INVITED REVIEW
Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels

Elizabeth A. Coppock, Jeffrey R. Martens, and Michael M. Tamkun

Departments of Physiology and Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523


    ABSTRACT
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ABSTRACT
INTRODUCTION
BACKGROUND
IDENTIFICATION OF KV CHANNEL...
POTENTIAL MECHANISMS OF KV...
KV CHANNEL REGULATION BY...
SUMMARY
REFERENCES

The hypoxia-induced membrane depolarization and subsequent constriction of small resistance pulmonary arteries occurs, in part, via inhibition of vascular smooth muscle cell voltage-gated K+ (KV) channels open at the resting membrane potential. Pulmonary arterial smooth muscle cell KV channel expression, antibody-based dissection of the pulmonary arterial smooth muscle cell K+ current, and the O2 sensitivity of cloned KV channels expressed in heterologous expression systems have all been examined to identify the molecular components of the pulmonary arterial O2-sensitive KV current. Likely components include Kv2.1/Kv9.3 and Kv1.2/Kv1.5 heteromeric channels and the Kv3.1b alpha -subunit. Although the mechanism of KV channel inhibition by hypoxia is unknown, it appears that KV alpha -subunits do not sense O2 directly. Rather, they are most likely inhibited through interaction with an unidentified O2 sensor and/or beta -subunit. This review summarizes the role of KV channels in hypoxic pulmonary vasoconstriction, the recent progress toward the identification of KV channel subunits involved in this response, and the possible mechanisms of KV channel regulation by hypoxia.

voltage-gated potassium channel; pulmonary artery; oxygen sensor; smooth muscle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
BACKGROUND
IDENTIFICATION OF KV CHANNEL...
POTENTIAL MECHANISMS OF KV...
KV CHANNEL REGULATION BY...
SUMMARY
REFERENCES

MEMBRANE POTASSIUM CHANNELS play an essential role in cellular homeostasis and nerve and muscle excitability. In vascular smooth muscle, voltage-gated K+ (KV) channels are integral in the regulation of membrane potential and, therefore, vascular tone (42, 60). Closure of vascular smooth muscle cell (VSMC) K+ channels, open at the resting membrane potential, causes membrane depolarization. This change in membrane potential activates voltage-gated Ca2+ channels, leading to an increase in intracellular Ca2+ concentration ([Ca2+]i), and vasoconstriction (60). Activation of VSMC K+ channels leads to hyperpolarization, inhibition of voltage-gated Ca2+ channels, and vasodilation (60). VSMCs have a high input resistance; therefore, even a small change in K+ channel activity can have a significant effect on membrane potential and, consequently, vascular tone (60, 92). Indeed, many factors that modulate vessel tone do so through activating or inhibiting VSMC K+ channels (42, 60, 92). This review focuses on the pulmonary arterial smooth muscle cell (PASMC) K+ channels thought to be inhibited by hypoxia, leading to pulmonary vasoconstriction.

In the systemic circulation, small arteries dilate in response to low levels of O2 (hypoxia) to increase O2 delivery to the tissues. In contrast, small resistance arteries in the pulmonary circulation constrict in response to alveolar hypoxia, a process known as hypoxic pulmonary vasoconstriction (HPV) (95). In the fetal circulation, HPV contributes to the high pulmonary arterial resistance, diverting blood through the ductus arteriosus (55). In the adult, HPV reduces the flow of blood through atelectatic or underventilated areas of the lung where ventilation is not adequate for oxygenation (55). In this manner, acute HPV is a mechanism that helps to match perfusion to ventilation, diverting blood flow away from poorly ventilated portions of the lung to maximize arterial saturation (107). When only a small region of the lung is hypoxic, HPV can occur without a significant effect on pulmonary arterial pressure (59). However, if hypoxia is generalized, as seen with many lung diseases and high-altitude exposure, the subsequent pulmonary vasoconstriction causes an increase in pulmonary arterial pressure, which can potentially lead to pulmonary hypertension, heart failure, and death (34).


    BACKGROUND
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ABSTRACT
INTRODUCTION
BACKGROUND
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POTENTIAL MECHANISMS OF KV...
KV CHANNEL REGULATION BY...
SUMMARY
REFERENCES

HPV

Since first described by von Euler and Liljestrand (103) in 1946, HPV has been extensively studied. However, the exact mechanisms underlying HPV remain unknown. Although endothelium-derived vasoactive factors are clearly important and appear to be necessary for the full expression of HPV in vivo [for a review, see Ward and Aaronson (105)], it seems likely that HPV is initiated, at least partially, through a mechanism intrinsic to PASMCs. Responsiveness to acute hypoxia has been demonstrated not only in isolated lungs (38, 54, 73, 102) but also in pulmonary arterial rings denuded of endothelium (3, 37, 49, 113) and in single PASMCs (3, 16, 27, 50, 63, 69, 72, 73, 112).

It is currently hypothesized that alveolar hypoxia acts to directly depolarize the PASMC membrane, thus causing an influx of Ca2+ through L-type voltage-gated Ca2+ channels and subsequent contraction. Indeed, in isolated PASMCs, acute hypoxia has been shown to significantly depolarize the membrane potential by ~15-20 mV (3, 31, 49, 112), leading to contraction of individual PASMCs (50). In 1976, McMurtry et al. (54) were the first to demonstrate that HPV is inhibited by the voltage-gated Ca2+ channel antagonists verapamil and SKF-525 in the isolated perfused lung. McMurtry (53) and others (97) also demonstrated that HPV is potentiated by BAY K 8644, a Ca2+ channel agonist. In 1985, Harder et al. (37) were the first to demonstrate that the hypoxia-induced constriction of small pulmonary arteries (<300 µm) was associated with membrane depolarization that could be inhibited by verapamil. More recently, Salvaterra and Goldman (83), using isolated adult PASMCs, and Cornfield et al. (16), using isolated fetal PASMCs, demonstrated that the hypoxia-induced increase in intracellular Ca2+ was inhibited by L-type Ca2+ channel blockers. These studies clearly illustrate the importance of Ca2+ influx through membrane voltage-gated Ca2+ channels in HPV. Indeed, Franco-Obregon and Lopez-Barneo (27) have shown that hypoxia causes a potentiation of the Ca2+ current in the majority of cells isolated from rabbit resistance pulmonary arteries. However, because these channels are generally closed at the resting membrane potential of PASMCs (27), it is likely that hypoxia acts first to depolarize the membrane by inhibiting the K+ channels involved in setting the resting membrane potential (107). Hypoxic inhibition of K+ channels was first demonstrated in 1988 by Lopez-Barneo et al. (47) in carotid body type I cells. Later, Post et al. (73) showed that hypoxia causes both an inhibition of whole cell K+ current and membrane depolarization in isolated PASMCs. An alternative, or perhaps an additional, hypothesis is that hypoxia causes the release of Ca2+ from intracellular stores independent of Ca2+ influx and that this rise in intracellular Ca2+ leads to the inhibition of membrane KV channels (31, 41, 72). The relative importance of these mechanisms is not clear; however, the aforementioned studies suggest that hypoxia acts through multiple pathways to induce pulmonary vasoconstriction.

Hypoxic Vasoconstriction Is Unique to Small Pulmonary Arteries

VSMCs show different responses to hypoxia according to the size and type of vessel from which they were isolated. Small resistance pulmonary arteries (third intrapulmonary artery or greater) constrict in response to hypoxia (3, 49, 72, 113), whereas large conduit pulmonary arteries (main pulmonary artery and right and left branches) usually do not respond or dilate slightly (3, 49, 72), although hypoxic responses have been described in some preparations (39, 113). In addition, the response of small pulmonary arteries is in marked contrast to that of small arteries isolated from the systemic circulation, which dilate in response to hypoxia (113). At the single-cell level, hypoxia causes small contractions (49, 88) and increased intracellular Ca2+ (88) in resistance PASMCs but has little or no effect in conduit PASMCs or VSMCs isolated from small systemic arteries (49, 88). Furthermore, hypoxia causes a significant depolarization of the membrane potential from approximately -51 to -37 mV in resistance PASMCs, whereas in conduit PASMCs, the membrane potential is not significantly affected (49). Proposed mechanisms for the contrasting vasoactive response to hypoxia between conduit and resistance pulmonary arteries and vessels from other vascular beds include differential expression of an O2-sensitive K+ channel (1, 3), differential expression of an O2-sensitive L-type Ca2+ channel (25, 28), and/or the presence of distinct O2-sensing mechanisms.

K+ Channels in the Pulmonary Vasculature

Four main classes of K+ channels have been described in vascular smooth muscle based on their biophysical and pharmacological properties: ATP sensitive (KATP), inward rectifier (KIR), large-conductance Ca2+-activated K+ (BKCa), and KV (42, 92). Of these, the KV channels are most likely to control the PASMC membrane potential and thus regulate vessel tone (23, 111).

KATP channels are sensitive to changes in the level of intracellular ATP and are therefore regulated by cellular metabolic status (43, 60). KATP currents are enhanced by ADP and inhibited by ATP. Antidiabetic sulfonylurea drugs such as glibenclamide inhibit this channel, whereas antihypertensive drugs such as minoxidil activate this channel (60). These channels probably do not contribute to HPV because glibenclamide has no effect on pulmonary perfusion pressure in normoxia or moderate hypoxia (38). However, these channels may serve as an important negative feedback mechanism, preventing excessive HPV under conditions of severe hypoxia when ATP levels are reduced (109). Furthermore, blockade of the endothelin subtype A receptor leads to activation of KATP channels and subsequent inhibition of HPV (84).

KIR channels conduct maximum current at membrane potentials negative to the K+ equilibrium potential and smaller outward currents at membrane potentials positive to the K+ equilibrium potential (60). This channel is very sensitive to inhibition by extracellular Ba2+. The presence of these channels in the pulmonary vasculature has not as yet been confirmed.

BKCa channels are sensitive to both membrane depolarization and increases in intracellular Ca2+, with increasing Ca2+ levels essentially shifting the activation curve in the hyperpolarizing direction. BKCa channels have a single-channel conductance of 250 pS in symmetrical K+ and are sensitive to blocking by tetraethylammonium (TEA), charybdotoxin (CTX), and iberiotoxin (60). BKCa channels play a role in control of the arterial smooth muscle membrane potential by serving as a negative feedback mechanism, regulating the degree of membrane depolarization and the constriction induced by increased cytoplasmic Ca2+ (14, 60). PASMC BKCa channels are activated at membrane potentials positive to the resting membrane potential [which explains why CTX does not depolarize PASMCs (3, 63, 111)]; thus under conditions of hypoxia, it is likely that the membrane depolarization and increased [Ca2+]i activate BKCa channels, leading to membrane hyperpolarization (75). Therefore, it is more likely that BKCa channels prevent excessive HPV rather than initiate it.

All VSMCs examined to date have at least one component of KV current, and many studies report more than one type of current (60). These currents include "delayed rectifier" (IK,DR) and "transient outward" (IK,TO) types (14). Recently, a third noninactivating KV current (IK,N), which is activated at the resting membrane potential, has also been described in rabbit, rat, and pig PASMCs (24, 62, 63). These channels provide an important K+ channel conductance in the physiological membrane potential range of pulmonary arteries (60). Indeed, several groups of investigators (3, 61, 63, 91, 115) have shown that the membrane potential in PASMCs is controlled by one or more subtypes of the delayed rectifier K+ channel. Pharmacologically, most KV currents can be isolated by selective inhibition to 4-aminopyridine (4-AP). The outward K+ current in PASMCs is inhibited by low doses of 4-AP (3, 61) but not by TEA (3) or CTX (3). In addition, 4-AP (3, 63, 111, 115) but not TEA (3, 63), CTX (3, 63, 111), iberiotoxin (63), or glibenclamide (111) depolarizes PASMCs. Furthermore, exposure to 4-AP but not to glibenclamide or CTX leads to an increase in [Ca2+]i (19). Taken together, these studies clearly emphasize the importance of KV channels in the regulation of PASMC membrane potential and pulmonary arterial tone.

K+ Channels in HPV

Agents that block K+ channels, such as 4-AP and TEA, depolarize the membrane (3, 72, 111), increase tension in pulmonary arterial rings (3, 73), and increase pulmonary arterial pressure in isolated perfused lungs (38). Hypoxia acts in a similar manner to inhibit whole cell outward K+ current (3, 72, 73) and cause membrane depolarization (3, 73, 112), constriction of small pulmonary arteries (3), and an increase in pulmonary arterial pressure (38). The fact that PASMC membrane depolarization and pulmonary arterial constriction can be mimicked by K+ channel blockers suggests that the depolarization induced by hypoxia is due to the inhibition of K+ channels open at the resting membrane potential.

Two types of PASMC K+ channels have been suggested to be modulated by hypoxia leading to HPV, BKCa (68, 73) and KV (which includes IK,DR and IK,N); however, the majority of electrophysiological and pharmacological evidence points to members of the KV family as the main PASMC O2 sensors [see McCulloch et al. (52) for further review]. The O2-sensitive K+ current described in most rat PASMC preparations is a delayed rectifier, which is sensitive to 4-AP and insensitive to CTX (6). Indeed, hypoxic inhibition of PASMC IK,DR has been shown in whole cell and single-channel studies in the presence of BKCa channel inhibitors (3, 64). Furthermore, hypoxia and 4-AP constrict resistance arteries, whereas CTX has no effect (3). In addition, pretreatment of isolated dog PASMCs with 4-AP (1 mM) but not with TEA (1 mM) prevents the hypoxic effect on outward K+ current (72). Taken together, these data suggest that KV channels that exhibit IK,DR and are sensitive to 4-AP but insensitive to CTX are responsible for the O2-sensitive K+ current in PASMCs. However, pretreatment with 4-AP does not always prevent, and may even potentiate, hypoxic constriction as demonstrated in rat and dog lungs (7, 38), rat resistance pulmonary arteries (3), and isolated pig PASMCs (88). Although these studies seem to argue against a role for KV channels in HPV, several points need to be considered. For instance, increased sensitivity to hypoxia has been demonstrated after an initial priming depolarization (98) and in the presence of agonist-induced pretone (106). Therefore, in some cases, pretreatment with 4-AP may act as a priming agent, causing decreased background K+ permeability and a potentiation of the hypoxic response. Additionally, because 4-AP sensitivity varies between KV channel subtypes and between species (17), the use of 4-AP as a pharmacological tool for determining the O2-sensitive K+ current is limited. For example, the heteromeric Kv2.1/Kv9.3 channel, which is a good candidate for at least one O2-sensitive K+ current, is insensitive to 4-AP at 1-10 mmol/l (69), the concentration used in the above-mentioned studies where 4-AP did not attenuate hypoxic constriction. Therefore, it is likely that the pulmonary arterial O2-sensitive K+ current represents an ensemble of current from distinct KV channel isoforms, some of which may be 4-AP insensitive. Additionally, the variability in the 4-AP sensitivity of the hypoxic response may be due to species-specific alpha -subunit combinations responsible for the hypoxia-sensitive current. Thus the variable response to hypoxia in the presence of 4-AP does not exclude a role for KV channels in HPV. Given that the majority of published studies suggest the involvement of a KV current in PASMC O2 sensing, the remainder of this review focuses on the role of KV channels in HPV.


    IDENTIFICATION OF KV CHANNEL GENES INVOLVED IN PULMONARY ARTERIAL O2 SENSING
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ABSTRACT
INTRODUCTION
BACKGROUND
IDENTIFICATION OF KV CHANNEL...
POTENTIAL MECHANISMS OF KV...
KV CHANNEL REGULATION BY...
SUMMARY
REFERENCES

Complexity and Diversity of KV Channels

The first complete nucleotide sequence encoding a KV channel was reported in 1987 with the cloning of the Drosophila Shaker channel (65). Low-stringency screening of Drosophila cDNA libraries with the Shaker cDNA led to isolation of the K+ channel cDNAs Shab, Shal, and Shaw that are derived from three distinct Drosophila genes (10). The sequences are homologous to Shaker, having ~40% identity. Each Drosophila gene has been shown to have one or more mammalian homologs (82), currently grouped into the Kv1, Kv2, Kv3, and Kv4 subfamilies. The Kv1 family, which has >60% homology to Shaker in the core region, is the largest, with at least seven members (12). In addition to the four mammalian subfamilies relating to Shaker, Shab, Shal, and Shaw, five additional subfamilies (Kv5-9) have also been described (81). Currently, over 30 KV channels have been cloned and expressed in heterologous expression systems (12). These channels often display differences in voltage sensitivity, current kinetics, and steady-state activation and inactivation (82).

KV channels exist as tetramers formed by 4 six-transmembrane-spanning alpha -subunits combining to form a functional channel (48). Not only can identical alpha -subunits combine to form a functional channel, but distinct alpha -subunits can also combine to form functional heteromeric channels both in vitro and in vivo (13, 81, 89). These heteromeric channels have unique properties that often represent a blend of the observed properties of the corresponding homomeric channels. Furthermore, several KV alpha -subunits are nonfunctional when expressed alone. For example, the Kv9.3 subunit, the most recently identified member of the mammalian KV family, does not form a functional homomeric channel itself but rather functions only in heteromeric complexes where it confers altered voltage sensitivity and activation kinetics (69).

Accessory beta -subunits can combine with KV alpha -subunits to add even more diversity to KV channel function (36). Currently, four KV beta -subunit gene families have been described (21, 22, 26, 56, 77). All are cytoplasmic proteins, ~40 kDa in mass, with a conserved core sequence and variable NH2 termini. KV beta -subunits have been shown to confer functional effects onto alpha -subunits, including both fast and slow inactivation, altered voltage sensitivity, and slowed deactivation (57, 101). Additionally, the beta -subunit may play a role as a cellular redox sensor because it appears to confer O2 sensitivity on the Kv4.2 channel in heterologous expression systems (71). Thus the roles of KV beta -subunits are many, and, indeed, they may be important not only in the functional modulation of KV alpha -subunits but also in the PASMC response to hypoxia.

Recent Progress Toward Identification of KV Channel alpha -Subunits Likely to Contribute to Pulmonary Arterial O2-Sensitive K+ Current

Although the electrophysiological and pharmacological profile of the O2-sensitive currents in the pulmonary artery have been extensively characterized, the molecular nature of these currents is only starting to be elucidated. Beginning in 1997, several groups of investigators (5, 40, 64, 69, 104, 116) examined the expression of KV channels in the pulmonary artery, antibody dissection of PASMC whole cell K+ current, and the O2 sensitivity of cloned KV channels expressed in heterologous expression systems in an attempt to identify the molecular components of the O2-sensivite KV current in PASMCs.

KV channel expression in the pulmonary vasculature. Wang et al. (104) used RT-PCR and immunoblotting to show that Kv1.5 and Kv1.2 mRNA and protein were expressed in primary cultures of rat PASMCs. Patel et al. (69), using degenerate RT-PCR, detected mRNA expression of Kv1.2, Kv1.3, Kv2.1, and a novel KV channel alpha -subunit, Kv9.3, in rat PASMCs. Despite several approaches, these investigators (69) failed to detect Kv1.5 and thus concluded that it was not expressed in the rat pulmonary artery. Archer et al. (5), using immunohistochemistry and immunoblotting of dissociated rat resistance PASMCs, reported expression of Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv2.1 but not of Kv1.4. About the same time, Yuan et al. (116), using RT-PCR of primary cultures of rat PASMCs, confirmed expression of Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1, and Kv9.3 mRNAs and reported mRNA expression of Kv1.4. Using immunoblotting, they detected Kv1.2, Kv1.4, and Kv1.5 but not Kv1.3. Hulme et al. (40) detected expression of Kv1.2 and Kv1.5 in PASMCs from rat lung tissue sections. Most recently, Osipenko et al. (64) reported expression of Kv3.1b protein in freshly dissociated PASMCs from rats and rabbits using immunocytochemistry. Additionally, our group has data suggesting that Kv1.2, Kv1.5, Kv2.1, Kv9.3, and Kv3.1b subunits are expressed in bovine resistance PASMCs (Coppock and Tamkun, unpublished observations). Taken together, these results strongly suggest the expression of the following alpha -subunits in resistance PASMCs: Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv9.3 (see Table 1). Not as convincingly, the presence of Kv1.1, Kv1.4, and Kv1.6 is also suggested.

                              
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Table 1.   Summary of candidate O2-sensitive KV channels expressed in the pulmonary vasculature

Antibody dissection of the O2-sensitive K+ current. Recently, antibodies against KV channels have been utilized in the intracellular patch pipette solution in an attempt to dissect the O2-sensitive K+ current in PASMCs during whole cell recordings under normoxic and hypoxic conditions. When Archer et al. (5) added an anti-Kv2.1 antibody to the patch pipette, they found inhibition of the outward K+ current and membrane depolarization in rat resistance PASMCs, suggesting that Kv2.1 plays a role in setting the resting membrane potential. When an anti-Kv1.5 antibody was added to the patch pipette, whole cell K+ current was significantly decreased, and both the hypoxia- and 4-AP-induced increase in [Ca2+]i in isolated PASMCs was attenuated (5). However, the anti-Kv1.5 antibody did not consistently depolarize the membrane in isolated PASMCs. Based on their findings, Archer et al. (5) hypothesized that hypoxic inhibition of Kv2.1 leads to membrane depolarization that shifts the resting membrane potential into a range where Kv1.5 is active and can thus be inhibited by hypoxia, leading to further inhibition of whole cell K+ current. Somewhat contrary to these results, others (31) found that application of anti-Kv1.5 in the patch pipette increased [Ca2+]i and caused membrane depolarization in rat PASMCs. Additionally, subsequent hypoxic challenge resulted in a further increase in intracellular Ca2+, with no effect on membrane potential. In related studies, Conforti et al. (15) used an antibody against Kv1.2 in the patch pipette to specifically inhibit Kv1.2 current from the whole cell current in rat pheochromocytoma (PC12) cells (a model for O2 sensing in the carotid body). When exposed to hypoxia, they found no additional K+ current inhibition, suggesting that the Kv1.2 current was responsible for the hypoxic effect in PC12 cells.

O2 sensitivity of cloned channels. The O2 sensitivity of several cloned KV channels has recently been studied in heterologous expression systems (15, 40, 64, 69). Investigators have primarily focused on those channels that, when expressed in heterologous expression systems, meet the profile of the PASMC O2-sensitive K+ current: delayed rectifier, sensitive to 4-AP, and insensitive to CTX.

KV2.1. Patel et al. (69) examined the O2 sensitivity of Kv2.1 expressed in COS-7 cells. In a subset of COS cells (21% of cells), Kv2.1 was reversibly inhibited by hypoxia by 34% (69). Hulme et al. (40) found similar results with Kv2.1 in mouse L cells. Hypoxia significantly inhibited Kv2.1 current by 23% at 60 mV in nearly all of the cells studied. In both studies, only the Kv2.1 currents that were activated at more positive potentials were significantly inhibited by hypoxia. Furthermore, little Kv2.1 current was detected at potentials more negative than -20 mV. In contrast, Conforti et al. (15) found the Kv2.1 current to be O2 insensitive when expressed in Xenopus oocytes. These data indicate that Kv2.1 is sensitive to hypoxia, at least in some expression systems. However, this sensitivity does not occur in the voltage range of the resting membrane potential of native PASMCs.

KV2.1/KV9.3. Patel et al. (69) cloned a novel subunit, Kv9.3, which does not form a functional channel itself but combines with Kv2.1 as evidenced by immunoprecipitation from metabolically labeled Xenopus oocytes and altered Kv2.1 biophysical and pharmacological properties (69). Most importantly, Kv9.3 causes a shift in the voltage dependence of activation into the voltage range of the resting membrane potential of PASMCs (40, 69), suggesting that the Kv2.1/Kv9.3 heteromeric channel may contribute to the regulation of PASMC resting membrane potential and thus the PASMC O2-sensitive K+ current. In fact, in a subset of COS cells (56% of cells), Kv2.1/Kv9.3 current was reversibly inhibited by hypoxia by 28% (69). Hulme et al. (40) found similar results when Kv2.1/Kv9.3 was expressed in mouse L cells. Hypoxia reversibly inhibited the Kv2.1/Kv9.3 current by 21% at 60 mV in all cells studied (40). In support of a role for the Kv2.1/Kv9.3 current in the physiological response of PASMCs to hypoxia, Kv2.1/Kv9.3 current was found to be sensitive to hypoxia in the voltage range of the PASMC resting membrane potential (40, 69).

KV1.5. The results from studies utilizing anti-Kv1.5 antibodies in the patch pipette have led some investigators (5) to hypothesize that Kv1.5 is an important component of the O2-sensitive K+ current in the pulmonary artery. Furthermore, Kv1.5 meets the profile of the O2-sensitive K+ current in PASMCs: delayed rectifier, sensitive to 4-AP, and insensitive to CTX (33). However, Kv1.5 is not sensitive to hypoxia when expressed as a homotetramer in L cells (40), COS-7 cells (64), or MEL cells (64). Therefore, it is unlikely that Kv1.5 homomeric channels contribute to the O2-sensitive current in PASMCs.

KV1.2. Although it is thought to underlie the CTX- and hypoxia-sensitive K+ current in PC12 cells (15, 117), Kv1.2, due to its sensitivity to CTX, has historically been ignored as an important player in PASMC O2 sensing (6). However, the O2 sensitivity of this channel was recently examined by Hulme et al. (40), who found that, when expressed in mouse L cells, the Kv1.2 current was significantly inhibited by hypoxia (19% inhibition at 80 mV), although only at depolarized potentials of >40 mV. In agreement with this observation, Conforti et al. (15) found that the Kv1.2 current was significantly inhibited by hypoxia in Xenopus oocytes. To the contrary, when expressed in B82 cells, the Kv1.2 current was not affected by hypoxia (64). Although these results suggest that the Kv1.2 current is O2 sensitive, at least in some preparations, because it does not activate in the voltage range of the PASMC resting membrane potential (40) and because of its sensitivity to CTX, it is unlikely that Kv1.2 homomeric channels underlie a O2-sensitive current in PASMCs.

KV1.2/KV1.5. When coexpressed, Kv1.2 and Kv1.5 assemble to form a functional heteromeric channel with kinetic and pharmacological properties distinct from those displayed by Kv1.2 and Kv1.5 homomeric channels (40, 80). This heteromeric channel produces a IK,DR that is sensitive to 4-AP but insensitive to CTX (40, 80). Noting that the Kv1.2/Kv1.5 current meets the characteristics of the O2-sensitive K+ current in PASMCs, Hulme et al. (40) examined the O2 sensitivity of this channel. When expressed in L cells, Kv1.2/Kv1.5 whole cell current was reversibly inhibited by hypoxia (18% reduction at 80 mV) (40). Furthermore, in support of a role for the Kv1.2/Kv1.5 current in the physiological response of PASMCs to hypoxia, this current was significantly inhibited by hypoxia in the voltage range of the PASMC resting membrane potential (for example, hypoxia inhibited the current at -40 mV by ~65%) (40).

KV3.1B. Most recently, the effects of hypoxia on Kv3.1b were examined by Osipenko et al. (64). Hypoxia significantly and consistently reduced the whole cell current amplitude in L929 cells expressing Kv3.1b channels by ~24% at 40 mV (64). Furthermore, an inhibitory effect of hypoxia was demonstrated at the single-channel level and in membrane patches excised from the cell (64). However, the inhibitory effect of hypoxia was only apparent at positive potentials. These results clearly demonstrate that Kv3.1b is O2 sensitive; however, when expressed alone as a homotetramer, it is not likely to be a component of the PASMC O2-sensitive K+ current.

Summary. The results from the aforementioned studies are summarized in Table 1. Kv1.1, Kv1.3, and Kv1.5 currents were not O2 sensitive in all cell systems studied (40, 64). The O2 sensitivity of Kv1.4 has not been examined; however, as seen in Table 1, there is some controversy as to whether it is even expressed in PASMCs, and because it produces an A-type current, it does not meet the biophysical profile of the O2-sensitive K+ current in PASMCs. Several investigators (5, 40, 69, 116) have detected Kv1.2 expression; however, its O2 sensitivity seems to vary markedly with expression systems (15, 40, 64, 69). Kv2.1 is expressed in PASMCs (5, 69, 116) and is found to be significantly inhibited by hypoxia in COS cells (69) and L cells (40) but not in Xenopus oocytes (15). In L cells, whole cell current from the Kv1.2/Kv1.5 heteromeric channel is significantly inhibited by hypoxia in the voltage range of the PASMC resting membrane potential (40). Although there is no direct evidence that this heteromeric channel is expressed in vivo, both individual subunits have been detected in PASMCs (5, 40, 116). The Kv2.1/Kv9.3 heteromeric channel is sensitive to hypoxia in the voltage range of the PASMC resting membrane potential in COS cells (69) and L cells (40). Both individual subunits are expressed in PASMCs (69, 116), and evidence for assembly in vitro has been demonstrated (69); however, there is no direct evidence that this heteromeric channel is expressed in vivo. Furthermore, when expressed in Xenopus oocytes, the Kv2.1/Kv9.3 current is significantly less sensitive to 4-AP than the Kv2.1 current alone (IC50, 4.5 mM for Kv2.1 vs. 31.6 mM for Kv2.1/9.3) (69), making the Kv2.1/Kv9.3 heteromeric channel much less sensitive to 4-AP than the O2-sensitive K+ current described in PASMCs. Kv3.1b is expressed in PASMCs and is O2 sensitive when expressed in L929 cells (64). In support of a role for Kv3.1b in O2 sensing in the pulmonary artery, data from our laboratory in the bovine pulmonary artery indicate that the protein expression of Kv3.1b markedly increases from the main conduit pulmonary artery to the third and fourth intrapulmonary resistance arteries (Coppock and Tamkun, unpublished observations). Because small resistance pulmonary arteries constrict in response to hypoxia, whereas large conduit pulmonary arteries do not (3, 49, 72), these findings strongly suggest a role for Kv3.1b in HPV.

Taken together, these studies support a potential role for Kv2.1/Kv9.3 and Kv1.2/Kv1.5 heteromeric channels in the physiological response of PASMC K+ channels to hypoxia. The Kv3.1b alpha -subunit is also a strong candidate; however, because (as a homotetramer) it is not activated at the resting membrane potential, it would have to be expressed as a heterotetramer with another alpha -subunit, or perhaps with a beta -subunit, which shifts the activation potential in a more hyperpolarized direction in vivo to account for the physiological response of PASMCs to hypoxia. Further studies are needed to address this question.

The results from the aforementioned studies, while giving us clues into the molecular identity of the O2-sensitive K+ currents in the pulmonary artery, are also somewhat confusing and contradictory. The explanation may lie in the heterologous expression systems used and the presence of an as yet unidentified O2 sensor that is endogenously expressed in some of these cells. For instance, Kv2.1 and Kv2.1/Kv9.3 currents responded to hypoxia in only a subset of COS-7 cells (69), whereas they responded to hypoxia in virtually all L cells studied (40). Furthermore, the Kv1.2 current was found to be sensitive to hypoxia in L cells (40) and oocytes (15), whereas in B82 cells, it was not (64), suggesting that the hypoxic inhibition of KV channels is more complicated than direct redox sensing by the KV channel alpha -subunits alone.

Role of the KV beta -Subunit in O2 Sensing

It has been hypothesized that the KV beta -subunit may play a role in cellular O2 sensing (35). In fact, when the conserved core of Kvbeta 2.1 was crystallized, bound NADPH was detected in its crystal structure (35). Furthermore, comparison of the three-dimensional structure of the beta -subunit and that of aldo-keto reductases illustrates striking similarity, suggesting that the beta -subunit may act to couple redox state to channel function (35).

Multiple KV beta -subunits have been detected in PASMCs. Yuan et al. (116), using RT-PCR of rat primary cultured resistance PASMCs, detected mRNA expression of Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 2.1. Data from our laboratory indicate that Kvbeta 1.1, Kvbeta 1.2, and Kvbeta 1.3 proteins are expressed in bovine PASMCs (Coppock and Tamkun, unpublished observations). Furthermore, our data also suggest that protein expression of these beta -subunits dramatically increases from the main conduit pulmonary artery to the third and fourth intrapulmonary resistance arteries. This is interesting in light of the fact that resistance pulmonary arteries constrict in response to hypoxia, whereas conduit pulmonary arteries do not (113), suggesting that KV beta -subunit expression may be partially responsible for the differential effects of hypoxia on conduit and resistance pulmonary arteries.

The KV beta -subunit has been shown to confer O2 sensitivity to certain KV alpha -subunits. Indeed, in HEK-293 cells, Kv4.2 alone was unaffected by hypoxia, but when coexpressed with Kvbeta 1.2, the current was significantly reduced (15.5% inhibition at 40 mV) (70). Recently, we compared the effects of hypoxia on Kv2.1 current in L cells and HEK-293 cells. We found that although the Kv2.1 current was significantly suppressed by hypoxia in L cells, it was not significantly inhibited in HEK-293 cells (Sakamoto N and Tamkun M, unpublished observations). Uebele et al. (100) have previously shown that mouse L cells contain an endogenously expressed beta -subunit, Kvbeta 2.1. Therefore, we determined whether this difference (the presence of Kvbeta 2.1) could account for the different O2 sensitivities of the Kv2.1 current expressed in L cells versus that in HEK-293 cells. We found that when Kv2.1 was coexpressed with Kvbeta 2.1 in HEK-293 cells, the whole cell K+ current was significantly inhibited by hypoxia (Sakamoto N and Tamkun M, unpublished observations). The results from this experiment are rather puzzling, however, because Kvbeta 2.1 does not immunoprecipitate with Kv2.1 (58). Although the mechanism by which KV beta -subunits confer O2 sensitivity onto some KV alpha -subunits in heterologous expression systems is unknown, much evidence points to a functional role for KV beta -subunits in PASMC O2 sensing.


    POTENTIAL MECHANISMS OF KV CHANNEL INHIBITION BY HYPOXIA
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There have been many hypotheses put forth to explain the hypoxic inhibition of KV channels in PASMCs, but the mechanism by which these channels sense low levels of O2 is still unclear. KV channels could be the O2 sensors themselves through the direct interaction of O2 with the channel, or they may be an effector, responding through some indirect mechanism (85). The indirect hypothesis seems more likely, however, because cloned KV channels are O2 sensitive in some heterologous expression systems but not in others. Perhaps they may be coexpressed with some endogenous O2 sensor that confers O2 sensitivity to the channel. As previously mentioned, Kv2.1 and Kv2.1/Kv9.3 channels were inhibited by hypoxia in only a subset of COS cells (69). Variable expression of and/or channel colocalization with such an endogenous O2 sensor would explain some of the apparent differences in the O2 sensitivities of cloned channels expressed in heterologous expression systems.

Membrane-Bound Heme-Linked Protein and/or NADPH Oxidase Mechanisms of O2 Sensing

One possible explanation is the existence of a membrane-bound heme-linked protein closely associated with the channel. In this scenario, two hypotheses have been put forth. The binding of O2 to the sensor could affect its conformation, which, in turn, affects the conformation of the KV channel and thus the K+ current. Alternatively, hypoxia could cause a decrease in the production of reactive oxygen species (ROS) generated by a membrane-bound NADPH oxidase, modulating the channel through a redox mechanism (see Refs. 6, 11, 86 for further reviews). In support of a membrane-bound O2 sensor, KV channel inhibition has been clearly demonstrated in excised patches in the absence of intracellular mediators in type I cells of the carotid body (30) as well as in heterologous expression systems (64, 70). Furthermore, carbon monoxide, which in biological systems is known to only react with heme proteins, significantly reverses the hypoxic inhibition of KV channels expressed in HEK cells (70) and in rabbit carotid body type I cells (46), suggesting that the sensor is a heme protein.

In the second hypothesis, the heme protein complex also contains an NADPH oxidase that rapidly generates superoxide and H2O2 under normoxic conditions, creating a relatively oxidized redox state (11). During hypoxia, however, there is decreased substrate for the oxidase leading to decreased production of ROS, a more reduced state, and inactivation of redox-dependent KV channels (11). Indeed, it is well known that K+ channel activity can be regulated by redox modulation in vitro (20, 79). In support of the NADPH oxidase hypothesis, reducing agents such as dithiothreitol, reduced glutathione (GSH), and NADH mimic the effects of hypoxia on PASMCs by decreasing K+ current (66, 67, 114), whereas oxidizing agents such as diamide and oxidized glutathione (GSSG) have the opposite effect, increasing K+ current (67, 76). NADPH oxidase is expressed in a variety of O2-sensitive tissues including pulmonary airway chemoreceptor cells [neuroepithelial body (NEB) cells] (110), carotid bodies (45), and PASMCs (51). Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, has been shown to significantly inhibit HPV in isolated rat lungs (94), rabbit lungs (32), and rat pulmonary arteries (96). However, because DPI has been shown to inhibit both K+ and Ca2+ currents (108), it has been criticized as being a useful tool in evaluating the importance of NADPH oxidase in O2 sensing (107). Furthermore, although DPI inhibits HPV, it does not cause sustained vasoconstriction under normoxic conditions (11).

Recently, two independent groups of investigators used a NADPH oxidase-deficient mouse model to examine the role of NADPH oxidase in O2 sensing. These groups studied the effects of hypoxia on K+ current in NEB cells (29) and PASMCs (4) from these mice and found very different results. Hypoxia had no effect on the K+ current in NEB cells from oxidase-deficient mice, whereas it significantly inhibited the K+ current in NEB cells from wild-type mice (29). Additionally, DPI significantly reduced the K+ current by 30% in wild-type NEB cells but had no effect in oxidase-deficient NEB cells. These results clearly support a role for NADPH oxidase as an O2 sensor in pulmonary airway chemoreceptors. In contrast to these results are the results of Archer et al. (4) in PASMCs using the same mouse model. They found the production of ROS to be significantly lower in the lungs of oxidase-deficient mice than in the lungs of wild-type mice. However, there was no significant difference in the hypoxic inhibition of K+ current between PASMCs from wild-type and oxidase-deficient animals, suggesting that NADPH oxidase is not necessary for O2 sensing in PASMCs. However, the NADPH gp91phox knockout mouse experiments do not exclude a role for a "low-output" NADPH oxidase isoform (32). These seemingly contradictory results emphasize the complexity of O2 sensing and strongly suggest a diversity of O2-sensing mechanisms between tissues.

Mitochondria as O2 Sensors

Multiple hypotheses relating to mitochondria as O2 sensors have been proposed (6, 11, 85). These include depletion of high-energy phosphates, a shift toward the reduced forms of redox couples such as NADH/NAD and GSH/GSSG, and an increase in mitochondrial ROS production. A logical potential mechanism is the depletion of intracellular ATP. In isolated rat lungs, inhibitors of oxidative ATP production caused a transient pressor response and a loss of reactivity to hypoxia, suggesting that depression of oxidative ATP elicits pulmonary vasoconstriction (78). Additionally, inhibitors of glycolysis, such as 2-deoxyglucose, and inhibitors of oxidative phosphorylation, such as rotenone, significantly inhibit outward K+ current in PASMCs (2, 114). However, these responses may not be due to simple depletion of ATP because the patch pipettes contained 5-10 mM ATP (2, 114). Additionally, HPV has been shown to occur very rapidly before any changes in ATP levels (8). Severe hypoxia has been shown to decrease ATP concentration; however, the decreased ATP levels result in the opening of KATP channels, increased K+ efflux, and vasodilation (109).

An additional hypothesis is that hypoxia inhibits the activity of cytochrome oxidase, which leads to an altered mitochondrial redox state and the generation of ROS (11, 85). In support of this hypothesis, hypoxia has been shown to cause a decrease in the maximum velocity of cytochrome oxidase (11) and an increase in the production of ROS (44, 51). However, in one report (51), the hypoxic generation of superoxide was attributed to a membrane-bound NADPH oxidase because DPI but not the mitochondrial inhibitor myxathiazole significantly attenuated this response.

Alternatively, hypoxia may cause an accumulation of redox couples such as NADH/NAD and GSH/GSSG and/or a decreased production of ROS (6). Mitochondria in the lung have been shown to make ROS in proportion to PO2 (99). Furthermore, rotenone, an inhibitor of the mitochondrial electron transport chain, mimics the effect of hypoxia by decreasing whole cell K+ current (2, 114), reducing lung ROS production (2, 114), and causing pulmonary artery constriction (3, 78). However, there is still much controversy regarding the different responses to metabolic inhibitors in different O2-responsive tissues (11) and some question as to whether hypoxia causes an increase (44, 51) or a decrease (6) in the production of ROS. Further studies are needed to examine these questions.

Intracellular Ca2+ Release as a Mechanism of KV Channel Inhibition by Hypoxia

There is accumulating evidence (31, 41, 72) that Ca2+ release from intracellular stores may initiate HPV. Hypoxia has been shown to cause a rapid, transient increase in [Ca2+]i in PASMCs that is unaffected by extracellular Ca2+ removal or blockade of voltage-gated Ca2+ channels but is significantly decreased by caffeine depletion of Ca2+ from the sarcoplasmic reticulum (83). Furthermore, ryanodine plus caffeine significantly inhibits HPV in dog resistance pulmonary arteries (41), and thapsigargin, cyclopiazonic acid, and ryanodine (used to deplete Ca2+ from intracellular Ca2+ stores) prevent HPV in rat pulmonary arterial resistance vessels (72). In PASMCs loaded with fura 2, hypoxic challenge, thapsigargin, cyclopiazonic acid, and ryanodine all resulted in a significant increase in [Ca2+]i and membrane depolarization (31). However, pretreatment with thapsigargin, cyclopiazonic acid, or ryanodine prevented a hypoxic challenge-induced increase in [Ca2+]i. Taken together, these studies suggest a predominant role for Ca2+ release from the intracellular stores in HPV. Delayed rectifier K+ channels are inhibited by [Ca2+]i in dog PASMCs (72). Additionally, application of an anti-Kv1.5 antibody within the patch pipette solution causes an increase in [Ca2+]i and membrane depolarization, whereas subsequent hypoxic challenge results in a further increase in [Ca2+]i, with no effect on membrane potential (31). These results led the researchers to hypothesize that hypoxia acts to activate Ca2+ release from the sarcoplasmic reticulum, leading to a rise in [Ca2+]i, which, in turn, causes the inhibition of KV channels and membrane depolarization (31, 72). Although this hypothesis is fairly well supported, it is conceivable that hypoxia acts through multiple pathways and multiple ion channels to produce HPV.


    KV CHANNEL REGULATION BY CHRONIC HYPOXIA
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Long-term hypoxic exposure leads to decreased PASMC KV channel current (62, 90, 91) and membrane depolarization (18, 74, 90, 91). Although acute hypoxia modulates KV channel current by affecting channel function, chronic hypoxic exposure is thought to modulate KV channel current by affecting channel expression. Indeed, Wang et al. (104) showed that prolonged exposure (24-72 h) of rat primary cultured PASMCs to hypoxia is associated with a downregulation of Kv1.2 and Kv1.5 alpha -subunit mRNA and protein. Therefore, they concluded that the diminished transcription and expression of KV alpha -subunits most likely results in fewer functional KV channels, a decrease in outward KV current, and subsequent membrane depolarization.

Chronic hypoxic exposure is associated with the regulation of a number of genes in a wide variety of cell types. Protein phosphorylation and/or redox modulation of transcription factors are thought to be part of the signaling pathway that translates a hypoxic stimulus into the regulation of gene expression (9). The mechanism by which KV channels are downregulated by chronic hypoxia is unknown but is likely to involve modulation of a hypoxia-inducible transcription factor. For instance, hypoxia-inducible factor-1 has been implicated in the regulation of an ever-growing number of genes, the products of which include endothelin-1, erythropoietin, nitric oxide synthase 2, and vascular endothelial growth factor (87). Additionally, accumulating evidence suggests that the c-fos and c-jun family of genes can also be induced by hypoxia (9). Preliminary data by Sweeney and Yuan (93) suggest that overexpression of c-jun significantly decreases whole cell KV current, making c-jun an attractive candidate for the transcriptional downregulation of KV channel expression during prolonged conditions of hypoxia. Whatever the mechanism for the hypoxic downregulation of KV channel expression, it is likely to be important in the pathogenesis of chronic hypoxic pulmonary hypertension because the consequent reduction in outward K+ current contributes to a sequence of events that ultimately lead to pulmonary hypertension (93).


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Recently, many advances have been made in the study of PASMC O2 sensing through the use of cloned channels, heterologous expression systems, and transgenic mice. Through these studies, it has become evident that KV channels play an important role in the response of PASMCs to hypoxia. Because the O2 sensitivity of some cloned channels varies with the cell type in which they are expressed, it not likely that KV alpha -subunits sense O2 directly; rather, they are most likely inhibited through some indirect mechanism that involves interaction with an unidentified O2 sensor and/or beta -subunit. In this manner, it is likely that the varied results represent multiple mechanisms and multiple effectors through which hypoxia is sensed throughout the body. Further work is needed to examine this possibility and to elucidate the many mechanisms by which KV channels "sense" and respond to acute and chronic hypoxia.


    ACKNOWLEDGEMENTS

We thank Dr. Naoya Sakamoto for technical assistance and Barbara Birks for review of this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-49330 (to M. M. Tamkun).

J. R. Martens was supported by National Institute of Child Health and Human Development Postdoctoral Fellowship 1-F32-HD-08496-01.

Address for reprint requests and other correspondence: M. M. Tamkun, Dept. of Physiology, Colorado State Univ., Fort Collins, CO 80523 (E-mail: tamkunmm{at}lamar.colostate.edu).


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