 1 M KOH). The bath solution was the same as that used for whole cell current recording.
4-Aminopyridine (4-AP; Sigma) was directly dissolved in PSS on the day
of use. The pH of the solution containing 4-AP was adjusted to 7.4 with
saturated HCl before each experiment. Charybdotoxin (ChTX; Accurate
Chemical and Scientific) was dissolved in water to make a stock
solution of 100 µM; an aliquot of the stock solution was diluted
1:1,000-10,000 in PSS to make a final concentration of 10-100
nM.
Total RNA isolation. Total RNA was
prepared from freshly isolated PA rings, primary cultured PASMC (on
days
6-8), and brain tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (12). Briefly, the cultured cells were washed with
phosphate-buffered solution (Sigma) and scraped into 1 ml/dish of
denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate,
0.5% sarcosyl, and 0.1 M 2-mercaptethanol, pH 7.0). The DNA was
sheared by propelling the solution through a 21-gauge needle 5-10
times. The total RNA was subsequently phenol extracted from the
homogenate by adding 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of
water-saturated phenol, and 0.2 ml of a chloroform-isoamyl alcohol
mixture (49:1). The samples were then mixed vigorously and centrifuged
at 10,000 g for 20 min. The RNA was
precipitated from the aqueous phase by isopropanol (1:1). The total RNA
yield was calculated from the absorption of the RNA preparation at 260 nm. The quality of the RNA was determined from the absorbance ratio of
the optical density (OD) at 260 nm to the OD at 280 nm (OD260/OD280 > 1.7) and by electrophoresis of the denaturated RNA samples through
a 1% agarose-formaldehyde gel (integrity of the 28S and 18S rRNA bands
stained with ethidium bromide). Isolated total RNA was dissolved in
diethyl pyrocarbonate water at 1 µg/µl and stored at
70°C.
RT-PCR. RT was performed with the
First-Strand cDNA Synthesis Kit (Pharmacia Biotech). Four micrograms of
total RNA were reverse transcribed with random hexamers
[pd(N)6 primer]. The
reaction mixture (33-µl final volume) contained 11 µl of the Bulk
First-Strand Reaction Mix [consisting of 45 mM Tris, pH 8.3, 68 mM KCl, 15 mM dithiothreitol, 9 mM
MgCl2, 0.08 mg/ml of BSA, 1.8 mM
each deoxynucleotide triphosphate (dNTP), and 55 units of
FPLCpure murine reverse
transcriptase], 1 µl of
pd(N)6, and 20 µl of the total
RNA solution (including 1-5 µg of total RNA). The reaction mixture
was incubated for 1 h at 37°C and heated at 90°C for 5 min to
inactivate the reverse transcriptase.
The sense and antisense PCR oligonucleotide primers chosen to amplify
cDNA are listed in Table 1. The sense and
antisense primers for the
KV-channel
-subunits of
KV1.1,
KV1.2,
KV1.3, KV1.4,
KV1.5,
KV1.6,
KV2.1, and
KV9.3 were designed from coding regions of RCK1 (GenBank accession no. X12589), BK2 (GenBank accession
no. J04731), KV3 (GenBank accession no. M31744), RCK4 (GenBank
accession no. X16002), KV1 (GenBank accession no. M27158), KV2 (GenBank
accession no. M27159), RSDRK1PC (GenBank accession no. X16476), and
Kv9.3 (GenBank accession no. AF029056), respectively (Table 1). The
sense and antisense primers for the
KCa-channel
-subunit of
Slo were designed from the coding
region of rat Slo
(rSlo; GenBank accession no. U55995). Sequences of the sense and antisense primers for
KV
1.1,
KV
2, and
KV
3 were kindly provided by Dr.
S. Reinhardt (University of Mainz, Germany) (Table 1). The fidelity and
specificity of the sense and antisense oligonucleotides were examined
with the BLAST program.
PCR was performed by a GeneAmp PCR system (Perkin-Elmer, Norwalk, CT)
with Taq polymerase and accompanying
buffers. Two to three microliters of the first-strand cDNA reaction
mixture were used in a 50-µl PCR consisting of 0.2 nM each primer, 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 2 mM
MgCl2, 200 M each dNTP, and 2 units of Taq DNA polymerase
(Perkin-Elmer). The cDNA samples were amplified in a DNA thermal cycler
(model 2400, Perkin-Elmer) under the following conditions: the mixture
was annealed at 50-61°C (1 min), extended at 72°C (2.0 min), and denatured at 94°C (1 min) for 30-35 cycles. This was
followed by a final extension at 72°C (10 min) to ensure complete
product extension. The PCR products were electrophoresed through a 1%
agarose gel, and amplified cDNA bands were visualized by ethidium
bromide staining.
To quantify the PCR products (the amounts of mRNA) of the
KV channels (
- and
-subunits), an invariant mRNA of
-actin was used as an internal
control. Immediately after each experiment, the OD values for each band
on the gel were measured by a gel documentation system (UVP, Upland,
CA). The OD values of K+-channel
signals were normalized to the OD values of the
-actin signals,
which are expressed in arbitrary units (the ratio of the
KV-channel mRNA levels to the
-actin mRNA levels) for quantitative comparison (66). Because PCR
amplification is an exponential process, the extent of amplification is
not only dependent on the initial amount of target mRNAs (or cDNAs) but
is also related to the efficiency and cycle number. Although invariant
-actin mRNA was used as an internal control, the possible difference in efficiencies between the primer pairs for
-actin and the target mRNAs can still lead to different yields of PCR products. Therefore, the PCR study provides only a relative comparison of the amounts of
mRNA.
Immunoblotting. The primary cultured
PASMC were washed with PBS, scraped into PBS (2 ml/dish), and
centrifuged at 3,500 rpm. The cell pellet as well as freshly isolated
rat heart (ventricular muscle only) and brain tissues was homogenized
in 10 mM HEPES-KOH (pH 7.0) containing the protease inhibitor cocktail
(Complete tablets, Boehringer Mannheim, Indianapolis, IN) with a
Polytron (Brinkmann) for 10 s at 7,000 rpm. Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL) with BSA as
a standard. The samples of homogenates were used for immunoblotting.
Proteins solubilized in SDS buffer were separated by SDS-PAGE. The 10%
gels were calibrated with prestained protein molecular-weight markers
(Bio-Rad, Richmond, CA). The proteins were then transferred to Hybond-C
extra nitrocellulose membrane (Amersham) as previously described (63).
The efficiency of the transfer was verified by Ponceau-S staining.
Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline
and 0.1% Tween 20. The blots were then incubated with the
affinity-purified polyclonal antibodies specific for
KV1.2-,
KV1.3-,
KV1.4 (Alomone Labs, Jerusalem,
Israel)-, KV1.5-, and
KV2.1 (Upstate Biotechnology, Lake
Placid, NY)-channel
-subunits for
1 h. The membranes were washed
(3 × 5 min) and incubated with anti-rabbit horseradish
peroxidase-conjugated IgG for 1 h, and an enhanced chemiluminescence
detection system (Amersham, Arlington Heights, IL) was used for
detection of the bound antibody.
Statistical analysis. The composite
data are expressed as means ± SE. Statistical analyses were
performed with paired and unpaired Student's
t-test and one-way ANOVA, as
indicated. Differences were considered to be significant when
P < 0.05.
 |
RESULTS |
Electrophysiological differentiation of rapidly inactivating and
delayed rectifier IK(V).
When cells were superfused with the
Ca2+-free, 1 mM EGTA-containing
bath solution and dialyzed with the
Ca2+-free, 10 mM EGTA-containing
pipette solution (with 5 mM ATP), IK(V) was
elicited by depolarization to test potentials ranging from
40 to
+80 mV (Fig. 1,
A and
B).
IK(Ca) and
IK(ATP) were minimized under these conditions (29, 46, 67). On depolarization to +80
mV, the current was rapidly activated and consisted of a transient
component
[IK(tr)]
and a steady-state component
[IK(ss)] (Fig. 1C). Changing the holding
potential from
70 to
40 mV abolished IK(tr) and
decreased IK(ss)
(Fig. 1C). Subtraction of the two current records obtained from different holding potentials revealed an
IK(tr) that
resembles the rapidly inactivating (A-type)
IK(V) and
isolated an
IK(ss) that is
similar to the non- or slowly inactivating delayed rectifier
IK(V) (16, 18,
46, 47, 67, 70). Both components of
IK(V),
IK(tr) and
IK(ss), were
significantly and reversibly inhibited by 5 mM 4-AP (Fig.
1D), whereas ChTX (10-20 nM)
had no effect on these currents (67).

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Fig. 1.
Electrophysiological differentiation of rapidly inactivating (A-type)
and delayed rectifier voltage-gated
K+
(KV) currents
[IK(V)]
and inhibitory effect of 4-aminopyridine (4-AP) on currents in
pulmonary arterial smooth muscle cells (PASMC) superfused and dialyzed
with Ca2+-free solution.
A: family of currents obtained by
depolarizing the cell from a holding potential of 70 mV to a
series of command potentials ranging from 40 to +80 mV. Pipette
(intracellular) solution contained 10 mM EGTA and 5 mM ATP.
B: current-voltage
(I-V)
relationship curve assembled from data shown in
A. Transient
IK(V)
[IK(tr)]
was measured at 10-50 ms. Steady-state
IK(V)
[IK(ss)]
was measured at 250-290 ms (test pulse duration was 300 ms).
C: predepolarization to 40 mV
inactivated
IK(tr).
Inset: current component that was
inactivated by predepolarizing the cells from 70 to 40 mV
before +80-mV test pulse was applied.
D: representative current trace
elicited by depolarizing the cell from a holding potential of 70
to +60 mV recorded before (Control), during (4-AP), and after
(Recovery) extracellular application of 5 mM 4-AP.
Inset: 4-AP-sensitive component of
IK(V).
|
|
Activation of IK(Ca) by increasing
[Ca2+]cyt.
When cells were superfused with the 1.8 mM
Ca2+-containing bath solution and
dialyzed with the 8.8 mM
Ca2+-containing pipette solution
(with 5 mM ATP and 10 mM EGTA present), both
IK(V) and
IK(Ca) were
elicited by depolarization to test potentials ranging from
40 to
+80 mV (Fig.
2A,
left). The activation kinetics of
the currents was slower than the
IK(V) shown in
Fig. 1, but the amplitude of the currents was much greater (compare Fig. 2 with Fig. 1). Extracellular application of 10 nM ChTX
significantly inhibited the currents from 9.1 ± 1.7 to 4.1 ± 1.1 nA (P < 0.001; n = 5 cells) at the test potential of
+80 mV (Fig. 2, A and
B), mainly due to the inhibitory
effect of ChTX on
IK(Ca).
Subtraction of the current records obtained before and during
introduction of ChTX revealed the ChTX-sensitive
IK(Ca) (Fig.
2A,
bottom). The relatively slow
activation kinetics of the ChTX-sensitive IK(Ca) appears to
be similar to the
IK(Ca) recorded
from the hSlo cRNA-injected
Xenopus oocytes (33).

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Fig. 2.
Whole cell Ca2+-activated
K+ currents in PASMC dialyzed with
pipette solution containing high concentration of free
Ca2+
([Ca2+]).
A: family of currents elicited by
depolarizing the cell from a holding potential of 70 mV to a
series of command potentials ranging from 40 to +80 mV when
[Ca2+] in pipette
solution was increased to ~2.65 µM (8.8 mM
CaCl2, 10 mM EGTA, and 5 mM ATP,
pH 7.2, at 24°C). Currents were recorded before (Control) and
during extracellular application of 10 nM charybdotoxin (ChTX).
ChTX-sensitive component of the currents was obtained by subtracting
the currents recorded during application of ChTX from the currents
recorded under control condition (Subtraction).
IK,
K+ current.
B:
I-V
relationship curves for current records shown in
A.
|
|
The dose giving half-maximal inhibition of ChTX for
IK(Ca) is
2-15 nM in smooth muscle cells (10, 40). Thus 10 nM ChTX did not
completely block
IK(Ca); the
remaining currents shown in Fig. 2A,
right, included both
IK(V) and
IK(Ca). Because
of the large fraction of
IK(Ca), the
remaining currents during application of 10 nM ChTX displayed similar
kinetics as
IK(Ca). The
large, long-lasting inward tail currents elicited under these
conditions might be due to the contamination of
Ca2+-regulated nonselective cation
(e.g., Na+) currents (10)
and/or Ca2+-activated
Cl
currents, which have
been described in these cells (68), although we do not know why the
currents were somehow inhibited by ChTX. It has been demonstrated that
many K+-channel blockers also
block the Ca2+-activated
nonselective cation channels (10). It is not clear, however, whether
ChTX can affect the Ca2+-activated
nonselective cation currents in PASMC.
Single-channel IK(Ca) and
IK(V) in PASMC.
In cell-attached membrane patches of PASMC superfused with a
Ca2+-containing solution, a
large-amplitude
IK was elicited
by depolarizing the patch to +70 mV (Fig.
3A). The
slope conductance of the large-amplitude IK, calculated
from the current-voltage relationships, was 214 pS
(n = 8 patches; Fig.
3C). Therefore, the large-amplitude
IK corresponds to
the large-conductance
IK(Ca) that is
activated by an increased
[Ca2+]cyt
(2, 7, 18, 19, 48, 73). In some cell-attached membrane patches of PASMC
superfused with a Ca2+-free
solution, several small-amplitude
IK were elicited
by depolarizing the patches to +70 mV (Fig.
3B). These small-amplitude
IK represent the
small-conductance
IK(V) that were
previously described in both animal and human PASMC (18, 19, 40, 46,
73). As shown in Fig. 3, B and
C, there appeared to be at least three classes of KV channels in PASMC
based on the calculated slope conductances: a 24-pS channel
(n = 14 patches;
a), a 40-pS channel (n = 9 patches;
b), and a 67-pS channel
(n = 8 patches;
c). It has been demonstrated that
there are multiple
IK(V) in colonic smooth muscle cells, with conductances of 82, 42, 8, and <4 pS (27).
These results indicate that smooth muscle cells functionally express
multiple KV channels.

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Fig. 3.
Single-channel IK
recorded from cell-attached membrane patches of PASMC superfused with
Ca2+-containing
(A) and
Ca2+-free
(B) solutions. Patch command
potential [holding potential (HP)] was held at +70 mV. A
large-amplitude current (A) and 3 small-amplitude currents
(Ba-Bc)
were observed on depolarization. Dotted lines, current levels when
channels are closed. Currents were recorded from different PASMC.
C:
I-V
relationship curves for large-amplitude current
{Ca2+-activated current
[IK(Ca)]}
and the 3 small-amplitude currents
[IK(V);
a-c].
Calculated slope conductances are 214 (n = 8 patches; ), 24 (n = 14 patches; ), 40 (n = 9 patches; ), and 67 pS
(n = 8 patches; ). Resting membrane
potential (Em)
of these cells is about 40 mV. Data are means ± SE.
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Functional roles of KV and
KCa channels in regulating
Em and
[Ca2+]cyt.
Application of 4-AP (5 mM) depolarized PASMC and elicited
Ca2+-dependent action potentials
(Fig.
4A).
Removal of extracellular Ca2+
abolished the transient action potentials but did not affect the
steady-state depolarization (data not shown). The
KCa-channel blocker ChTX (20 nM),
however, had a negligible effect on the resting
Em (Fig.
4B). Although the inability of ChTX
to elicit depolarization may be partially due to a high-buffering
capacity of intracellular Ca2+, it
is more likely that KCa channels
are relatively closed under resting conditions.

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Fig. 4.
Effect of 4-AP and ChTX on
Em
(A and
B) and cytosolic free
[Ca2+]
([Ca2+]cyt;
C-E)
in PASMC. Em was
measured using current-clamp mode in cells before, during, and after
application of 5 mM 4-AP (A) or 20 nM ChTX (B) in presence of
extracellular Ca2+.
C:
[Ca2+]cyt
measured in peripheral areas (small boxes labeled
a-c
in black-and-white 360-nm image).
Right: corresponding
[Ca2+]cyt
record obtained from each of the cells shown at
left.
D: summarized data showing change in
[Ca2+]cyt
induced by 4-AP [5 mM; peak and plateau (Plat)] or ChTX (20 and 100 nM). Data are means ± SE;
n = 41 cells.
*** P < 0.001 vs. basal
[Ca2+]cyt
levels. E: pseudocolor images showing
[Ca2+]cyt
before (a), during [peak
(b) and Plat
(c)] and after
(d) application of 4-AP as well as
[Ca2+]cyt
before (e), during [20
( f ) and 100 (g) nM], and after
(h) application of ChTX. The 360-nm
excitation image of the cells used for measuring
[Ca2+]cyt
(a-h)
is shown in C
(left) to clarify size of cells.
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In intact PASMC devoid of dialysis with the pipette solution, 4-AP (5 mM) reversibly increased
[Ca2+]cyt
(Fig. 4, C,
D, and
Ea-Ed)
due, apparently, to the evoked membrane depolarization and
Ca2+ action potentials. After
application of 4-AP, a slight shift of the resting
[Ca2+]cyt
was noted (compare Fig. 4Ea with Fig.
4Ee), especially in the perinuclear
areas where the sarcoplasmic reticulum is mainly located (20, 65). This
suggests that more Ca2+ may be
sequestered into the sarcoplasmic reticulum after application of 4-AP,
and slow leakage of the stored
Ca2+ to the cytosol resulted in
the small drift of the resting
[Ca2+]cyt
in perinuclear areas. Application of the
KCa-channel blocker ChTX (20 or
100 nM) had no effect on
[Ca2+]cyt
(Fig. 4C,
right,
D, and
Ee-h).
The cells used for measuring [Ca2+]cyt
were then stained with anti-
-smooth muscle actin antibody, and the
results indicated that all of the cells were smooth muscle cells.
The results for
[Ca2+]cyt
in the nondialyzed PASMC (Fig. 4,
C-E)
are consistent with the findings for
Em in the
dialyzed PASMC (Fig. 4A), indicating
that the IK(V) is
the major determinant of the
Em and thus the
[Ca2+]cyt
under resting conditions. KCa
channels, although relatively closed under resting conditions,
certainly contribute to the regulation of
Em,
Ca2+ homeostasis, and vascular
tone when
[Ca2+]cyt
is increased (7, 16, 17, 29, 38, 46, 67).
Identification of KV- and
KCa-channel
-subunit gene
transcripts in PASMC.
Based on the known sequences, K+
channels fall into three superfamilies: the voltage-gated channels,
Ca2+-activated channels, and
inward rectifier channels. The subsequent experiments were designed to
identify the K+-channel genes
expressed in PASMC.
RT-PCR was used to test the functional expression of
KV and
KCa channels in PASMC. Equal
amounts of total mRNA isolated from primary cultured PASMC were
reversely transcribed and amplified with oligonucleotide primers (Table
1) specifically designed for the
-subunits
[KV1.1 (RCK1),
KV1.2 (BK2),
KV1.3 (KV3),
KV1.4 (RCK4),
KV1.5 (KV1),
KV1.6 (KV2), and
rSlo] as well as for the
-subunits (KV
1.1,
KV
2, and
KV
3). The
K+-channel PCR products were
separated on a 1% agarose gel and visualized by staining the gel with
ethidium bromide. On the basis of the predicted size of the PCR
products, PASMC contain KV1.1 (594 bp), KV1.2 (295 bp),
KV1.4 (322 bp),
KV1.5 (1,111 bp),
KV1.6 (394 bp), and
rSlo (864 bp) gene transcripts (Fig.
5A).
Because the RT-PCR procedures used in these experiments were not
quantitative, these results did not provide any information regarding
the quantity of the particular
KV-channel mRNAs (see
Quantitative comparison of
KV- and
KCa-channel gene transcripts in
PASMC).

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Fig. 5.
RT-PCR analysis of KV- and
Ca2+-activated
K+
(KCa)-channel -subunit mRNAs
isolated from PASMC. A: PCR-amplified
products displayed in agarose gel stained with ethidium bromide for
KV1.1 (594 bp;
a),
KV1.2 (295 bp;
b),
KV1.4 (322 bp;
c),
KV1.5 (1,111 bp;
d),
KV1.6 (394 bp;
e), and rat
slowpoke gene
(rSlo; 864 bp;
f ) in PASMC.
B and
C: PCR-amplified products for
KV1.3 (515 bp) and conserved
region of all KV channels
(KV-all; 227 bp), respectively, in
PASMC (PA) and brain tissues (Br). First-strand cDNAs synthesized from
5 µg of total RNA (extracted from PASMC or Br) were amplified with
specific sense and antisense primers for the respective -subunits
(Table 1). M, molecular-weight marker; +, cDNA added; , cDNA not
added. Same results were repeated in 3-5 independent
experiments.
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With the use of the primer specifically designed for the
KV1.3
-subunit, the PCR product
(predicted size is 515 bp) was not detected in PASMC but was perceived
in brain tissue (Fig. 5B). To ensure
that the cDNAs used for the amplification of
KV1.3 channels were not defective,
the same cDNAs of PASMC were used in a PCR with another primer that was
designed for the conserved region of
KV channels
(KV-all in Table 1). As shown in
Fig. 5C, a 227-bp PCR product was
detected in both PASMC and brain tissue, indicating that the mRNAs and
cDNAs were both intact. These results suggest that PASMC may not
express KV1.3 channels. Because
KV1.3 is a KV channel that is sensitive to
ChTX (11), these data are consistent with the previous observation by
Yuan (67) that ChTX negligibly affected the native
IK(V) in PASMC.
Identification of KV-channel
-subunit gene transcripts in PASMC.
KV-channel
-subunits were
recently cloned from rat brain
(KV
1.1,
KV
2, and
KV
3) and human heart
(KV
1.2 and
KV
1.3) (15, 24, 26, 50).
Association of the
-subunits with
KV-channel pore-forming
-subunits significantly contributes to the
KV-channel diversity and its
various functions in vivo (24, 26, 36, 50, 56). Figure
6 illustrates that PASMC also express
KV
1.1, KV
2, and
KV
3; the predicted sizes of the
PCR products are 150, 141, and 178 bp, respectively.

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Fig. 6.
RT-PCR analysis of KV-channel
-subunit mRNA in PASMC and Br. PCR-amplified products are displayed
in agarose gel stained with ethidium bromide for
KV 1.1 (150 bp;
A),
KV 2 (141 bp;
B), and
KV 3 (178 bp;
C). First-strand cDNAs, synthesized
from 5 µg of total RNAs extracted from PASMC and Br, were amplified
with specific sense and antisense primers for the respective
-subunits (Table 1). Same results were repeated in 3-4
independent experiments.
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In another set of experiments, a sham cDNA control (i.e., the RNA that
was treated exactly like the RT reactions except that no reverse
transcriptase was used) was included in the PCR experiments to ensure
that all the RNA samples prepared from PASMC were not contaminated by
DNA. As shown in Fig. 7, even though RNA
was used for PCR (i.e., RT reaction was blocked by eliminating reverse transcriptase from the reaction mixture) as a no-RT control, there were
no PCR products detected when the specific primers for
K+-channel
- and
-subunits
were used.

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Fig. 7.
RT-PCR analyses of KV1.2,
KV1.4,
rSlo, and
KV 1.1 mRNA in PASMC.
PCR-amplified products are displayed in agarose gel stained with
ethidium bromide for KV1.2 (295 bp; A),
KV1.4 (322 bp;
B),
rSlo (864 bp;
C),
KV 1.1 (150 bp;
D), and -actin (244 bp;
A-D).
+, RT performed in presence of reverse transcriptase (cDNA); ,
RT performed in absence of reverse transcriptase (RNA). Same results
were repeated in 3-4 independent experiments.
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Quantitative comparison of KV-
and KCa-channel gene transcripts in PASMC.
In previous experiments by Wang et al. (66), the quantity of PCR
products for
-actin and KV1.2
correlated linearly with the change in cycle numbers between 23 and 28 cycles when 3.0 µg of total RNA and 3.0 µl of cDNA were used in
RT-PCR. Under conditions of 25 cycles and 3.0 µg of total RNA being
used for amplifying the messages in PCR, the change in the cDNA level
of
-actin and KV
3 between
1.5 and 5.0 µl correlated linearly with the amount of the PCR
products. When 3.0 µl of cDNA and 25 cycles were used in the PCR, the
change in total RNA levels between 1.5 and 5.0 µg also correlated
linearly with the quantity of the PCR products of
-actin,
KV1.2, and
KV
3. These results indicate
that the experimental protocol for RT-PCR, 3 µg of total RNA for RT and 3 µl of cDNA and 25 cycles for PCR, was appropriate to quantify the mRNA levels of the KV channels
(66).
Total RNA was extracted from the primary cultured PASMC. After RT, the
same amount of first-strand cDNA was used in PCR, consisting of the
specific primers for K+ channels
and
-actin. Gene transcription (mRNA levels) of the KV-channel
-subunits
(KV1.1,
KV1.2,
KV1.4,
KV1.5, and
KV1.6), KCa-channel
-subunit
(rSlo), and
KV-channel
-subunits
(KV
1.1, KV
2, and
KV
3) was examined and compared,
whereas the
-actin mRNA level was used as the control
level (Fig. 8). The mRNA levels of
KV1.2,
KV1.5, and
rSlo were significantly greater than
those of KV1.1,
KV1.4, and
KV1.6 (Fig.
8A), whereas the mRNA level of KV
1.1 was significantly greater
than those of KV
2 and
KV
3 (Fig. 8B). These results suggest that the
transcriptional levels of various
K+-channel
- and
-subunits
are different in PASMC. KV1.2,
KV1.5, and
rSlo appear to be the major
K+ channels expressed in PASMC.

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Fig. 8.
Quantitative comparison of
KV-channel - and -subunit
and KCa-channel -subunit mRNA
levels in PASMC. A: PCR-amplified
products displayed in agarose gels for
KV1.1 (594 bp),
KV1.2 (295 bp),
KV1.4 (322 bp),
KV1.5 (283 bp),
KV1.6 (394 bp),
rSlo (864 bp), and -actin (244 bp)
when first-strand cDNAs, synthesized from total RNA extracted from
PASMC, were amplified with specific sense and antisense primers (Table
1). Bottom: data that were normalized
to amount of -actin expressed as means ± SE (experiments were
repeated 4 times independently). B:
PCR analyses for KV 1.1 (150 bp), KV 2 (141 bp),
KV 3 (178 bp), and -actin
when first-strand cDNAs were amplified with specific sense and
antisense primers (Table 1). Bottom:
data that were normalized to amount of -actin expressed as means ± SE (experiments were repeated 3 times independently).
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|
The quantitative RT-PCR experiments, however, were performed with
different primers for the respective channel genes, and different
primers may have different binding efficiencies with the corresponding
channel cDNAs. Therefore, the data may not reflect the true differences
in the transcriptional levels of the various channels in PASMC.
Identification of KV- and
KCa-channel gene transcripts in freshly
isolated PA rings.
Total RNA was extracted from freshly isolated, endothelium-denuded PA
rings. After RT, the same amount of first-strand cDNA was used in PCR,
consisting of the specific primers for
K+ channels (Table 1) and
-actin. Consistent with the results obtained from primary cultured
PASMC, the gene transcripts of KV1.1,
KV1.2,
KV1.4,
KV1.5,
KV1.6, and
rSlo as well as those of KV
1.1,
KV
2 and
KV
3 were also detected in the
isolated PA rings (Fig. 9).

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Fig. 9.
RT-PCR analyses of KV-channel -
and -subunit and KCa-channel
-subunit mRNAs in freshly isolated pulmonary arteries.
A: PCR-amplified products displayed in
agarose gels for KV1.1 (594 bp),
KV1.2 (295 bp),
KV1.4 (322 bp),
KV1.5 (283 bp),
KV1.6 (394 bp), and
rSlo (864 bp) when first-strand cDNAs,
synthesized from total RNA extracted from rat endothelium-denuded
pulmonary arteries, were amplified with specific sense and antisense
primers (Table 1). B: PCR analysis for
KV 1.1 (150 bp),
KV 2 (141 bp), and
KV 3 (178 bp) when first-strand
cDNAs were amplified with the specific sense and antisense primers for
KV-channel -subunits (Table
1).
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Identification of KV-channel proteins by
immunoblotting in PASMC.
The expression of three KV-channel
proteins (KV1.2,
KV1.4, and
KV1.5) from the
Shaker family was verified by
immunoblotting (Fig. 10). In control
experiments, the immunoblot was incubated in rabbit normal
serum; the serum did not cross-react with
KV-channel proteins (data not
shown).

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Fig. 10.
Western blotting analyses of KV1.2
(A)-,
KV1.3
(B)-,
KV1.4
(C)-, and
KV1.5
(D)-channel proteins in PASMC.
Immunoblots of PASMC (PA), Br, and heart (H) tissue proteins (10 µg/lane) were incubated with affinity-purified
anti-KV1.2,
anti-KV1.3,
anti-KV1.4, and
anti-KV1.5 polyclonal antibodies.
Nos. on left, molecular-mass markers.
Control blot, incubated with rabbit normal serum, was blank and is not
shown.
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|
The anti-KV1.2 antibody recognized
a single band at ~75 kDa in PASMC (Fig.
10A). A band of almost the same size
was also detected in brain and heart tissues (Fig.
10A). The predicted molecular mass of the
KV1.2-channel protein is 56.7 kDa
(35). The slower mobility of the
KV1.2-channel protein on
immunoblots was probably caused by extensive glycosylation of this
protein; similar results were also observed by other investigators
(57). The anti-KV1.3 antibody did
not recognize a band in PASMC and heart ventricular muscle (Fig.
10B). However, a double band at
~68 kDa and a single band at ~82 kDa were detected in brain tissue
(Fig. 10B). The molecular sizes of
the two bands are in good agreement with the results (68 and 88 kDa) by
researchers from the Alomone Labs (from which we purchased the
KV1.3 antibody) but are larger
than the predicted molecular mass (58.4 kDa) (61). This is likely due
to the glycosylation of the
KV1.3-channel protein, which is a
typical feature for most of the KV
channels (57). These results are consistent with RT-PCR experiments
that showed that KV1.3 may not be
expressed in PASMC and heart ventricles (61). The
anti-KV1.4 antibody recognized a
single band at ~72 kDa in PASMC and brain and heart samples (Fig.
10C). The size of this band is in
good agreement with the predicted molecular mass of the
KV1.4-channel protein (73.4 kDa; Ref. 60) but is smaller than the
KV1.4-channel proteins identified by Maletic-Savatic et al. (95 kDa; Ref. 31) and Takimoto et al. (96 kDa; Ref. 62). The discrepancies in the sizes of the KV1.4-channel protein detected by
immunoblots can be due to different experimental conditions used for
sample preparation and immunoblotting (e.g., gel percentage, sample
boiling). The anti-KV1.5 antibody recognized a sharp band at ~63 kDa in all three tested samples (PASMC
and brain and heart tissues) (Fig.
10E). This result is consistent with
the predicted molecular mass (66.3 kDa) of the KV1.5-channel protein in rat brain
(61) and canine colonic smooth muscle (43).
Identification of KV2.1 and
KV9.3 channels in PASMC and freshly isolated
PA.
Patel et al. (45) recently cloned
KV2.1 and a novel
KV channel,
KV9.3, from rat PASMC. Expression
of KV2.1 and coexpression of
KV2.1 and
KV9.3
(KV2.1/KV9.3)
into COSm6 cells significantly altered the resting
Em (from +4 to
31 and
51 mV, respectively). KV2.1 and
KV2.1/KV9.3
were both reversibly inhibited by hypoxia and activated by
intracellular ATP, suggesting that the
KV2.1/KV9.3 heteromultimer in PASMC may play an important role in hypoxia-mediated membrane depolarization (3, 42, 45, 47, 48, 59, 71) and pulmonary
vasoconstriction (28, 37).
With the use of the primers specifically designed for
KV2.1 and
KV9.3 (45), 496- and 569-bp PCR
products were identified from both PASMC (Fig.
11A)
and the endothelium-denuded PA rings (Fig.
11B). The expression of
KV2.1 was confirmed in PASMC by
Western blot analysis with the polyclonal antibody for
KV2.1. The
anti-KV2.1 antibody recognized a
single band at ~108 kDa (58) in PASMC (Fig.
11C). A band at the same size was
also detected in rat brain and heart (ventricle) tissues (Fig.
11C). The predicted molecular mass
of the KV2.1 channel from the
deduced primary sequence is 95 kDa (58). The increased size of
KV2.1 in PASMC and brain and heart
tissues is probably due to posttranslational modification (58).
KV9.3 is an electrically silent
KV-channel
-subunit that modulates the electrophysiological properties of the functional KV-channel
KV2.1 (45). Whether
KV9.3 is heteromultimerized with other subfamilies of KV-channel
-subunits (e.g., KV1) is
unknown.

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Fig. 11.
Identification of KV2.1 and
KV9.3 in PASMC and pulmonary
arterial (PA) rings. A and
B: RT-PCR-amplified products displayed
in agarose gels for KV2.1 (496 bp;
left) and
KV9.3 (569 bp;
right). First-strand cDNAs,
synthesized from total RNA extracted from rat PASMC
(A) and endothelium-denuded PA rings
(B), were amplified with specific
sense and antisense primers (Table 1). +, RT performed in presence of
reverse transcriptase (cDNA); , RT performed in absence of
reverse transcriptase (RNA). Same results were repeated in 3 independent experiments. C: Western
blot analysis of KV2.1 in PASMC.
Immunoblots of PASMC (PA; 11 µg/lane), Br (5 µg/lane), and H (18 µg/lane) tissue proteins were incubated with affinity-purified
anti-KV2.1 polyclonal antibody.
Nos. on left, molecular-mass markers.
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 |
DISCUSSION |
Activity of K+ channels, by
governing Em,
plays an important role in the regulation of
[Ca2+]cyt
and pulmonary vascular tone. Inhibition of
KV channels by 4-AP or hypoxia
depolarizes PASMC, thereby increasing
[Ca2+]cyt
and causing pulmonary vasoconstriction (3, 16, 23, 42, 45-48, 59,
67). Activation of KV channels by
nitric oxide hyperpolarizes PASMC, thus inhibiting the evoked increase
in
[Ca2+]cyt
and inducing pulmonary vasodilation (4, 73). In this study, two
kinetically distinct
IK(V),
IK(tr) [the
A-type
IK(V)] and
IK(ss) [the
non- or slowly inactivating delayed rectifier IK(V)], as
well as a large-amplitude slowly activating
IK(Ca) were
described in PASMC using electrophysiological approaches.
Because IK(tr) is
almost completely inactivated at a potential close to the resting
Em (approximately
40 mV) in PASMC,
IK(tr) is mainly
involved in regulating duration of the action potential and limiting
agonist-induced depolarization.
IK(ss), however, is active at the resting
Em (19 ± 3 pA
at
40 mV; n = 28 patches) and
thus plays an important role in controlling the resting
Em (16-19,
29, 47, 59, 67). In vascular smooth muscle cells, including PASMC, the
resting
[Ca2+]cyt
ranges from 50 to 150 nM, and most
KCa channels are closed when
[Ca2+]cyt
is
300 nM at 0 mV (2, 46). Thus, under resting conditions in which
[Ca2+]cyt
is ~100 nM and
Em is
approximately
40 mV, KCa
channels are largely inactive. However, when
[Ca2+]cyt
in PASMC is increased and the cells are depolarized, activation of
KCa channels provides a critical
negative feedback pathway to control vascular tone and
stimulation-induced active tension in the pulmonary vasculature (7,
46).
Molecular identification of KV channels
in PASMC.
There are at least eleven subfamilies of the
KV-channel
-subunits
KV1
(KV1.1-KV1.7,
Shaker),
KV2
(KV2.1-KV2.2,
Shab),
KV3 (KV3.1-KV3.4,
Shaw),
KV4
(KV4.1-KV4.3,
Shal),
KVLQT, EAG
(ether-a-go-go), KV5
(KV5.1),
KV6
(KV6.1),
KV7,
KV8
(KV8.1), and
KV9
(KV9.1-KV9.3) that have been cloned in mammals (11). Four of these,
KV5,
KV6, KV8, and
KV9, are known to be electrically
silent KV-channel modulatory
-subunits, whereas the remainders are functional
KV-channel
-subunits (with
electrical activity) (11, 14, 25, 45, 49, 54, 55, 74). In addition,
there are three subfamilies of the
KV-channel
-subunits
KV
1
(KV
1.1-KV
1.3),
KV
2
(KV
2.1), and
KV
3
(KV
3.1) (15). At the molecular
level, the native K+ channels are
heteromultimers composed of four large pore-forming
-subunits and
four smaller cytoplasmic
-subunits
(
4
4)
(11, 26). The biophysical properties of
K+ channels encoded by certain
-subunits can be dramatically altered by association with
-subunits (24, 26, 50, 56).
By using RT-PCR, five KV-channel
-subunit genes from the Shaker
subfamily (KV1.1,
KV1.2,
KV1.4,
KV1.5, and
KV1.6), a
KV-channel
-subunit gene from
the Shab subfamily
(KV2.1), and a modulatory
-subunit gene from the KV9
subfamily (KV9.3) as well as a
KCa-channel
-subunit gene
(rSlo) were identified in PASMC. In
addition, transcripts of three
-subunits,
KV
1.1,
KV
2, and
KV
3, were also identified in
PASMC. By using immunoblotting, expression of
KV1.2-,
KV1.4-, KV1.5-, and
KV2.1-channel
-subunits was
confirmed. The specifically cross-reacting bands of
KV1.2-,
KV1.4-,
KV1.5-, and
KV2.1-channel proteins have
molecular masses of ~75, 72, 63, and 108 kDa, respectively, which are
comparable in size to the respective
KV-channel bands from the brain
and heart (43, 57, 58, 60, 61). These Western blotting results also
indicate that the KV1.2,
KV1.4, KV1.5, and
KV2.1 channels expressed in PASMC
are immunologically similar to the corresponding
KV channels expressed in the
brain.
The data from the present study suggest that the native
K+ channels in PASMC are encoded
by multiple
- and
-subunit genes. The homo- and/or
heteromultimeric assembly of
-subunits as well as the association of
- and
-subunits both contribute to the remarkable diversity of
K+ channels and the numerous
electrophysiological and pharmacological properties of
K+ channels in vivo (11, 26, 50,
52).
Although extensively studied in the brain (26, 61) and heart (6, 15,
51), the molecular basis for KV
channels in smooth muscle has only recently been elucidated (1, 22, 43, 45). In canine colonic smooth muscle (22, 43),
KV1.2 and KV1.5 have been cloned, and the
sequences revealed significant homology to
KV1.2 and
KV1.5 in the brain and heart. The
colonic KV1.5
(cKV1.5) was also detected in the
canine PA (43). By using a Northern blot and PCR, Adda et al. (1)
recently identified KV1.1,
KV1.2, and
KV1.5 transcripts in human airway
smooth muscle cells. The electrophysiological and functional studies
indicated that the gene products of
KV1.1,
KV1.2, and
KV1.5 play an important role in
regulating airway smooth muscle contractility in humans (1).
The Shab
KV channel
KV2.1 and a novel
KV-channel
-subunit,
KV9.3, have been recently cloned
in rat PASMC (45). The
KV2.1/KV9.3 heteromultimer, expressed in COSm6 cells or
Xenopus oocytes, opens at the voltage
range of the resting
Em in PASMC and
is regulated by oxygen tension and intracellular ATP (45).
Biophysical and pharmacological properties of
KV channels.
In PASMC, the native
IK(V) is composed
of at least an A-type
IK(V) and a non-
or slowly inactivating delayed rectifier
IK,V. Both of the currents are
activated at relatively negative potentials (
40 to
30
mV), and the single-channel conductance is 5-75 pS (17-19,
27, 47, 73). 4-AP is a potent blocker of
KV channels (41, 43); the
IC50 is 0.3-0.7 mM at 10 mV
(18, 41), and the dissociation constant
(Kd) ranges
from 0.2 to 9 mM for expressed KV
channels (11). KV channels,
however, are relatively insensitive to ChTX, iberiotoxin, and a low
dose of tetraethylammonium (TEA;
1 mM) (10, 40).
Of the seven mammalian Shaker
KV channels that have been
biophysically characterized, only
KV1.4 displays rapid (N-type)
inactivation, and the remainder
(KV1.1,
KV1.2,
KV1.3,
KV1.5,
KV1.6, and
KV1.7) are non- or slowly
inactivating delayed rectifiers (11). Inactivation of the A-type
IK(V)
[IK(tr)]
can be explained by the "ball-and-chain" model (11, 26, 50, 53).
In KV channels, the
ball corresponds to the first 20 NH2-terminal amino acids in the
channel
-subunit protein (53) or the associated
KV
1 subunit (50). Oxidation of
a cysteine residue located in the ball sequence of the
KV1.4 channel or the
KV
1 subunit eliminates, whereas
application of the reducing agent glutathione restores, rapid
inactivation of this channel (50, 53). The rapid inactivating property
and the sensitivity to hypoxia and the reducing agents of the native IK(tr) in PASMC
(71, 72) as well as the long
NH2-terminal amino acid sequence
of the KV1.4 gene-encoded protein
product (11) support the contention that
IK(tr) is
generated by KV1.4 channels.
Nevertheless, the possibility that
-subunits are associated with
delayed rectifier KV-channel
-subunits in PASMC cannot be excluded because the association
confers the fast N-type inactivation on the slowly inactivating,
delayed rectifier KV channels
(50).
The cloned delayed rectifier KV
channels share some electrophysiological and pharmacological properties
with the native KV channels
described in smooth muscle cells. The activation threshold of
KV1.1,
KV1.2,
KV1.5, and
KV2.1/KV9.3
is
50 to
30 mV, and the single-channel conductance is
8-30 pS (11, 22, 43, 45). The cloned delayed rectifier channels
are sensitive to 4-AP; the Kd ranges from
0.2 to 9 mM (11). Similarly, the activation threshold of the native
delayed rectifier
IK(V) in PASMC is
approximately
50 to
30 mV (47, 67), and the currents are
also sensitive to 4-AP; the
Kd ranges from
0.2 to 10 mM (40, 52). The single-channel conductance of the delayed
rectifier KV channels, however, is somehow greater (20-104 pS) (17-19, 40, 47, 73) than that of
the cloned channels (11, 22, 43, 45).
Although we have demonstrated that both
- and
-subunits are
expressed in PASMC, their stoichiometry and whether the native A-type
KV channels and/or delayed
rectifier KV channels in PASMC are
homogenous (monomer) or heteromultimeric (multimer) tetramers are
unclear and need further study. In this study, the
KV1-family
-subunits were
emphatically examined in PASMC because the
KV-channel
-subunits only bind
to the Shaker-related subfamily
(KV1
-subunits) (56).
Other KV-channel genes that were
not examined in the study are also very likely involved in encoding the
channels that contribute to the functionally and kinetically distinct
IK(V) in PASMC.
Biophysical and pharmacological properties of
KCa channels.
KCa channels (BK or maxi-K
channels) have been described in smooth muscle cells obtained from a
variety of systemic and pulmonary arteries and veins (2, 4, 10, 46).
These channels are often activated by relatively more positive test
potentials when the membrane is exposed to a physiological
[Ca2+]cyt
(50-150 nM) (2, 10, 18, 29).
IK(Ca) can be
significantly blocked by ChTX
(IC50 = 2 nM),
iberiotoxin (IC50 = 10 nM), or a
low dose of TEA (IC50 = 0.16 mM)
and is extremely sensitive to
[Ca2+]cyt
(2, 10). Other characteristics of
KCa channels include their large
conductance (200-250 pS with a symmetrical
K+ gradient) (2, 10, 18, 41) and
relatively slow activation kinetics (33).
Correspondingly, the cloned KCa
channels (hSlo,
mSlo, or
dSlo) expressed in
Xenopus oocytes also have a large
conductance (200-282 pS) and relatively positive activation
threshold (23-32 mV when
[Ca2+]cyt
is 10 M; Refs. 8, 33).
IK(Ca) in the
hSlo cRNA-injected Xenopus oocytes is also very sensitive
to ChTX, iberiotoxin [40 nM ChTX or 20 nM iberiotoxin almost
abolishes the hSlo
IK(Ca); Ref.
33], and TEA (IC50 = 0.14 mM; Refs. 8, 33). The significant similarities of the
electrophysiological and pharmacological properties between the native
IK(Ca) and the
IK(Ca) derived
from the Slo gene suggest that rat
PASMC express an rSlo gene that gives
rise to the large-conductance
IK(Ca). Because
KCa channels are also composed of
- and
-subunits and the latter contributes to the pharmacological
and biophysical properties of
IK(Ca) (21, 36), further study is needed to elucidate whether
rSlo and
KV
1 (or KV
2 and
KV
3)-subunit encoding products
are associated in PASMC.
Function of KV and
KCa channels in regulating
Em,
[Ca2+]cyt,
and pulmonary vasomotor tone.
Functionally, the non- or slowly inactivating delayed rectifier
KV channels are more likely the
major determinants in controlling the resting
Em and thus the
resting
[Ca2+]cyt
and tonic tension (16-19, 23, 29, 46, 47, 67).
KCa channels, in contrast,
comprise a negative-feedback pathway in regulating the
Em when the
[Ca2+]cyt
is increased and thus govern phasic or active tension (7, 19, 29, 46).
Indeed, inhibition of KV channels
by 4-AP depolarized PASMC, raised
[Ca2+]cyt,
and significantly increased PA pressure and vascular resistance in an
isolated perfused lung (23), whereas inhibition of
KCa channels by a low dose of TEA
and ChTX negligibly affected the Em,
[Ca2+]cyt,
or PA pressure (23). The same results were also obtained from an
isolated, endothelium-denuded human PA (46).
It has been recently demonstrated that
KV channels are regulated by
oxygen tension (3, 30, 45-48, 59, 66, 71), cellular metabolism
(45, 72), redox status (3, 50, 53, 72), and nitric oxide (4, 73).
Hypoxia-induced inhibition of KV channels in PASMC has been demonstrated to be an important trigger of
hypoxic pulmonary vasoconstriction (3, 30, 42, 45-48, 59, 66, 71).
Furthermore, dysfunctional KV
channels have been described in PASMC from patients with primary
pulmonary hypertension (69) and in renal arterial smooth muscle cells
from genetically hypertensive rats (32). Thus a defect in
KV channels may also play an
important role in the pathogenesis of primary pulmonary hypertension
(69) and genetic systemic hypertension (32).
Due to the large conductance, activation of
KCa channels by
Ca2+ sparks (originated by
Ca2+ release from the
ryanodine-sensitive sarcoplasmic reticulum) has been demonstrated to
cause relaxation of arterial smooth muscle (38). Because the voltage-
and Ca2+-dependent gating for
KCa channels are synergistic, a
small shift of Em
in the direction of depolarization dramatically increases Ca2+ sensitivity of the channel;
conversely, a small increase in
[Ca2+]cyt
markedly increases the sensitivity of the channel to the Em (10). This
property of KCa channels explains
why KCa channels play a critical
role in regulating vasomotion when
[Ca2+]cyt
is increased and cells are depolarized.
Summary and conclusion. The
KV-channel
-subunit genes of
the Shaker-related subfamily
(KV1.1,
KV1.2,
KV1.4,
KV1.5, and
KV1.6), the
KV-channel
-subunit gene of the
Shab subfamily
(KV2.1), the KV-channel modulatory
-subunit
gene (KV9.3), and the
KCa-channel
-subunit gene
(rSlo) are expressed in PASMC. In
addition, the KV-channel
-subunits KV
1.1,
KV
2, and
KV
3 are also expressed in
PASMC. The 4-AP-sensitive non- or slowly inactivating delayed rectifier
IK(V), apparently
attributed to all of the KV1.1,
KV1.2, KV1.5,
KV1.6,
KV2.1, and
KV2.1/KV9.3
gene products, is a critical determinant of resting
Em and
[Ca2+]cyt
in PASMC. The 4-AP-sensitive A-type
IK(V), probably
conferred by the KV1.4 channel
and/or the delayed rectifier
KV channels (KV1.1,
KV1.2,
KV1.5, and
KV1.6) associated with the
KV-channel
-subunits, plays an
important role in limiting depolarization and controlling duration of
the action potentials in PASMC. The ChTX-sensitive,
Ca2+-activated
IK(Ca), likely
endowed by the rSlo gene product,
plays a central role in triggering and maintaining repolarization of the Em when the
myocytes are stimulated and the
[Ca2+]cyt
is increased. Association of the cytoplasmic
-subunits with the
pore-forming
-subunits of the
KV and
KCa channels and
heteromultimerization between different
K+-channel
-subunits
significantly contribute to the diversity of
K+ channels in vivo. Sensitivity
of the native KV and
KCa channels to hypoxia, redox
status change, and metabolic inhibition may be conferred by
KV-channel
-subunits
and/or other modulatory
-subunits.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge A. M. Aldinger and J. E. Seiden for
technical assistance; Dr. M. L. Tod for critical review of the manuscript; and Drs. X. Liu, D.-X. Zhou, S. Reinhardt, Q. Zhu, E. Limen, and F. Xia for generous advice on the molecular biological experiments.
 |
FOOTNOTES |
This project was supported by National Heart, Lung, and Blood Institute
Grants HL-54043, HL-02659, and HL-32276 and grants from the American
Heart Association-Maryland Affiliate, the Primary Pulmonary
Hypertension (PPH) Cure Foundation, and the PPH Research Foundation.
X.-J. Yuan is an Established Investigator of the American Heart
Association, a Parker B. Francis Fellow in Pulmonary Research, and a
recipient of the Giles F. Filley Memorial Award and the Research Career
Enhancement Award from the American Physiological Society.
Address for reprint requests: X.-J. Yuan, Division of Pulmonary and
Critical Care Medicine, Univ. of Maryland School of Medicine, 10 S. Pine St., Suite 800, Baltimore, MD 21201.
Received 14 May 1997; accepted in final form 7 January 1998.
 |
REFERENCES |
1.
Adda, S.,
B. K. Fleischmann,
B. D. Freedman,
M.-F. Yu,
D. W. P. Hay,
and
M. I. Kotlikoff.
Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle.
J. Biol. Chem.
271:
13239-13243,
1996[Abstract/Free Full Text].
2.
Albarwani, S.,
B. E. Robertson,
P. C. G. Nye,
and
R. Z. Kozlowski.
Biophysical properties of Ca2+- and Mg2+-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat.
Pflügers Arch.
428:
446-454,
1994[Medline].
3.
Archer, S. L.,
J. Huang,
T. Henry,
D. Peterson,
and
E. K. Weir.
A redox-based O2 sensor in rat pulmonary vasculature.
Circ. Res.
73:
1100-1112,
1993[Abstract].
4.
Archer, S. L.,
J. M. Huang,
V. Hampl,
D. P. Nelson,
P. J. Shultz,
and
E. K. Weir.
Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
91:
7583-7587,
1994[Abstract].
5.
Atkinson, N. S.,
G. A. Robertson,
and
B. Ganetzky.
A component of calcium-activated potassium channels encoded by Drosophila slo locus.
Science
253:
551-555,
1991[Medline].
6.
Barry, D. M.,
J. S. Trimmer,
J. P. Merlie,
and
J. M. Nerbonne.
Differential expression of voltage-gated K+ channel subunits in adult rat heart: relation to functional K+ channels?
Circ. Res.
77:
361-369,
1995[Abstract/Free Full Text].
7.
Brayden, J. E.,
and
M. T. Nelson.
Regulation of arterial tone by activation of calcium-dependent potassium channels.
Science
256:
532-535,
1992[Medline].
8.
Butler, A.,
S. Tsunoda,
D. P. McCobb,
A. Wei,
and
L. Salkoff.
mSlo, a complex mouse gene encoding "Maxi" calcium-activated potassium channels.
Science
261:
221-224,
1993[Medline].
9.
Butler, A.,
A. Wei,
K. Baker,
and
L. Salkoff.
A family of K+ channel genes in Drosophila.
Science
243:
943-947,
1989[Medline].
10.
Carl, A.,
H. K. Lee,
and
K. M. Sanders.
Regulation of ion channels in smooth muscles by calcium.
Am. J. Physiol.
271 (Cell Physiol. 40):
C9-C34,
1996[Abstract/Free Full Text].
11.
Chandy, K. G.,
and
G. A. Gutman.
Voltage-gated K+ channels.
In: Ligand- and Voltage-Gated Ion Channels, edited by R. A. North. Boca Raton, FL: CRC, 1995, p. 1-71.
12.
Chomczynski, P.,
and
S. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
13.
Clapp, L. H.,
and
A. M. Gurney.
ATP-sensitive K+ channels regulate resting potential of pulmonary arterial smooth muscle cells.
Am. J. Physiol.
262 (Heart Circ. Physiol. 31):
H916-H920,
1992[Abstract/Free Full Text].
14.
Drewe, J. A.,
S. Verma,
G. Frech,
and
R. H. Joho.
Distinct spatial and temporal expression patterns of K+ channel mRNAs from different subfamilies.
J. Neurosci.
12:
538-548,
1992[Abstract].
15.
England, S. K.,
V. N. Uebele,
J. Kodali,
P. B. Bennett,
and
M. M. Tamkun.
A novel K+ channel
-subunit (hKv
1.3) is produced via alternative mRNA splicing.
J. Biol. Chem.
270:
28531-28534,
1995[Abstract/Free Full Text].
16.
Evans, A. M.,
O. N. Osipenko,
and
A. J. Gurney.
Properties of a novel K+ current that is active at resting potential in rabbit pulmonary artery smooth muscle cells.
J. Physiol. (Lond.)
496:
407-420,
1996[Abstract].
17.
Fleischmann, B. K.,
R. J. Washabau,
and
M. I. Kotlikoff.
Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells.
J. Physiol. (Lond.)
469:
625-638,
1993[Abstract].
18.
Gelband, C. H.,
and
J. R. Hume.
Ionic currents in single smooth muscle cells of the canine renal artery.
Circ. Res.
71:
745-758,
1992[Abstract].
19.
Gelband, C. H.,
and
J. R. Hume.
[Ca2+]i inhibition of K+ channels in canine renal artery: novel mechanism for agonist-induced membrane depolarization.
Circ. Res.
77:
121-130,
1995[Abstract/Free Full Text].
20.
Goldman, W. F.,
S. Bova,
and
M. P. Blaustein.
Measurement of intracellular Ca in cultured arterial smooth muscle cells using fura-2 and digital imaging microscopy.
Cell Calcium
11:
221-231,
1990[Medline].
21.
Hanner, M.,
W. A. Schmalhofer,
P. Munujos,
H.-G. Knaus,
G. J. Kaczorowski,
and
M. L. Garcia.
The
subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybodotoxin.
Proc. Natl. Acad. Sci. USA
94:
2853-2858,
1997[Abstract/Free Full Text].
22.
Hart, P. J.,
K. E. Overturf,
S. N. Russell,
A. Carl,
J. R. Hume,
K. M. Sanders,
and
B. Horowitz.
Cloning and expression of a KV1.2 class delayed rectifier K+ channel from canine colonic smooth muscle.
Proc. Natl. Acad. Sci. USA
90:
9659-9663,
1992[Abstract].
23.
Hasunuma, K.,
D. M. Rodman,
and
I. F. McMurtry.
Effects of K+ channel blockers on vascular tone in the perfused rat lung.
Am. Rev. Respir. Dis.
144:
884-887,
1991[Medline].
24.
Heinemann, S. H.,
J. Rettig,
H.-R. Graack,
and
O. Pongs.
Functional characterization of KV channel
-subunits from rat brain.
J. Physiol. (Lond.)
493:
625-633,
1996[Abstract].
25.
Hugnot, J.-P.,
M. Salinas,
F. Lesage,
E. Guillemare,
J. De Weille,
C. Heurteaux,
M.-G. Mattei,
and
M. Lazdunski.
Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.
EMBO J.
15:
3322-3331,
1996[Abstract].
26.
Isom, L. L.,
K. S. De Jongh,
and
W. A. Catteral.
Auxiliary subunits of voltage-gated ion channels.
Neuron
12:
1183-1194,
1994[Medline].
27.
Koh, S. D.,
J. D. Campbell,
A. Carl,
and
K. M. Sanders.
Nitric oxide activates multiple potassium channels in canine colonic smooth muscle.
J. Physiol. (Lond.)
489:
735-743,
1995[Abstract].
28.
Kozlowski, R. Z.
Ion channels, oxygen sensation and signal transduction in pulmonary arterial smooth muscle.
Cardiovasc. Res.
30:
318-325,
1995[Medline].
29.
Leblanc, N.,
X. Wan,
and
P. M. Leung.
Physiological role of Ca2+-activated and voltage-dependent K+ currents in rabbit coronary myocytes.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1523-C1537,
1994[Abstract/Free Full Text].
30.
Lopez-Barneo, J.,
J. R. Lopez-Lopez,
J. Urena,
and
C. Gonzalez.
Chemotransduction in the carotid body: K+ current modulated by PO2 in type chemoreceptor cells.
Science
241:
580-582,
1988[Medline].
31.
Maletic-Savatic, M.,
N. J. Lenn,
and
J. S. Trimmer.
Differential spatiotemporal expression of K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro.
J. Neurosci.
15:
3840-3851,
1995[Abstract].
32.
Martens, J. R.,
and
C. H. Gelband.
Alterations in rat interlobar artery membrane potential and K+ channels in genetic and nongenetic hypertension.
Circ. Res.
79:
295-301,
1996[Abstract/Free Full Text].
33.
McCobb, D. P.,
N. L. Fowler,
T. Featherstone,
C. J. Lingle,
M. Saito,
J. E. Krause,
and
L. Salkoff.
A human calcium-activated potassium channel gene expressed in vascular smooth muscle.
Am. J. Physiol.
269 (Heart Circ. Physiol. 38):
H767-H777,
1995[Abstract/Free Full Text].
34.
McCormack, T.,
and
K. McCormack.
Shaker K+ channel
subunits belong to an NAD(P)H-dependent oxidoreductase superfamily.
Cell
79:
1133-1135,
1994[Medline].
35.
McKinnon, D.
Isolation of a cDNA clone coding for a putative second potassium channel indicates the existence of a gene family.
J. Biol. Chem.
264:
8230-8236,
1989[Abstract/Free Full Text].
36.
McManus, O. B.,
L. M. H. Helms,
L. Pallanck,
B. Ganetzky,
R. Swanson,
and
R. J. Leonard.
Functional role of the
subunit of high conductance calcium-activated potassium channels.
Neuron
14:
645-650,
1995[Medline].
37.
McMurtry, I. F.,
H. S. Stanbrook,
and
S. Rounds.
The mechanism of hypoxic pulmonary vasoconstriction: a working hypothesis.
In: Oxygen Transport to Human Tissues, edited by J. A. Loeppky,
and M. L. Riedesel. New York: Elsevier/North Holland, 1982, p. 77-91.
38.
Nelson, M. T.,
H. Cheng,
M. Rubart,
L. F. Santana,
A. D. Bonev,
H. J. Knot,
and
W. J. Lederer.
Relaxation of arterial smooth muscle by calcium sparks.
Science
270:
633-637,
1995[Abstract].
39.
Nelson, M. T.,
J. B. Patlak,
J. F. Worley,
and
N. B. Standen.
Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone.
Am. J. Physiol.
259 (Cell Physiol. 28):
C3-C18,
1990[Abstract/Free Full Text].
40.
Nelson, M. T.,
and
J. M. Quayle.
Physiological roles and properties of potassium channels in arterial smooth muscle.
Am. J. Physiol.
268 (Cell Physiol. 37):
C799-C822,
1995[Abstract/Free Full Text].
41.
Okabe, K.,
K. Kitamura,
and
H. Kuriyama.
Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery.
Pflügers Arch.
409:
561-568,
1987[Medline].
42.
Osipenko, O. N.,
A. M. Evans,
and
A. M. Gurney.
Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O2-sensing potassium current.
Br. J. Pharmacol.
120:
1461-1470,
1997[Abstract].
43.
Overturf, K.,
S. N. Russell,
A. Carl,
F. Vogalis,
P. J. Hart,
J. R. Hume,
K. M. Sanders,
and
B. Horowitz.
Cloning of and characterization of a KV1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1231-C1238,
1994[Abstract/Free Full Text].
44.
Papazian, D. M.,
T. L. Schwarz,
B. L. Tempel,
Y. N. Jan,
and
L. Y. Jan.
Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.
Science
237:
749-753,
1987[Medline].
45.
Patel, A. J.,
M. Lazdunski,
and
E. Honore.
Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes.
EMBO J.
16:
6615-6625,
1997[Abstract/Free Full Text].
46.
Peng, W.,
S. V. Karwande,
J. R. Hoidal,
and
I. S. Farrukh.
Potassium currents in cultured human pulmonary arterial smooth muscle cells.
J. Appl. Physiol.
80:
1187-1196,
1996[Abstract/Free Full Text].
47.
Post, J. M.,
C. H. Gelband,
and
J. R. Hume.
[Ca2+]i inhibition of K+ channels in canine pulmonary artery, novel mechanisms for hypoxia-induced membrane depolarization.
Circ. Res.
77:
131-139,
1995[Abstract/Free Full Text].
48.
Post, J. M.,
J. R. Hume,
S. L. Archer,
and
E. K. Weir.
Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction.
Am. J. Physiol.
262 (Cell Physiol. 31):
C882-C890,
1992[Abstract/Free Full Text].
49.
Post, M. A.,
G. E. Kirsch,
and
A. B. Brown.
Kv2.1 and electrically silent Kv6.1 potassium channel subunits combine and express a novel current.
FEBS Lett.
399:
177-182,
1996[Medline].
50.
Rettig, J.,
S. H. Heinemann,
F. Wunder,
C. Lorra,
D. N. Parcej,
J. O. Dolly,
and
O. Pongs.
Inactivation properties of voltage-gated K+ channels altered by presence of
-subunit.
Nature
369:
289-294,
1994[Medline].
51.
Roberds, S. L.,
and
M. M. Tamkun.
Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart.
Proc. Natl. Acad. Sci. USA
88:
1798-1802,
1991[Abstract].
52.
Rudy, B.
Diversity and ubiquity of K channels.
Neuroscience
25:
729-749,
1988[Medline].
53.
Ruppersberg, J.,
M. Stocker,
O. Pongs,
S. Heinemann,
R. Frank,
and
M. Koenene.
Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation.
Nature
352:
711-714,
1991[Medline].
54.
Salinas, M.,
J. De Weille,
E. Guillemare,
M. Lazdunski,
and
J.-P. Hugnot.
Modes of regulation of Shab K+ channel activity by the Kv8.1 subunit.
J. Biol. Chem.
272:
8774-8780,
1997[Abstract/Free Full Text].
55.
Salinas, M.,
F. Duprat,
C. Heurteaux,
J.-P. Hugnot,
and
M. Lazdunski.
New modulatory
subunits for mammalian Shab K+ channels.
J. Biol. Chem.
272:
24371-24379,
1997[Abstract/Free Full Text].
56.
Sewing, S.,
J. Roeper,
and
O. Pongs.
Kv
1 subunit binding specific for Shaker-related potassium channel subunits.
Neuron
16:
455-463,
1996[Medline].
57.
Sheng, M.,
M.-L. Tsaur,
Y. N. Jan,
and
L. Y. Jan.
Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain.
J. Neurosci.
14:
2408-2417,
1994[Abstract].
58.
Shi, G.,
A. K. Kleinklaus,
N. V. Marrion,
and
J. S. Trimmer.
Properties of Kv2.1 K+ channels expressed in transfected mammalian cells.
J. Biol. Chem.
269:
23204-23211,
1994[Abstract/Free Full Text].
59.
Smirnov, S. V.,
T. P. Robertson,
J. P. T. Ward,
and
P. I. Aaronson.
Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells.
Am. J. Physiol.
266 (Heart Circ. Physiol. 35):
H365-H370,
1994[Abstract/Free Full Text].
60.
Stuhmer, W.,
J. P. Ruppersberg,
K. H. Schroter,
B. Sakmann,
M. Stocker,
K. P. Giese,
A. Perschke,
A. Baumann,
and
O. Pongs.
Molecular basis of functional diversity of voltage gated potassium channels in mammalian brain.
EMBO J.
8:
3235-3244,
1989[Abstract].
61.
Swanson, R.,
J. Marshall,
J. S. Smith,
J. B. Williams,
M. B. Boyle,
K. Folander,
C. J. Luneau,
J. Antanavage,
C. Oliva,
S. A. Buhrow,
C. Bennett,
R. B. Stein,
and
L. K. Kaczmarek.
Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain.
Neuron
4:
929-939,
1990[Medline].
62.
Takimoto, K.,
R. Gealy,
A. F. Fomina,
J. S. Trimmer,
and
E. S. Levitan.
Inhibition of voltage-gated K+ channel gene expression by the neuropeptide thyrotropin-releasing hormone.
J. Neurosci.
15:
449-457,
1995[Abstract].
63.
Towbin, H.,
and
J. Gordon.
Immunoblotting and dot immunobinding
current status and outlook.
J. Immunol. Methods
72:
313-340,
1984[Medline].
64.
Trimmer, J. S.
Immunological identification and characterization of a delayed rectifier K+ channel in rat brain.
Proc. Natl. Acad. Sci. USA
88:
10764-10768,
1991[Abstract].
65.
Wahl, M.,
R. G. Sleight,
and
E. Gruenstein.
Association of cytoplasmic free Ca2+ gradients with subcellular organelles.
J. Cell. Physiol.
150:
593-609,
1992[Medline].
66.
Wang, J.,
M. Juhaszova,
L. J. Rubin,
and
X.-J. Yuan.
Hypoxia inhibits gene expression of voltage-gated K+ channel
-subunits in pulmonary artery smooth muscle cells.
J. Clin. Invest.
100:
2347-2353,
1997[Abstract/Free Full Text].
67.
Yuan, X.-J.
Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes.
Circ. Res.
77:
370-378,
1995[Abstract/Free Full Text].
68.
Yuan, X.-J.
Role of calcium-activated chloride current in regulating pulmonary vasomotor tone.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L959-L968,
1997[Abstract/Free Full Text].
69.
Yuan, X.-J., A. M. Aldinger, J. B. Orens,
J. V. Conte, and L. J. Rubin. Dysfunctional
voltage-gated potassium channels in the pulmonary artery smooth muscle
cells of patients with primary pulmonary hypertension (Abstract).
Circulation 94, Suppl.: I-48, 1996.
70.
Yuan, X.-J.,
W. F. Goldman,
M. L. Tod,
L. J. Rubin,
and
M. P. Blaustein.
Ionic currents in rat pulmonary and mesenteric arterial myocytes in primary culture and subculture.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L107-L115,
1993[Abstract/Free Full Text].
71.
Yuan, X.-J.,
W. F. Goldman,
M. L. Tod,
L. J. Rubin,
and
M. P. Blaustein.
Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L116-L123,
1993[Abstract/Free Full Text].
72.
Yuan, X.-J.,
M. L. Tod,
L. J. Rubin,
and
M. P. Blaustein.
Deoxyglucose and reduced glutathione mimic the effects of hypoxia on K+ and Ca2+ conductances in pulmonary artery cells.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L52-L63,
1994[Abstract/Free Full Text].
73.
Yuan, X.-J.,
M. L. Tod,
L. J. Rubin,
and
M. P. Blaustein.
NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.
Proc. Natl. Acad. Sci. USA
93:
10489-10494,
1996[Abstract/Free Full Text].
74.
Zhao, B.,
F. Rassendren,
B.-K. Kaang,
Y. Furukawa,
T. Kubo,
and
E. R. Kandel.
A new class of noninactivating K+ channels from Aplysia capable of contributing to the resting potential and firing patterns of neurons.
Neuron
13:
1205-1213,
1994[Medline].
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