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Cardiovascular Research 2002 55(2):349-360; doi:10.1016/S0008-6363(02)00411-X
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

KV2.1 channels mediate hypoxic inhibition of IKV in native pulmonary arterial smooth muscle cells of the rat

Dayle S Hogga, Andrew R.L Daviesa, Gordon McMurrayb and Roland Z Kozlowskia,*

aDepartment of Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
bUniversity Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK

* Corresponding author. Tel.: +44-117-954-6875; fax: +44-117-925-2659 roland.kozlowski{at}bristol.ac.uk

Received 28 November 2001; accepted 4 March 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To determine whether, in native pulmonary arterial smooth muscle cells (PASMC), KV2.1 delayed-rectifying K+ channels are central to the process of hypoxic pulmonary vasoconstriction. Methods: In this study, we tested for the presence of KV2.1 channel transcripts in rat small pulmonary arteries using RT-PCR, and for the protein itself using immunolocalisation. The contribution of KV2.1 channels to whole-cell KV currents (IKV) and their role in hypoxic inhibition of IKV in native PASMC was investigated utilising patch-clamp recordings. Results: KV2.1 mRNA expression and AbKV2.1 (anti-KV2.1 antibody) protein immunoreactivity were both present in small pulmonary arteries. Dialysis of PASMC with AbKV2.1 significantly attenuated IKV by 67% at +50 mV. Hypoxia (~20–30 mmHg) inhibited IKV by ~70% at +50 mV. Ablation of currents associated with KV2.1 using AbKV2.1 caused a marked reduction in the amplitude of IKV. Hypoxia in the presence of the antibody did not affect the magnitude of IKV. Conclusions: These results indicate that KV2.1 channel subunits exist within small pulmonary arteries and conduct a significant part of IKV within native PASMC. Furthermore, application of AbKV2.1 abolishes hypoxic inhibition of IKV in native PASMC suggesting that KV2.1 channels play a pivotal role in mediating hypoxic pulmonary vasoconstriction.

KEYWORDS Arteries; Hypoxia/anoxia; K-channel; Myocytes; Pulmonary circulation

This article is referred to in the Editorial by Insuk So and Yung E. Earm (pages 233–235) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In the lungs, efficient gas exchange is maintained by a mechanism known as hypoxic pulmonary vasoconstriction (HPV), which serves to match local perfusion with ventilation, thereby optimising the arterial blood–gas content. Central to this process is inhibition of a K+ conductance, resulting in membrane depolarisation and the subsequent activation of L-type Ca2+ channels, leading to Ca2+ influx and ultimately vasoconstriction [1]. Hypoxic inhibition of K+ channels has been shown to be a membrane-delimited process [2–4] which occurs in the pulmonary arterial smooth muscle itself [5,6]. A number of possible candidates have emerged for the K+ conductance inhibited by hypoxia. The majority of studies agree that it is a member of the delayed-rectifier (KV) channel family [5,7–9], although a low threshold, oxygen-sensitive, non-inactivating K+ current has been identified in pulmonary arterial smooth muscle cells (PASMC) of some species [10–12].

The KV channel family is very diverse and 23 gene products encoding KV channel {alpha} subunits [13–15] and 3 KV channel β subunits [14] have been identified. In addition, further diversification is afforded by both homomeric and heteromeric tetramerisation and alternative splicing by some KV channel {alpha} subunits and some KV channel β subunits [13,14]. This has led to conflicting reports demonstrating the identity of the KV subunits responsible for the sensing or transduction of the hypoxic response. Molecular candidates proposed for the channel subunits responsible for oxygen sensing include KV1.2 [16–18], KV1.5 [16,19], KV2.1 [17,19,20], KV3.1b [6] and auxiliary subunits which can confer oxygen sensitivity such as KVβ1.2 [4] and KV9.3 [17,20]. These studies demonstrate hypoxic inhibition of K+ currents formed from either cloned homomeric or heteromeric channels. While various combinations of these channel subunits expressed in cell-lines may have important implications for oxygen sensing by K+ channels per se, it reveals little about oxygen sensing of the K+ currents in native PASMC.

KV channel mRNA expression analysis in pulmonary arterial smooth muscle has demonstrated the presence of many KV channel subunits [21,22], including the Shaker (KV1) subfamily, KV1.1, KV1.2, KV1.3, KV1.4, KV1.5, KV1.6 and KV1.7 [21,22], Shab (KV2) subfamily KV2.1 and KV2.2 [21,22], Shaw (KV3) subfamily KV3.3 and KV3.4 [22], Shal (KV4) subfamily KV4.1, KV4.2 and KV4.3 [22], auxiliary subunits KV5.1, KV6.1, KV9.1, KV9.2 and KV9.3 [21,22] and the KV β-subunits, KVβ1 and KVβ2 [21,22]. However, KV3.4 and KV4.1 channel subunits have only been found in smooth muscle of small pulmonary arteries while those of KV6.1 only in main pulmonary arteries. Discrepancies appear to exist in the literature regarding expression of a number of Shaker subunits. Patel and colleagues [20], were unable to detect mRNA expression of KV1.4, KV1.5 and KV1.6 in contrast to others [21,22] and others [21] have failed to detect KV1.3 mRNA expression in primary cultures of PASMC in spite of reported expression of this gene product [20,22]. Localisation and immunoblotting studies have illustrated the presence of KV1.1 [19], KV1.2 [16,17,19,21], KV1.3 [19], KV1.4 [21], KV1.5 [16,17,19,21,23], KV1.6 [19] and KV2.1 [19,21] protein immunoreactivity in rat pulmonary arterial smooth muscle and KV3.1b [6] in rabbit pulmonary arteries.

The hypoxia sensitive K+ current in pulmonary arterial smooth muscle has been proposed to be a 4-aminopyridine (4-AP)-sensitive [5,24], charybdotoxin (CTX)-insensitive [20], non-inactivating KV current [9,24]. Cloning and expression of homomeric KV1.2 [17,25–27], KV1.5 [17,26,28–32], and KV2.1 [17,31,33] channels reveal they all display slowly inactivating or non-inactivating KV currents that are sensitive to block by low concentrations of 4-AP. Both KV2.1 [34] and KV1.5 [26] have been shown to be CTX-insensitive, while KV1.2 [26] shows some inhibition to low concentrations of CTX. Although the pharmacological profile of KV1.2 does not strictly follow the profile for the hypoxia sensitive K+ channel, KV1.2 can form a heteromeric channel with KV1.5 that is insensitive to CTX [17]. From these observations it would appear that KV1.2, KV1.5 and KV2.1 might play a role in the hypoxic inhibition of IKV. Electrophysiological recordings of cloned KV channels have demonstrated that homomeric KV1.5 channels are not sensitive to hypoxia when expressed in L cells [17], COS cells [6] or MEL cells [6]. However, currents recorded from cloned KV2.1 channels were reversibly inhibited when expressed in both in mouse L-cells [17] and COS-7 cells [20], although this was only observed in ~20% of cells examined. Although this identifies KV2.1 channels as potential oxygen sensors, it reveals little about the hypoxia sensitivity of KV2.1 channels in native PASMC and their contribution to IKV.

The aim of the present study was to determine whether mRNA and protein expression of the KV2.1 channel subunit exists within small pulmonary arteries of the rat using RT-PCR and immunolocalisation protocols, respectively. Patch-clamp electrophysiology, utilising subunit specific antibodies, was then used to confirm the contribution KV2.1 channels to IKV and its functional role in mediating hypoxic inhibition of IKV within native PASMC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 RT-PCR
KV2.1 transcripts were detected in whole brain and small pulmonary artery mRNA preparations using RT-PCR as described previously [22]. The forward primer, 5'-agaaggatgaggacgacaccaag-3', and reverse primer, 5'-aagtgctgtggactagacgaggac-3', were used to amplify a 514 bp product corresponding to positions 1067 to 1580 of the published rat KV2.1 cDNA sequence (GenEMBL accession number X16476 [GenBank] ). PCR products were visualised using ethidium bromide-stained agarose gel electrophoresis and the identity verified by DNA sequencing. DNA sequencing was carried out with a fluorescent sequencing system (ABI Prism® BigDyeTM terminators) by the DNA Sequencing Facility, Department of Biochemistry, University of Oxford. The absence of contaminating genomic DNA was demonstrated by the absence of PCR products when the reverse transcriptase enzyme was omitted from the reaction.

2.2 Immunolocalisation
Male Wistar rats (~200 g; Harlan) were killed by an overdose of intraperitoneal sodium pentobarbitone (Rhone Merieux) and cervically dislocated, the lungs removed and placed in ice-cold PSS. Lobes of rat lungs were then dissected into quarters and embedded in Tissue Tek® optimal cutting temperature compound (OCT compound) on cork discs and snap frozen in liquid nitrogen cooled isopentane. Subsequently, 12 µm sections were cut on a microtome (Bright 5030, Bright Instrument Company Ltd.) within a cryostat chamber (–23 °C) and thaw mounted onto VectabondTM (Vector Laboratories Inc.)-treated slides. Sections were then fixed for 20 min using 4% paraformaldehyde in 0.1 mol/l PBS, consisting of (in mmol/l): 145.4 NaCl, 9.2 Na2HPO4, 6.4 NaH2PO4; pH 7.4 (NaOH), before being washed in Tris(hydroxymethyl)methylammonium chloride (Tris)-buffered saline (TBS), consisting of (in mmol/l): 256 NaCl, 50 Tris–HCl; pH 7.4 (NaOH). Standard avidin–biotin peroxidase immunohistochemical protocols were employed for antibody localisation. Briefly, endogenous peroxidase activity and avidin–biotin binding sites were abolished by incubation in 0.3% hydrogen peroxide for 20 min and the use of an avidin–biotin blocking kit (Vector Laboratories Inc.), respectively. Primary antisera, rabbit AbKV2.1 (Alomone Laboratories Ltd.), was applied for 24 h in a humid incubation chamber at 4 °C diluted to 1:200 in TBS containing 0.5% normal goat serum and 0.3% Triton X-100. Sections were then washed in TBS and the biotinylated secondary antibody (goat anti-rabbit) applied for 30 min, in a humid incubation chamber at room temperature. After further washing, sites of antibody localisation were visualised with the use of the Vectastain® Elite ABC peroxidase kit (Vector Laboratories Inc.) utilising 3,3'-diaminobenzidine as a chromogen and haematoxylin as a nucleophilic counterstain. Sections were then dehydrated and mounted before viewing under a microscope (Leica DMRB, Leica UK Ltd.) with an attached digital camera (Nikon Coolpix 950).

2.3 Cell isolation and electrophysiological recordings
Male Wistar rats were killed as described above and the heart and lungs removed en bloc. Following removal of the small pulmonary arteries (200–400 µm in diameter) from the lung, smooth muscle cells were isolated using a dispersion procedure previously described [35].

Membrane currents were recorded utilising the patch-clamp technique [36] using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). To examine the effects of hypoxia and antibody administration to IKV, cells were voltage-clamped at –50 mV. Electrodes were fabricated from borosillicate glass capillaries (Harvard Apparatus) using a vertical microelectrode puller (PP-83, Narishige Scientific Instruments). Data were filtered at 5 kHz, digitised at 24 kHz and recorded off-line with a modified DAT recorder (Sony DTC-A8). Data were analysed using pClamp software (version 6.04, Axon Instruments Inc.).

Membrane potentials of PASMCs were recorded in current-clamp mode and were found to be –40±1.0 mV (n = 71). To investigate the contribution of K+ currents associated with KV2.1 channel subunits, whole-cell patch-clamp experiments were carried out. Ionic conditions, based on the work of others [9], were adjusted to isolate IKV and minimise the contribution of currents carried by L-type Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels. Cells were perfused at room temperature (~22 °C) with a nominally Ca2+-free bath solution that contained (in mmol/l): 130 NaCl, 10 NaHCO3 4.2 KCl, 1.2 KH2PO4, 0.5 MgCl2, 5.5 D-glucose, 10 HEPES; pH 7.4 (NaOH) and the patch pipette contained (in mmol/l): 140 KCl, 1 MgCl2, 2.3 CaCl2, 5 EGTA, 10 HEPES; pH 7.3 (KOH). The intracellular free Ca2+ concentration, calculated from the computer program EQCAL (Biotools) based on the equations of Fabiato [37], was found to be 70 nmol/l. To ablate K+ currents associated with KV2.1, a subunit specific antibody (rabbit AbKV2.1, Alomone Laboratories Ltd.) was added to the pipette solution (dilution of 1:100), according to Archer et al. [19]. In order to determine if the effects of AbKV2.1 were specific, experiments were carried out using an antibody directed at KV1.5 channels (rabbit AbKV1.5, Alomone Laboratories Ltd.). AbKV1.5 (1:100 dilution) was found to have no significant effect on the mean IKV at +50 mV (see below) when compared to control conditions. The PO2 of the extracellular bathing solution was monitored using an O2-sensitive microelectrode (ISO2, World Precision Instruments). Hypoxic solutions were achieved by bubbling bath solutions with 100% N2 to achieve a PO2 of 20–30 mmHg.

Antibody effects on IKV were assessed following application of voltage-steps from –100 to +60 mV in 10 mV increments from a holding potential of –50 mV at 1 and 15 min (T1 and T15). Mean data were assessed by comparison of the steady-state current at +50 mV (I50) and are shown as mean±standard error of the mean, normalised to the peak outward current evoked at +60 mV in the voltage-step protocol at T1. The half-maximal activation potential (V0.5) of normalised difference currents were determined by fitting a Boltzman function of the form:


Formula

where k represents the slope factor, V is the membrane potential and V0.5 is the voltage at which 50% of channels are open.

2.4 Statement
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.5 Statistics
Statistical comparisons were made using an unpaired Student's t-test with differences considered significant at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 KV2.1 channel subunit mRNA expression and immunolocalisation of AbKV2.1 in rat small pulmonary arteries
RT-PCR protocols were employed to identify the potential oxygen-sensitive K+ channel KV2.1 in pulmonary arteries. Using specific primer pairs to regions of KV2.1, mRNA expression of this channel was identified in both rat small pulmonary arteries and brain (Fig. 1). Both products were found to be the same size and have the same sequence.


Figure 1
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  		Fig. 1 KV2.1 transcripts were identified in brain (Br) and small pulmonary artery (PA) mRNA using RT-PCR with subunit-specific primers, in the absence (–) or presence (+) of reverse transcriptase. Products were visualised by ethidium bromide-stained agarose gel electrophoresis and size determined by comparison to a 100 bp DNA ladder.

 
Since reports suggest that the KV channel subunits KV2.1 are involved in oxygen sensing in small pulmonary arteries, it was important to demonstrate immunolocalisation of these channel subunits within these arteries. Immunohistochemical staining of rat lung sections revealed diffuse staining of the smooth muscle layer for KV2.1 channel protein immunoreactivity in small pulmonary arteries (Fig. 2A). A representative example of control experiments showing the absence of non-specific staining of the secondary antibody in the absence of AbKV2.1 is shown in Fig. 2B.


Figure 2
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  		Fig. 2 Immunolocalisation of AbKV2.1 in sections of rat lung. (A) In small pulmonary arteries, localisation of the KV2.1 protein is present in the smooth muscle layer. Areas in brown represent localisation of AbKV2.1. (B) Representative example of a control experiment showing the absence of non-specific staining with the secondary antibody in the absence of AbKV2.1. Note the complete absence of any brown immunolocalisation of the channel proteins. Areas in blue represent nucleophilic staining with haematoxylin. Bar=100 µm. SM=smooth muscle.

 
3.2 Contribution of channels formed from KV2.1 channels subunits to IKV
To ensure that BKCa channels were not activated during our voltage-step protocol under conditions to isolate IKV, whole-cell patch-clamp experiments were carried out in the absence and presence of Iberiotoxin (IbTX; Alomone Laboratories Ltd.), a selective BKCa channel inhibitor [38]. The mean normalised current at I50 under control conditions (0.92±0.019; n = 5) was not significantly affected by application of 20 nM IbTX (0.85±0.090; n = 5) indicating that BKCa channels were not activated under these conditions. Whole-cell recordings at T1 and T15 induced by the voltage-step protocol elicited currents that showed a relatively rapid activation and little time-dependent inactivation during the course of the voltage-step (Fig. 3A). At T15, the voltage-step protocol induced a family of membrane currents that displayed similar characteristics to those induced at T1 but with a slightly smaller current magnitude at more depolarised potentials caused by time-dependent run-down (Fig. 3A). Data summarising the effects of run-down on IKV are displayed in Fig. 3B. At T15, I50 was reduced by ~11% (n = 4) and was not significantly different from the I50 recorded at T1 (n = 9; Fig. 3B): indicating no significant ‘run-down’ of IKV in pulmonary arterial myocytes after 15 min dialysis with the intracellular pipette solution under these conditions.


Figure 3
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  		Fig. 3 Effects of ‘run-down’ on IKV. Membrane currents were recorded under whole-cell voltage-clamp in nominally Ca2+-free conditions. (A) From a holding potential of –50 mV, voltage-steps, from –100 to +60 mV in 10 mV increments (see inset) evoked a family of membrane currents at T1, which were attenuated slightly at T15. (B) Mean I50 (normalised to the mean steady-state current recorded at +60 mV at T1) summarising the effects of time-dependent ‘run-down’ on IKV at T1 and T15 after the ‘whole-cell’ configuration was determined. Note that at 15 min after rupture of the cell membrane, there is no significant difference in the I50 when compared to that at T1.

 
To assess the contribution of currents associated with the KV2.1 channel subunit to IKV, the effects of intracellularly applied AbKV2.1 were examined. Membrane currents in response to voltage-steps showed marked attenuation after 15 min dialysis with AbKV2.1 when compared to those observed at T1 (Fig. 4A). The family of difference currents, revealing the AbKV2.1 sensitive current (IAbKV2.1), displayed relatively rapid activation and little inactivation (Fig. 4A). The mean steady-state I–V relationship revealed significant reduction in the steady-state outward current at potentials positive to and including –30 mV (Fig. 4B). In the presence of AbKV2.1, the I50 at T15 was reduced by ~67% (n = 3; P<0.0001) when compared to the I50 at T1. To eliminate any errors induced by a possible contribution of ‘run-down’, the currents observed at T15 in the presence of AbKV2.1 were compared to the current recorded at T15 in the control group. Normalised I50 at T15 after dialysis with AbKV2.1 was also significantly attenuated when compared to the normalised I50 observed at T15 under control conditions (Fig. 7). The AbKV2.1 induced inhibition of IKV was specific since boiled (95 °C for 35 min) AbKV2.1 had no significant effect on the I50 of IKV (Fig. 7). The mean steady-state I–V relationship of IAbKV2.1 was typical of a KV current (Fig. 4C). In order to compare the kinetics of IAbKV2.1 with those of cloned KV2.1 channels expressed in cell-lines, the V0.5, +5.9±3.4 mV (n = 3), was determined by fitting a Boltzman function to normalised current amplitudes evoked by the voltage-step protocol. The close similarity of the biophysical profile of IAbKV2.1 with currents from cloned KV2.1 channels indicate the functional presence of KV2.1 channels in native PASMC.


Figure 4
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  		Fig. 4 Effects of AbKV2.1 on IKV. (A) Under whole-cell voltage-clamp conditions, membrane currents were evoked in response to voltage-steps from –100 to +60 mV in 10 mV increments from a holding potential of –50 mV (see inset). The left panel shows a family of membrane currents 1 min after whole-cell status had been established. The right panel demonstrates a typical family of membrane currents after 15 min dialysis with AbKV2.1 antibody. A family of difference currents, representing IAbKV2.1 is illustrated in the lower panel. (B) Mean steady-state I–V relationship for families of membrane currents induced by the voltage-step protocol after 1 min (n = 6) and 15 min (n = 3) dialysis with AbKV2.1. Currents are normalised to peak outward current after 1 min. (C) Mean steady-state I–V relationship for IAbKV2.1 (n = 3). * indicates P<0.05 when compared to control.

 

Figure 7
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  		Fig. 7 Summary of the effects of AbKV2.1 and hypoxia on IKV. Data displaying mean normalised I50 recorded after 15 min (T15) under control conditions (n = 3; PO2~150 mmHg), hypoxic conditions (n = 6; PO2~35 mmHg), after dialysis with AbKV2.1 (n = 3), after dialysis with AbKV2.1 in the presence of hypoxia (n = 5) and after dialysis with boiled (95 °C for 30 min) AbKV2.1 (n = 5). Note that dialysis with AbKV2.1, application of hypoxia and dialysis with AbKV2.1 in the presence of hypoxia are all significantly attenuated compared to control. This confirms that the reduction in current is not due to ‘run-down’. It is also worth noting that all of these currents display almost identical magnitudes. * indicates P<0.05 and ** indicates P<0.005 when compared to the control I50 after 15 min.

 
3.3 Hypoxic inhibition of IKV
To identify the hypoxia-sensitive component of IKV (IKVO2), whole-cell patch-clamp experiments were carried out. Upon application of hypoxia just prior to T15 in order that the PO2 was 20–30 mmHg at T15, outward currents at T15 in response to voltage-steps were significantly and reversibly reduced from those activated under normoxic conditions at T1 (Fig. 5A). This is clearly illustrated by the steady-state I–V relationship which shows significant hypoxia induced inhibition of IKV at all membrane potentials positive to –30 mV at T15 when compared to normoxic conditions at T1 (Fig. 5B). Under hypoxic conditions at T15, the mean I50 was significantly reduced by ~70% when compared to the control at T1 (Fig. 5B). Comparison of the mean normalised current at T15 under hypoxic conditions with those induced at T15 under control conditions, in order to eliminate any errors induced by ‘run-down’, also showed a significant inhibition of the outward current upon application of hypoxia (Fig. 7). In order to examine the biophysical profile of IKVO2, the hypoxia-insensitive current at T15 was subtracted from the total current under normoxic conditions at T1. The difference current (Fig. 5A, lower panel), is a relatively rapidly activating current with little time-dependent inactivation, a result consistent with the findings of others [9,24]. The I–V relationship for the mean steady-state difference current revealed a V0.5 of +6.5±3.3 mV (n = 5): a value very close to V0.5 calculated for IAbKV2.1 (+5.9±3.4 mV; n = 3). The normalised I50 of IKVO2 (0.55±0.05; n = 6) was not significantly different to that of IAbKV2.1 (0.62±0.04; n = 3). Together these data suggest that hypoxia inhibits a component of IKV that displays similar electrophysiological characteristics to IAbKV2.1.


Figure 5
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  		Fig. 5 Effects of hypoxia on IKV. (A) Under whole-cell voltage-clamp conditions, families of membrane currents were evoked in response to voltage-steps from –100 to +60 mV in 10 mV increments from a holding potential of –50 mV (see inset), under normoxic conditions (PO2~150 mmHg) after 1 min (left panel) and under hypoxic conditions (PO2~35 mmHg) after 15 min (right panel). The lower panel shows the difference current representing the hypoxia-sensitive current (IKVO2). (B) Mean steady-state I–V relationship for families of currents evoked by the voltage-step protocol at 1 min in normoxic conditions (n = 11) and after attenuation by hypoxic challenge at 15 min (n = 5). (C) Mean steady-state I–V relationship for IKVO2 (n = 5). In (B) and (C) current is shown normalised to peak outward current under control conditions. * indicates P<0.005 when compared to control.

 
3.4 Contribution of channels formed from KV2.1 channel subunits to hypoxic inhibition of IKV
In order to confirm whether hypoxia was inhibiting a component of current associated with KV2.1 channel subunits, experiments were carried out to assess the effects of hypoxia on IKV after inhibition of IAbKV2.1. Cells were dialysed with AbKV2.1 until T15, a time frame shown to allow inhibition of IAbKV2.1 (see Fig. 4A, B). Hypoxia was applied just prior to T15 so that the PO2 was between 20 and 30 mmHg at T15. Whole-cell voltage-clamp experiments revealed a family of outward currents that displayed significant attenuation at T15 in the combined presence of both AbKV2.1 and hypoxia when compared to the control at T1 (Fig. 6A, left and right panels). The current difference revealed that the combined AbKV2.1 and hypoxia sensitive currents (Fig. 6A, lower panel) displayed an almost identical profile to those of IAbKV2.1 and IKVO2 (see lower panels, Figs. 4A and 5AGo, respectively). A mean I–V relationship demonstrating the current inhibited by the combination of AbKV2.1 and hypoxia, revealed significant attenuation of currents activated at potentials including and positive to –30 mV (Fig. 6B): a characteristic similar to inhibition of IKV by either AbKV2.1 or hypoxia (Fig. 4B and 5BGo, respectively). The mean I50 was reduced by ~60% in the presence of the combination of AbKV2.1 and hypoxia at T15 when compared to control conditions at T1 (Fig. 6B). Furthermore, these currents inhibited by the combination of both AbKV2.1 and hypoxia had a V0.5 of +1.2±5.3 mV (n = 5), which was not significantly different from the V0.5 of either IAbKV2.1 (+5.9±3.4 mV; n = 3) or IKVO2 (+6.5±3.3; n = 6). To eliminate any errors induced by possible ‘run-down’, the mean I50 at T15 in the presence of hypoxia and AbKV2.1 was compared to control values at T15 and was confirmed to be significantly attenuated. It is interesting to note that the current inhibited by the combination of AbKV2.1 and hypoxia had an almost identical normalised I50 (0.52±0.08; n = 5) to those found in experiments in the presence of either AbKV2.1 (0.62±0.04; n = 3) or hypoxia (0.55±0.05; n = 6) alone. This would indicate that AbKV2.1 and hypoxia are inhibiting the same component of IKV and thus hypoxic inhibition of KV2.1 channels may be responsible for hypoxic attenuation of IKV. To further illustrate this point, comparison of the I50 at T15 under control conditions and following dialysis with AbKV2.1, in the absence and presence of hypoxia (Fig. 7) revealed a similar value for I50 confirming the involvement of KV2.1 in oxygen sensing.


Figure 6
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  		Fig. 6 Effects of AbKV2.1 and hypoxia on IKV. Membrane currents were recorded using the whole-cell voltage-clamp technique. Membrane currents were recorded in response to voltage-steps from –100 to +60 mV in 10 mV increments from a holding potential of –50 mV (see inset). (A) Typical currents recorded using the voltage-step protocol described above illustrate a family of currents recorded at 1 min under normoxic conditions (PO2~150 mmHg; left panel) and at 15 min after dialysis with AbKV2.1 and in the presence of hypoxia (PO2~35 mmHg; right panel). The lower panel shows a family of difference currents, representing the currents that were sensitive to the combination of both AbKV2.1 and hypoxia. (B) Mean steady-state I–V relationship for currents evoked by the voltage-step protocol after 1 min (n = 10) and at 15 min after dialysis with AbKV2.1 and in the presence of hypoxia (n = 5). (C) Mean steady-state I–V relationship for the current sensitive to the combination of AbKV2.1 and hypoxia (n = 5). For (B) and (C) current is shown normalised to peak outward current under control conditions. * indicates P<0.05 when compared to control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this manuscript we describe the presence of KV2.1 channel subunit mRNA using RT-PCR. We also describe the presence of the channel protein in small pulmonary arteries of the rat using immunolocalisation protocols. Furthermore, electrophysiological studies demonstrate that KV2.1 subunits form channels that contribute significantly to IKV and we show for the first time that their ablation abolishes hypoxic inhibition of IKV in native PASMC.

Patch-clamp experiments illustrated that the IKV showed relatively rapid activation and little time-dependent inactivation, a current profile that has been attributed to activation of members of the KV channel family [9,28,31]. The magnitude of IKV at T15 was slightly attenuated when compared to that recorded at T1, although not significantly different. This phenomenon is known as ‘wash-out’ or ‘run-down’, which has been attributed to loss of intracellular cytosolic factors [39]. Ablation of currents associated with KV2.1 channel subunits using AbKV2.1 revealing IAbKV2.1 demonstrated that channels possessing KV2.1 channel subunits conduct significant components of IKV in PASMC. This is in direct agreement with another study that has demonstrated significant inhibition of IKV after application of an antibody directed towards KV2.1 channel subunits [19]. The V0.5 of IAbKV2.1 and its characteristic biophysical profile of relatively rapid activation and little inactivation shows a close similarity to the profile of cloned homomeric KV2.1 channels expressed in Xenopus oocytes [31], transfected mammalian cells [33], mouse L cells [17] and COS cells [20], confirming the functional presence of this channel.

From the data presented in this study it cannot be concluded whether the currents inhibited by AbKV2.1 are a product of homomeric or heteromeric channels, or perhaps a combination of both. Indeed, it is known that many of the KV channel family subtypes may form heteromultimers [40], but it is believed that their assembly is subfamily specific [41]. Since only one other member of the Shab (KV2) family, KV2.2, has been shown to exist in pulmonary arterial smooth muscle at the mRNA level [22], the number of channel products is limited. However, under certain conditions, KV2.1 and KV2.2 channel subunits have been shown to heteromultimerise [42]. It would therefore be interesting to determine if these channels show co-localisation within native PASMC. Comparison of the V0.5 of IAbKV2.1 with that of cloned homomeric KV2.1 channels demonstrate very similar activation kinetics [31], which may indicate that KV2.1 channels exist as homomultimers in native PASMC. It is worth noting, however, that the kinetics of a KV2.1/KV2.2 heteromultimer are also similar to those of a KV2.1 homomultimer [42]. Interestingly, the V0.5 of cloned KV2.1 channels in other studies were found to be more depolarised [17,20,33], although this may be explained by differences between the kinetics of native channels when compared to those of channels cloned and expressed in cell lines due to their modulation by auxiliary subunits. Alternatively the modulatory subunit KV9.3 (expressed in pulmonary arterial smooth muscle [20,21]) can form a heteromultimer with KV2.1 and modulate the activation potential of cloned homomeric KV2.1 channels to more hyperpolarised potentials [17,20].

The component of IKV inhibited by hypoxia (IKVO2) in PASMC displayed kinetics that were mimicked by IAbKV2.1. Not only were the V0.5 of activation virtually identical, but also the magnitude of IKVO2 and IAbKV2.1. From this data alone it may be suggested that the KV channel subunits inhibited by AbKV2.1 may provide a possible candidate for the molecular identity of the hypoxia sensitive K+ channel in native pulmonary arterial myocytes. The observation that KV2.1 may be the molecular nature of the hypoxia sensitive KV channel in pulmonary arterial smooth muscle is in direct agreement with previous studies that have demonstrated cloned homomeric KV2.1 channels to possess intrinsic oxygen sensitivity [17,20]. Consistent with this, application of hypoxia after antibody ablation of currents associated with KV2.1 channels revealed the absence of hypoxic inhibition of IKV. Furthermore, comparison of the difference currents recorded after application of hypoxia in the presence of AbKV2.1, IKVO2 and IAbKV2.1 showed that they were all identical, implying that IAbKV2.1 and IKVO2 were indeed the same current. This is the first demonstration that KV2.1 channels in native pulmonary arterial smooth muscle are responsible for mediating hypoxic inhibition of IKV.

KV2.1 channel subunits have also been identified in coronary, renal and cerebral arteries [19] and the IKV characterised in the smooth muscle of these tissues do not demonstrate hypoxic inhibition. In addition, the mechanism of hypoxic inhibition of IKV in pulmonary arteries has been shown to be membrane associated [2–4,6,43]. Together these observations imply that although KV2.1 channels are directly involved in the transduction of hypoxic inhibition of IKV in PASMC, it is likely that a membrane-associated subunit may be responsible for sensing and/or conferring oxygen sensitivity to KV2.1 channels. Several modulatory subunits that can modulate KV2.1 channels have been shown to exist in pulmonary arterial smooth muscle either at the level of mRNA or protein expression, including KV9.3 [20,21], KV5.1 [22] and KV6.1 [22]. It would therefore be interesting to evaluate the role of auxiliary subunits, such as KV9.3, KV5.1 and KV6.1, or heteromultimeric assembly of this channel by using their subunit-specific antibodies. KV9.3, an electrically silent subunit when expressed alone, can form a heteromultimer with KV2.1. Indeed it has been shown that cloning and expression of this heteromultimer in COS cells [20] and mouse L cells [17] produces an oxygen sensitive K+ current that activates close to the resting membrane potential of PASMC. Furthermore, a recent study has demonstrated that the mRNA expression of both KV9.3 and KV2.1 channel subunits are decreased during chronic hypoxia, however, in contrast only protein expression of KV2.1 channel subunits was demonstrated to decrease during chronic hypoxia [44]. It would be tempting to speculate that it is this subunit that confers oxygen sensitivity to KV2.1 channels in native PASMC. The possibility that KV2.1 channels in native PASMC may be modulated by either KV5.1 or KV6.1 subunits also cannot be excluded either since their coexpression with KV2.1 channel subunits has been demonstrated [45,46]. Furthermore, both KV5.1 and KV6.1 channel subunits have also been shown to modulate the activation potential of KV2.1 currents [46]. Since these channel subunits have been shown to exist in native PASMC and are able to modulate the channel kinetics of KV2.1, a possible role in conferring oxygen sensitivity should not be ignored.

From the data described in this study, it is clear that currents associated with KV2.1 channels in native PASMC mediate hypoxic inhibition of IKV. Uncovering the role of KV2.1 channels in mediating hypoxic inhibition of IKV in native PASMC is likely to have important implications in further elucidating the transduction process and oxygen sensor in HPV.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by the British Heart Foundation and the Wellcome Trust.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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