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(Circulation Research. 2000;86:534.)
© 2000 American Heart Association, Inc.

Cellular Biology

Potential Role for Kv3.1b Channels as Oxygen Sensors

O. N. Osipenko, R. J. Tate, A. M. Gurney

From the Department of Physiology and Pharmacology (O.N.O, A.M.G.) and Molecular Biology Laboratory (R.J.T.), Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.

Correspondence to Alison M. Gurney, Department of Physiology and Pharmacology, University of Strathclyde, 27 Taylor St, Glasgow, UK G4 0NR. E-mail a.m.gurney{at}strath.ac.uk

	Abstract

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Abstract—Hypoxia inhibits voltage-gated K channels in pulmonary artery smooth muscle (PASM). This is thought to contribute to hypoxic pulmonary vasoconstriction by promoting membrane depolarization, Ca²⁺ influx, and contraction. Several of the K-channel subtypes identified in pulmonary artery have been implicated in the response to hypoxia, but contradictory evidence clouds the identity of the oxygen-sensing channels. Using patch-clamp techniques, this study investigated the effect of hypoxia on recombinant Kv1 channels previously identified in pulmonary artery (Kv1.1, Kv1.2, and Kv1.5) and Kv3.1b, which has similar kinetic and pharmacological properties to native oxygen-sensitive currents. Hypoxia failed to inhibit any Kv1 channel, but it inhibited Kv3.1b channels expressed in L929 cells, as shown by a reduction of whole-cell current and single-channel activity, without affecting unitary conductance. Inhibition was retained in excised membrane patches, suggesting a membrane-delimited mechanism. Using reverse transcription–polymerase chain reaction and immunocytochemistry, Kv3.1b expression was demonstrated in PASM cells. Moreover, hypoxia inhibited a K⁺ current in rabbit PASM cells in the presence of charybdotoxin and capsaicin, which preserve Kv3.1b while blocking most other Kv channels, but not in the presence of millimolar tetraethylammonium ions, which abolish Kv3.1b current. Kv3.1b channels may therefore contribute to oxygen sensing in pulmonary artery.

Key Words: hypoxia • pulmonary artery myocyte • K⁺ channel • Kv3.1 • Kv1

	Introduction

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Oxygen sensing is an important property of the pulmonary circulation, with hypoxia causing vasoconstriction and an increase in pulmonary vascular resistance. Oxygen-sensing K channels have been proposed to play a role in this response.^{1 2} Thus, hypoxia causes membrane depolarization in pulmonary arterial myocytes by inhibiting a noninactivating, voltage-gated K⁺ current.^{3 4} This is thought to promote Ca²⁺ influx, smooth muscle contraction, and hence, vasoconstriction. The molecular correlates of the O₂-sensing current are unclear, and their identification is complicated by the expression of multiple K-channel genes in pulmonary artery.^{5 6 7} An additional complexity is that two O₂-sensitive K⁺ currents may exist in pulmonary artery smooth muscle (PASM) cells; these are a delayed rectifier and a distinct low-threshold, voltage-gated current, which may arise from different genes.² Of the genes so far detected, there is evidence for the involvement of Kv2.1, Kv9.3, Kv1.2, and Kv1.5, although some of the evidence is contradictory.^{5 6 7 8}

Hypoxia was found to inhibit recombinant rat Kv2.1 (rKv2.1) channels expressed in COS cells⁵ and mouse L cells,⁸ and the effect was enhanced by coexpression with a silent Kv9.3 subunit cloned from pulmonary artery.^{5 8} Moreover, certain biophysical and pharmacological properties of the heteromeric Kv2.1/Kv9.3 current resembled the delayed rectifier K⁺ current recorded from rat PASM cells.⁵ Hypoxia also inhibited rKv1.2 channels expressed in L cells, as well as heteromeric rKv1.2/human Kv1.5 (hKv1.5) channels, but not homomeric Kv1.5 channels.⁸ The evidence that Kv1.2 or Kv1.5 contributes to the O₂-sensitive K⁺ currents of pulmonary artery is, however, equivocal. One report suggests that Kv1.2 contributes insignificantly and that Kv1.5 channels are not expressed in pulmonary artery,⁵ but others strongly support both the existence of Kv1.5 in pulmonary artery and a role in O₂ sensing.^{6 7} A major role for these subunits in pulmonary artery O₂ sensing is, however, hard to reconcile with the prominent expression of Kv1.2 and Kv1.5, sometimes together, in nonpulmonary blood vessels,^{9 10} which respond differently to hypoxia.

It is not known how many more Kv subunits are expressed in pulmonary artery or whether other K channels can sense O₂. The pharmacology of the O₂-sensitive currents in pulmonary artery provides clues to their molecular identity. For example, the O₂-sensitive delayed rectifier is blocked by low concentrations of 4-aminopyridine^{3 11} but not by charybdotoxin (CTX) or dendrotoxin,⁵ properties characteristic of Kv1.5 and Kv2.1. This pharmacology is also a feature of Kv3.1 channels,¹² which have not been investigated in pulmonary tissue. These channels are preferentially distributed to the central nervous system,¹³ but they have been found in other tissues^{14 15} and in brain areas that display O₂-sensitive delayed rectifiers.^{13 16 17 18 19}

The mechanisms by which hypoxia inhibits recombinant channels have yet to be explored. Because hypoxia caused inhibition of Kv2.1 and Kv2.1/Kv9.3 currents in only a subset of cells,⁵ inhibition of these channels appears to involve an indirect mechanism. Hypoxia inhibits delayed rectifier K⁺ currents in carotid body,²⁰ central neurons,¹⁶ and pheochromocytoma (PC12) cells¹⁵ by a mechanism that is retained in excised membrane patches, suggesting an O₂ binding site on or closely associated with the channel. In PASM, membrane-delimited factors, reduced intracellular ATP levels, changes in cellular redox state, and channel inhibition by intracellular Ca²⁺ have all been proposed.^{1 2 3 11} It remains to be established exactly how pulmonary artery K channels are modulated by hypoxia.

Hypoxic inhibition of Kv2.1 channels is established. This study investigated the effects of acute hypoxia on other subunits found in pulmonary arteries, namely Kv1.1, Kv1.2, and Kv1.5, after transient or stable expression in different mammalian cell lines. Because Kv3.1 channels share a pharmacology similar to that of O₂-sensitive currents in a number of tissues, we also investigated their modulation by hypoxia and their potential contribution to the O₂-sensitive delayed rectifier of pulmonary artery.

	Materials and Methods

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Cell Isolation, Culture, and Transfection
Male rats (65 g) or rabbits (2 to 2.5 kg) were killed by sodium pentobarbitone overdose (60 mg · kg^-1 IP rats, IV rabbits). The first intrapulmonary artery branch was removed and myocytes were isolated similarly to previous studies.²¹ Rat adrenal PC12, COS-7, and Chinese hamster ovary (CHO) cells were grown in 6.5% CO₂ in DMEM containing 5% to 10% FCS, 100 mg/mL streptomycin, and 100 U/mL penicillin. B8, MEL, and L929 cells stably expressing rKv1.2, hKv1.5, and murine Kv3.1b (mKv3.1b), respectively, were cultured as previously described.¹² COS-7 and CHO cells were transfected with pBK-CMV-rKv1.1 or pBK-CMV-rKv1.5 using a standard DEAE-dextran method. On the following day they were transferred to 24-well plates containing glass coverslips for electrophysiology, which was performed 24 to 48 hours later.

Electrophysiology
Cells were superfused with physiological salt solution (PSS) containing (in mmol/L) NaCl 124, KCl 5, MgCl₂ 1, CaCl₂ 1.8, glucose 10, and HEPES 21 (pH 7.3). Pipettes for whole-cell recording contained (in mmol/L) KCl 120, MgCl₂ 2, EGTA 5, and HEPES 10 (pH 7.2; total K⁺, 140 mmol/L). For single-channel recording, pipettes contained PSS or intracellular solution, and cells were superfused with intracellular solution to null the membrane potential. Resting potentials and K⁺ currents were recorded and analyzed as previously described.⁴ Single-channel activity was estimated as NP_o, where N was the maximum number of simultaneous openings at 50 mV and P_o the open probability. COS-7, CHO, B82, MEL, and L929 cells had capacitances of 50±6 pF (n=26), 9±2 pF (n=5), 21±2 pF (n=59), 7±1 pF (n=50), and 36±17 pF (n=26), respectively. Input resistances were 3±1, 7±2, 5±1, 9±3, and 5±1 G, respectively.

The O₂ tension near a cell was monitored with an O₂ electrode (WPI Inc). Hypoxic PSS, prepared by equilibration with N₂ gas, provided a PO₂ of 14±2 mm Hg (n=20) compared with 125±6 mm Hg in control solution.

Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Total RNA extracted from isolated cells was treated with RQ1 RNase-free DNase (Promega), and 1-µg aliquots were reverse transcribed using SuperScript II RNase H reverse transcriptase (Life Technologies). PCR amplification used Taq polymerase and paired oligonucleotide primers specific for Kv3.1.¹⁵ PCR was performed in 100 µL containing (in mmol/L) dNTPs 0.2 (Amersham Pharmacia Biotech), MgCl₂ 1.5, KCl 50, and Tris-Cl buffer (pH 8.8) 10; 0.1% Triton X-100; 1 µmol/L each primer; 2 µL of reverse transcription reaction; and 2.5 U Dynazyme II DNA polymerase (Flowgen). Cycling parameters were 95°C for 10 minutes followed by 35 cycles at 50°C for 30 seconds, 65°C for 100 seconds, and 65°C for 10 minutes. Products were resolved by agarose gel electrophoresis, purified, and verified by sequencing.

Immunofluorescence
Myocytes were fixed with 0.5% glutaraldehyde and incubated for 10 minutes with 1 mg/mL NaBH₄ in PBS followed by 0.1% Triton X-100, 0.2% BSA, and 1.5% blocking serum. Subsequent incubation for 1 hour with a polyclonal anti-Kv3.1b antibody (dilution 1:50), raised against residues 567 to 585 of the rat isoform (Alomone Laboratories), was followed by 1 hour with 1:200 FITC-conjugated, goat anti-rabbit antibody (Sigma). Duplicates were processed without primary antibody or with antigen peptide (1:3). Fluorescence images were obtained with a Bio-Rad confocal microscope (MRC-1024 MP) using 488 nm excitation and 522 nm detection.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Before transfection, COS-7 cells had a resting membrane potential of -13±2 mV (n=19) and failed to display time-dependent outward current when the membrane potential was stepped to +60 mV from a holding potential of -80 mV. The resting potential in cells transfected with rKv1.1 (-45±4 mV, n=5) was significantly (P<0.0001) hyperpolarized compared with wild-type cells, and depolarizing steps evoked outward currents at potentials above -50 mV, with a normalized amplitude of 109±53 pA/pF (n=6) at +40 mV. As shown in Figure 1A, these currents were not inhibited by hypoxia. A small enhancement was observed, such that the current at +40 mV under hypoxic conditions averaged 114±2% (n=3) of control. Currents recorded from CHO cells expressing rKv1.1 were smaller (11±5 pA/pF at +40 mV, n=5) and not significantly altered by hypoxia (n=3).

View larger version (9K): [in this window] [in a new window]   Figure 1. K+ currents recorded from COS-7 cells transiently expressing rKv1.1 (A) or rKv1.5 (B), B82 cells stably expressing rKv1.2 (C), and L929 cells stably expressing mKv3.1b (D) under control conditions and after 5 minutes of exposure to hypoxia. Voltage steps to +40 mV from -80 mV at 5-second intervals are shown. E, Voltage dependence of L929 mKv3.1b currents before (•) and after () 5 minutes of hypoxia.

The resting potential of COS-7 cells transfected with rKv1.5 was -17±3 mV (n=14), not significantly different from that of wild-type cells. Depolarizing steps evoked outward currents at potentials above -30 mV, with a normalized amplitude of 10±3 pA/pF (n=18) at +40 mV. These currents were not consistently altered by hypoxia (Figure 1B), remaining at 102±12% (n=6) of the control amplitude. MEL cells expressing hKv1.5 had a resting potential of -25±2 mV (n=33) and displayed kinetically similar currents (68±11 pA/pF at +40 mV, n=46). These cells also failed to respond consistently to hypoxia, so that the current amplitude under hypoxic conditions averaged 98±12% (n=22) of the control amplitude.

B82 cells expressing rKv1.2 had a resting potential of -28±1 mV (n=30) and displayed outward currents in response to depolarizing steps above -30 mV (322±39 pA/pF at +40 mV, n=38). Hypoxia had no consistent effect on these currents (Figure 1C), which remained at 99±4% (n=19) of the control amplitude.

In contrast, hypoxia consistently reduced current amplitude in L929 cells expressing mKv3.1b channels (Figure 1D). These cells had a resting potential of -21±2 mV (n=21) and an outward current that activated above -20 mV and was 203±19 pA/pF (n=24) at +40 mV. After 5-minute exposure to hypoxia, the average current amplitude at +40 mV was only 76±4% (n=22) of that observed in control conditions. The effect was apparent only at positive potentials (Figure 1E), and hypoxia failed to influence the resting membrane potential. The effect of hypoxia on Kv3.1b currents was also observed at the single-channel level. Figure 2 shows records from cell-attached membrane patches, studied with a physiological transmembrane K⁺ gradient before and during exposure to hypoxia and after reintroduction of the control solution. Patches were clamped at -60 mV and stepped to +40 mV for 720 ms at 5-second intervals. In the patch illustrated, hypoxia reduced the number of active channels from 2 to 1 and NP_o from 0.32 to 0.02, without altering the single-channel current. On returning to control conditions, NP_o was restored to 0.32, and the simultaneous opening of 2 channels could again be seen. In 4 separate patches, hypoxia reduced NP_o by 45%, from 0.47±0.09 to 0.26±0.14. The ensemble-average current, constructed from 60 consecutive records, was reduced by 59±15%.

View larger version (24K): [in this window] [in a new window]   Figure 2. Single-channel activity in cell-attached membrane patches under control conditions, after 5 minutes of hypoxia, and 5 minutes after return to normoxia. Each panel shows the voltage protocol at the top and ensemble average of 60 consecutive sweeps at the bottom. Pipette contained PSS. Leak and capacitance currents are subtracted. Upward deflections from 0 current, the closed-channel level (c), represent outward current. Patch contained 2 active channels.

The inhibitory effect of hypoxia on Kv3.1b channels was retained in membrane patches excised from the cell. In the excised, inside-out patch illustrated in Figure 3A, hypoxia reduced the number of active channels from 3 to 1 and NP_o from 0.36 to 0.03. This effect was reproduced in 9 patches, in which over a 5-minute period hypoxia reduced NP_o by 66±8% from 0.11±0.04 to 0.04±0.01 and reduced the ensemble-averaged current by 71±5%. Only partial recovery of channel activity was observed (Figure 3A) because of channel rundown. In patches superfused for 5 minutes with control solution, NP_o fell by 24±13% (n=15), from 0.12±0.03 to 0.07±0.01, with a 27±9% reduction in the ensemble-averaged current. This loss of channel activity was significantly smaller (P<0.001) than that observed in the presence of hypoxia, implying that hypoxia reduced activity by 42%.

View larger version (28K): [in this window] [in a new window]   Figure 3. A, Single-channel activity in excised, inside-out membrane patches under control conditions, after 5 minutes of hypoxia, and 5 minutes after return to normoxia. Each panel shows the voltage protocol at the top and ensemble average of 60 consecutive sweeps at the bottom. Pipette contained PSS. Leak and capacitance currents are subtracted. Upward deflections from 0 (c) represent outward current. Three active channels were present. B, Amplitude histogram constructed from 20 consecutive records from another patch. The gaussian fit is shown with peaks at 0, 1.8, and 3.6 pA. C, Channel current-voltage relationship recorded during ramp depolarizations (-60 to +60 mV) as illustrated. Slope conductances were 19 (upper trace) and 32 (lower trace) pS. [K+ ] on either side of the membrane (i, inner; o, outer) is indicated next to each trace.

Only 1 type of channel was recorded from L929 cells expressing mKv3.1b. In a physiological K⁺ gradient, the single-channel current at +40 mV was 2pA in excised (Figure 3B) and cell-attached (Figure 2) patches. Ramp depolarizations from -60 to +60 mV revealed a linear current-versus-voltage relationship (Figure 3C) and a slope conductance of 16±1 pS (n=4) in the presence of 140 mmol/L intracellular K⁺ and 5 mmol/L extracellular K⁺. When the K⁺ gradient was reversed, the slope conductance was similar (14 pS, n=1), and in symmetrical 140 mmol/L K⁺, it was 31±1 pS (n=4). Similar values were obtained in cell-attached patches, as follows: 34±1 pS (n=3) in symmetrical 140 mmol/L K⁺ and 24 pS (n=1) in a physiological K⁺ gradient. In all cases, the single-channel current reversed direction near the calculated K⁺ equilibrium potential. Hypoxia had no effect on the conductance or reversal potential.

Expression of Kv3.1 Channels in Pulmonary Artery Myocytes and PC12 Cells
RT-PCR amplified a product from L929 cells expressing mKv3.1b of the predicted size (414 bp; n=4). The same primers amplified a similar product from PC12 cells (n=4), as previously described,¹⁵ and from rabbit PASM cells (Figure 4). Each product was confirmed to have the predicted sequence for amplified Kv3.1. This result was reproduced in 4 separate RT-PCR reactions on RNA extracted from PASM cells isolated from 10 rabbits.

View larger version (64K): [in this window] [in a new window]   Figure 4. Ethidium bromide visualization of RT-PCR products using primers for Kv3.1. Left lane shows molecular weight markers. Other lanes show products from L929 cells expressing mKv3.1, PC12 cells, and rabbit pulmonary artery myocytes. Kv3.1 PCR product has a predicted size of 414 bp.

Immunocytochemistry provided further evidence for Kv3.1 expression in PASM cells. Figure 5 shows that cells from both rabbit and rat fluoresced strongly when exposed to the anti-Kv3.1b antibody. This was prevented by the Kv3.1b antigen (Figure 5B) and by omission of the Kv3.1b antibody, implying specific binding. These experiments were repeated on each of 3 preparations from each species, and specific antibody staining was found in 100% of cells. Staining was also observed in 3 preparations of L929 cells.

View larger version (21K): [in this window] [in a new window]   Figure 5. Fluorescence (top) and transmitted-light (bottom) images of isolated rabbit (A) or rat (C) pulmonary artery myocytes treated with the anti-Kv3.1b antibody alone and rabbit myocytes treated with the antibody plus Kv3.1b antigen (B). Bar=10 µm.

Hypoxia Inhibits a Kv3.1-Like Current in Pulmonary Artery Myocytes
Tetraethylammonium (TEA) ions potently block Kv3.1 channels¹² and at 1 mmol/L reduced Kv3.1b current in L929 cells by 92±2% (n=3). At the same concentration, TEA reduced the K⁺ current recorded at +40 mV from rabbit PASM cells by 32±8% (n=5). Figure 6A shows that hypoxia had no effect on K⁺ currents recorded in the presence of 1 mmol/L TEA (n=5), although in the same cell, hypoxia reduced the current in the absence of TEA. Pronounced inhibition (56±8%, n=7) was observed when cells were exposed to hypoxia in the presence of 100 nmol/L CTX and 100 µmol/L capsaicin (Figure 6B), which are expected to enhance any contribution of Kv3.1 channels to the K⁺ current.¹⁴ Figure 6C confirms that 100 µmol/L capsaicin had little effect on Kv3.1b current in L929 cells but almost abolished Kv1.5 current in MEL cells. CTX similarly had no effect on Kv3.1 current in L929 cells but reduced Kv1.5 current by 70%. CTX and capsaicin together reduced the current recorded from PASM cells by 70±8% (n=7).

View larger version (13K): [in this window] [in a new window]   Figure 6. K+ currents recorded from rabbit pulmonary artery myocytes obtained before and during exposure to hypoxia, in the presence of 1 mmol/L TEA (A) or 100 nmol/L CTX and 100 µmol/L capsaicin (B). Capsaicin (100 µmol/L) caused minimal inhibition of Kv3.1b current in L929 cells (C) but almost abolished Kv1.5 current in MEL cells (D). Voltage steps to +40 mV from -80 mV at 5-second intervals.

	Discussion

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The results show that hypoxia inhibits Kv3.1b channels in a membrane-delimited manner and provide evidence that these channels are expressed and respond to hypoxia in PASM cells. We therefore propose that inhibition of Kv3.1b channels contributes to the effect of hypoxia on delayed rectifier K⁺ current in these cells. In contrast, hypoxia failed to inhibit Kv1.1, Kv1.2, or Kv1.5 channels, implying that channels of the Kv1 family are not primary sensors of O₂.

Hypoxia consistently and reversibly inhibited Kv3.1b current in the whole-cell, cell-attached, and excised-patch configurations. A membrane-delimited mechanism was indicated by retention of the effect in excised membrane patches. Thus, O₂ could act directly on the channel protein, although an endogenous regulator that coassembles with Kv3.1b in L929 cells cannot be ruled out. Evidence exists for direct modulation of K channels by hypoxia in native cells, including PC12 cells,¹⁵ central nervous system neurons,¹⁶ and type I carotid body cells.²⁰ Direct modulation has also been suggested, but not demonstrated, in PASM. The conductance of single Kv3.1b channels reported here is close to previous measurements in L929 cells¹⁴ (27 pS in 140 mmol/L symmetrical K⁺) and close to values reported for O₂-sensing K⁺ channels in PC12 cells¹⁵ (20 pS), carotid body²⁰ (20 pS), and rat pulmonary artery¹¹ (25 pS).

The finding that Kv3.1b channels are expressed in PASM suggests that they contribute to the O₂-sensitive K⁺ currents of these cells. The RT-PCR experiments did not specifically demonstrate Kv3.1b expression, as the primers would also have amplified Kv3.1a. These are alternatively spliced variants of the Kv3.1 gene that differ only at the cytoplasmic end²² and are commonly expressed in the same cells.²³ Nonetheless, immunostaining with an antibody directed against the cytoplasmic end of the Kv3.1b protein provided clear evidence for its expression in identified myocytes. The strongest evidence that Kv3.1 channels contribute to O₂ sensing in rabbit PASM is the inhibition of hypoxia-sensitive delayed rectifier K⁺ current by millimolar TEA, but not by CTX or capsaicin. Hypoxia was previously found to have no effect on delayed rectifier current in rabbit⁴ (but not rat¹¹ ) pulmonary artery when TEA was present. TEA also blocked hypoxia-sensitive K⁺ currents in other cell types.^{17 24 25 26} At the concentration used here, TEA is expected to block BK_Ca, Kv1.1, Kv1.2, and Kv3.1 channels but to have no effect on Kv1.5 and minimal effect on Kv2.1 or Kv2.1/Kv9.3 channels.^{5 12} The effects of TEA in rabbit pulmonary artery are therefore consistent with an O₂-sensing role for Kv3.1 but argue against a major contribution from Kv1.5 or Kv2.1/Kv9.3 channels. CTX is a potent blocker of BK_Ca, Kv1.2, and Kv1.5 channels, whereas capsaicin, at the concentration used, blocks most channels of the Kv1 family while minimally affecting Kv3.1 channels.¹² Preservation of the O₂-sensitive current of rabbit PASM cells in the presence of CTX and capsaicin therefore strengthens the argument in favor of Kv3.1 and against a role for Kv1 channels in O₂ sensing.

The role that Kv3.1b channels play in pulmonary arteries is unclear. Their voltage threshold for activation is outside the range of membrane potentials encountered in resting pulmonary artery myocytes.^{2 4} Consequently, the resting potential of L929 cells expressing only Kv3.1 channels was rather positive compared with –50 mV in PASM cells.⁴ In fact, of the channels studied, only Kv1.1 activated near this voltage range and gave rise to resting potentials comparable with those of the pulmonary artery. Thus, inhibition of Kv3.1b channels by hypoxia would not cause membrane depolarization, as observed in L929 cells. Functional effects of hypoxia on these channels would become apparent only if cells were already depolarized. Interestingly, in rat small pulmonary arteries, hypoxia induced depolarization due to K⁺ current inhibition only after a "priming" depolarization caused by 20 mmol/L K⁺, current injection, or endothelin-1.²⁷ Kv3.1b channels may therefore serve to amplify the primary response to hypoxia. Alternatively, pulmonary artery Kv3.1b may form heteromeric channels with subunits of the Kv1²⁸ or silent Kv5-Kv9 families,²⁹ resulting in altered kinetics and voltage dependence. Perhaps Kv3.1b can assemble with Kv9.3, recently cloned from pulmonary artery.⁵ As found for Kv2.1 channels,⁵ this might confer properties more consistent with the low-threshold, noninactivating K⁺ current that mediates hypoxia-induced depolarization.⁴

Kv3.1 genes are highly conserved across species, suggesting an important physiological function.¹³ They are mainly thought to enable rapid spiking in the central nervous system by limiting action potential duration and refractory period.^{13 18 30 31} O₂ sensing could be an additional function in central neurons, which are highly vulnerable to hypoxia. Several brain areas that express abundant Kv3.1b channels also display voltage-gated K⁺ currents that are inhibited by hypoxia. Thus, hypoxia suppressed K⁺ currents in cells from rat substantia nigra, neocortex, and striatum,^{16 17} all of which express Kv3.1b.^{13 18 19 23} By inhibiting Kv3.1 channels, hypoxia would cause action potential broadening and impair the ability to respond to high-frequency stimulation.¹³ This could provide a mechanism for adapting neuronal behavior to local environmental changes in PO₂. The expression of Kv3.1 in sympathetic neuron-like PC12 cells suggests that they may also contribute to O₂ sensing in the peripheral nervous system.

The lack of effect of hypoxia on Kv1 channels agrees with recent studies on Kv1.5 and the related Shaker channel.³² In contrast to our results, however, hypoxia did inhibit rKv1.2 channels expressed in L cells.⁸ Moreover, hypoxia inhibited Kv2.1/Kv9.3 current expressed in all L cells⁸ but in only a subset (56%) of COS cells.⁵ Perhaps L cells contain an endogenous O₂ sensor, lacking in B82 and COS cells, that can couple to K channels and modulate their activity. A potential mediator is the Kvß2.1 subunit, which is expressed endogenously in L cells⁸ and coassembles with Kv1.2 subunits. Another ß subunit, Kvß1.2, was found to confer O₂ sensitivity on Kv4.2, but not Shaker channels, in HEK293 cells.³² Several indirect mechanisms have been proposed to explain O₂ sensing by K channels. These include activation of a membrane-bound sensor, such as NADPH oxidase, to generate reactive oxygen species and modulation of intracellular metabolites such as ATP and Ca²⁺.^{1 2 3 11} Such mechanisms could account for cell-dependent variation in the responses of Kv2.1/Kv9.3 and Kv1.2 to hypoxia. Our results do not rule out a role for these channels in pulmonary O₂ sensing but imply that they are not primary O₂ sensors.

	Acknowledgments

 
Funding was provided by the British Heart Foundation. We thank Dr J.M. Nerbonne (Washington University, Saint Louis, Mo) for supplying Kv1.5 DNA; Prof J.M. Allen and Dr A.P. Barrie for help with transfections; Dr E. Rowan (Strathclyde University) for providing cell lines; and T. McShane, C. Meideros, and B. Fathi-Hafshejani for maintaining the cell cultures.

Received September 1, 1999; accepted January 7, 2000.

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