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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2001 as doi:10.1096/fj.00-0649fje. |
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2
* Vascular Biology Group, Department of Medicine, Division of Cardiology, University of Alberta, Alberta, Canada;
Department of Physiology, University of Alberta, Edmonton, Canada;
Cardiovascular Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
Department of Physiology, Charles University, Second Medical School, Prague; and
Department of Pathology, University of Alberta, Edmonton, Canada,
2Correspondence: Department of Medicine, University of Alberta, WMC 2C2.36, 8440 112th St., Edmonton, Alberta, Canada T6G 2B7. E-mail: sarcher{at}cha.ab.ca
SPECIFIC AIMS
Hypoxic pulmonary vasoconstriction (HPV), a vasomotor mechanism that matches regional perfusion to ventilation, is initiated by the inhibition of 4-aminopyridine (4-AP) -sensitive, voltage-gated, potassium channels (Kv), resulting in membrane depolarization, opening of voltage-gated calcium channels, and vasoconstriction. We used gene targeting to create mice lacking a candidate O2-sensitive channel, Kv1.5, to evaluate the hypothesis that loss of Kv1.5 would impair HPV.
PRINCIPAL FINDINGS
1. Gene targeting selectively deleted Kv1.5 channels
The 4-AP-sensitive mouse K + channel gene
mKv1.5 was cloned and a targeting construct was engineered
by Barry London. The construct consisted of a 5' arm of the promoter
and 5'UTR of mKv1.5, the rat Kv1.1 K +
channel (rKv1.1) tagged with the 9 amino acid hemagglutinin tag (HA)
and cloned into the SMAI site of mKv1.5 located at position
-6, a neomycin resistance cassette (NeoR),
mKv1.5 starting at an XbaI site in the 3'UTR, and
the thymidine kinase gene for negative selection. Homologous
recombination with this construct should yield rKv1.1 driven by the
mKv1.5 promoter, although the effect of the NeoR
cassette is unknown and any 3' regulatory elements may be lost. These
issues may explain why the rKv1.1 was not expressed in the lung (even
though the gene was present and rKv1.1 was expressed in the heart; data
not shown). Electroporation of embryonic stem cells, blastocyst
injections, and matings to obtain mice heterozygous and homozygous for
the targeted allele were performed to create SWAP mice. Male and female
SWAP heterozygotes were backcrossed two generations into C57BL/6 and
mated to yield the 3- to 8-month-old SWAP homozygotes, heterozygotes,
and wild-type littermate controls used in these experiments. The Animal
Welfare Committees of the Universities of Alberta and Pittsburgh (where
the mice were made by B.L.) approved the experiments. SWAP mice are
indistinguishable from wild-type mice, having a normal appearance, life
expectancy, and gender distribution. The ratio of the weight of the
left ventricle plus septum to the right ventricle (LV+S/RV), a measure
of right ventricular hypertrophy, was not different between the
wild-type and SWAP groups (3.4±0.1 and 3.3±0.1, respectively, p NS).
PCR and RT-PCR confirmed the absence of Kv1.5 DNA and mRNA in SWAP
mice. SWAP mice had more Kv1.1 DNA than did wild-type mice on PCR, but
the additional rKv1.1 gene was not transcribed and the total Kv1.1 mRNA
levels in the lung did not differ between groups. Kv1.5 protein was
absent in the SWAP PAs (see Fig. 2B
), but Kv1.1 protein
levels were not increased. Immunoblots using antibodies against the HA
epitope unique to the rKv1.1 transgene did not detect any rKv1.1
protein in SWAP lungs. Thus, although the SWAP mouse was designed as a
gene replacement model, it functions as a targeted deletion or
knockout. RT-PCR did not detect compensatory change in mRNA levels for
other K + channels.
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2. Mice lacking Kv1.5 have significant impairment of HPV
HPV was significantly reduced in SWAP lungs (Fig. 1
). There was a trend toward reduced constriction to angiotensin II in
the SWAP mice, but this reduction did not reach statistical
significance (SWAP 0.9±0.2 vs. wild-type 2.2±0.9, P=0.15).
Normoxic perfusion pressure at baseline was the same (14±1 mmHg) in
both SWAP and wild-type mice. In PA rings, the magnitude of HPV was
greatest in wild-type mice, intermediate in heterozygotes, and
abolished in SWAP rings (Fig. 1)
.
|
Pulmonary artery smooth muscle cells (PASMCs) from SWAP mice lack
IKv1.5
SWAP PASMCs had lower normoxic current density than did
wild-type PASMCs (Fig. 2E
), consistent with their loss of Kv1.5 
-subunits (Fig. 2B
). Subtraction analysis showed that
IKv1.5 activated at physiologically relevant
membrane potentials (
-40 mV) (Fig. 2G
). SWAP PASMCs had
less hypoxia-sensitive IK than wild-type cells
(Fig. 2C
, D
) and depolarized less to hypoxia (hypoxic
EM+12 vs.+22%, respectively,
P<0.05). SWAP PASMCs were also slightly depolarized in
normoxia relative to wild-type cells (Fig. 2F
). As expected
after the uncompensated loss of a 4-AP-sensitive channel, SWAP PASMCs
displayed less 4-AP (1 mM) -sensitive current and depolarized less in
response to 4-AP than wild-type cells (21 vs. 33%, respectively,
P<0.05). The sensitivity to iberiotoxin was similar among
groups.
CONCLUSIONS
The primary finding of this study is that deletion of Kv1.5
-subunits channels from the mouse pulmonary vasculature (Fig. 2)
significantly reduces the 4-AP- and O2-sensitive
portions of IK in PASMCs (Fig. 2)
and impairs HPV
(Figs. 1
and 3)
. Several candidate channels have been proposed to
initiate HPV, based on electrophysiological and pharmacological
similarities between their characteristics in expression systems and
properties of the O2-sensitive K
+ current in PASMCs (e.g., Kv1.2, Kv1.5, Kv2.1,
and Kv3.1b). The O2-sensitive current in PASMCs
is rapidly activating, slowly inactivating, voltage-gated, and
inhibited by 4-AP (1–5 mM) but not charybdotoxin or glyburide. This
profile tends to exclude rapidly inactivating channels (e.g., Kv1.4 and
Kv4.3), 4-AP-resistant channels (e.g., the BKCa
channel), and charybdotoxin-sensitive channels (e.g., homotetrameric
Kv1.2 and Kv1.6 channels). Although the SWAP mouse was designed as a
gene replacement model, the lack of expression of rKv1.1 in the lung
indicates the mouse functions as a knockout. Supporting the causal role
of loss of Kv1.5 in the impairment of HPV, there was a direct
relationship between the Kv1.5 gene dose and the magnitude of HPV, with
SWAP heterozygotes having a degree of HPV intermediate between SWAP
homozygotes and wild-type mice (Fig. 1D
). As expected,
elimination of Kv1.5 is accompanied by a reduction in normoxic PASMC
current density (Fig. 2E
), slight membrane depolarization
(Fig. 2F
), and reduced sensitivity of
IK to hypoxia (Fig. 2C
, D
) and 4-AP.
Subtraction analysis showed that the difference current between
wild-type and SWAP PASMCs, presumably the current conducted by Kv1.5
(IKv1.5), activates at physiologically relevant
membrane potentials (Fig. 2G
). Consistent with the
postulated role of Kv1.5 as a major 4-AP- and hypoxia-sensitive K
+ channel in PASMCs, IKv1.5
is largely inhibited by hypoxia (Fig. 2G
) or 1 mM 4-AP.
Expression of other K + channels found in mouse
lung (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, Kv2.1 Kv9.3, Kv3.1b, Kir3.1,
and Kir6.1 -subunits) did not change in compensation for the loss of
Kv1.5. Thus, it appears that the changes observed in cellular
electrophysiology largely reflect the loss of Kv1.5, as shown
schematically in Fig. 3
. There is still residual normoxic Kv current in SWAP PASMCs and
persistence (at a reduced level) of hypoxia-sensitive K
+ current and membrane depolarization (Fig. 2D
). This is associated with some residual HPV in isolated
SWAP lungs (Fig. 1B
). The residual HPV may reflect the
actions of other mechanisms, such as endothelin synthesis, or may be
the result of hypoxic inhibition of other K +
channels.
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Other evidence suggests a role for Kv1.5 in HPV. First, acute inhibition of Kv1.5 in rat PASMCs, accomplished by intracellular administration of an anti-Kv1.5 antibody, reduces IK and inhibits hypoxia sensitivity. Second, expression of Kv1.5 protein and the size of IK are selectively decreased in PASMCs during chronic hypoxia, a condition that impairs acute HPV. What of the other candidate channels? A recent report by Hulme et al., using mammalian expression systems, indicates the Kv1.2/Kv1.5 heterotetramers are inhibited by hypoxia, as are Kv2.1/Kv9.3 heterotetramers. It was recently shown that Kv3.1b channels are present in PASMCs. Although the hypoxic sensitivity of Kv3.1b has been shown in expression systems, its role in HPV is unknown. Kv3.1b channels display greater sensitivity to TEA than does the endogenous IK of PASMCs. Whereas Osipenko et al. found that 1 mM TEA inhibited the electrophysiological effects of hypoxia on PASMCs, most groups find that TEA (<5 mM) enhances HPV and causes little constriction or depolarization.
Much of the research on the molecular identity of
O2-sensitive K + channels
is performed in expression systems (Xenopus or mammalian).
An advantage of the gene targeting strategy used in the current study
is the ability to study both the physiological and electrophysiologic
effects of deletion of a single channel in an intact animal that
normally manifests HPV and whose PASMCs are otherwise unaltered. In
expression systems, the absence of the normal variety of Kv channels
-subunits found in vivo limits the formation of heterotetramers and
may thereby dramatically change the activation threshold and kinetics
of the transfected channel. In addition, expression systems may lack or
variably express ß-subunits and kinases that alter channel kinetics
and/or expression in important ways. Finally,
O2-responsive tissues may have unique redox
oxygen sensors that provide the proximal signal linking
PO2 to K + channel gating.
Although certain K + channels display intrinsic
redox sensitivity due to their sulfhydryl groups, there is evidence
that O2 may be sensed in PASMCs in part through
changes in cytosolic redox status as controlled by NAD(P)H oxidase or
the mitochondrial electron transport chain. It is unlikely that these
putative redox sensors, if they even exist in expression systems, are
the same as those in PASMCs. Significant work remains to be done using
this model. For example, immunohistochemistry shows that PA endothelial
cells also express Kv1.5. We did not systematically explore the role of
Kv1.5 in the endothelium in this initial study. Such a study will be
important, particularly in light of the potential contribution of the
endothelium to HPV.
In conclusion, our finding of a central role for a channel(s)
containing Kv1.5 -subunits in HPV is consistent with the widely
conserved role K + channels play in other
O2-sensitive tissues, including the carotid body,
the neuroepithelial bodies, the ductus arteriosus, and adrenomedullary
(PC-12) cells.
FOOTNOTES
1 To read the full text of this article, go
to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0649fje ; to
cite this article, use FASEB J. (June 18, 2001)
10.1096/fj.00-0649fje 
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