Potential identification of the O2-sensitive K+ current in a human neuroepithelial body-derived cell line

I. O'Kelly1,2, R. H. Stephens1, C. Peers2, and P. J. Kemp1

1 School of Biomedical Sciences and 2 Institute for Cardiovascular Research, University of Leeds, Leeds LS2 9JT, United Kingdom

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Whole cell recording of H-146 cells revealed that the outward K+ current was completely inhibited by quinidine (IC50 ~17 µM). In contrast, maximal concentrations of 4-aminopyridine (4-AP; >= 10 mM) reversibly blocked only ~60% (IC50 ~1.52 mM). Ten millimolar 4-AP had no effect on the inhibition by hypoxia, which reduced current density from ~27 to ~13 pA/pF, whereas 1 mM quinidine abolished the hypoxic effect. In current clamp, 10 mM 4-AP depolarized the cell by ~18 mV and hypoxia caused further reversible depolarization of ~4 mV. One millimolar quinidine collapsed the membrane potential and abrogated any further hypoxic depolarization. RT-PCR revealed expression of the acid-sensitive, twin P domain K+ channel TASK but not of TWIK, TREK, or the known hypoxia-sensitive Kv2.1, which was confirmed by sequencing and further PCR with primers to the coding region of TASK. However, a reduction in extracellular pH had no effect on K+ current. Thus, although the current more closely resembles TWIK than TASK pharmacologically, structurally the reverse appears to be true. This suggests that a novel acid-insensitive channel related to TASK may be responsible for the hypoxia-sensitive K+ current of these cells.

potassium channels; chemoreceptor; hypoxia; TASK

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

AEROBIC METABOLISM is dependent on an adequate supply of O2, and rapid adaptation to changes in the partial pressures of inspired atmospheric gases is crucial to survival. Changes in arterial PO2, PCO2, and pH are constantly monitored by the carotid and aortic bodies, and perturbation of these parameters, particularly PO2, results in the activation of a high-gain feedback pathway that rapidly restores arterial PO2 to normal levels (10). Put simply, reduced PO2 results sequentially in depolarization of carotid body glomus cells and Ca2+-dependent transmitter release, leading to an increase in carotid sinus nerve activity. This, in turn, causes activation of the medullary respiratory center and a concomitant increase in ventilation (9, 10). Of central importance to this homeostatic mechanism is the efficiency of gaseous exchange in the lung, and for this process to occur optimally, it is imperative that alveolar ventilation and pulmonary blood perfusion rates are matched (VA/Q matching); this is brought about by changes in local vasomotor tone. In contrast to the situation in the systemic circulation, the pulmonary capillary bed responds to decreases in PO2 by constricting (23). Efficiency of gas transfer is maintained, therefore, by ensuring that only well-ventilated areas of the lung are afforded a large blood flow.

In addition to the mechanism of hypoxic vasoconstriction, it has been proposed that specialized cells in the small airways of the lung can sense acute changes in PO2 and regulate both local blood flow, via paracrine peptide transmitter release into the circulation, and ventilation pattern, through activation of ascending neural pathways. The primary candidate for this O2-sensing cell type resides in the neuroepithelial body (NEB) of the lung (24). NEBs are discrete clusters of cells situated throughout the airway and, being localized at the bronchiolar bifurcations, are well positioned to detect PO2 levels in inspired gases (3). Evidence for their role and importance in O2 sensing is incomplete. However, their prominence in the neonatal lung and the association of pathological respiratory conditions (such as apnea of prematurity and sudden infant death syndrome) with NEB cell hyperplasia strongly suggest that NEBs are involved in both the initiation of breathing in the neonate and respiratory control in the adult (4).

In common with carotid body glomus cells, NEB cells contain an array of transmitters (21), including amines and peptides, and are commonly identified by positive staining for serotonin [5-hydroxytryptamine (5-HT)] (17). In the lung, 5-HT may be both a neurotransmitter (activating NEB afferent fibers) and, when released into the pulmonary circulation, a regulator of local vasomotor tone and, therefore, of VA/Q matching (14). There is evidence to indicate that NEB cells release 5-HT in hypoxia (13), and this has prompted investigations into the cellular mechanisms underlying hypoxia-evoked secretion from NEB cells. Using NEB cells isolated from fetal rabbit lungs, Youngson et al. (24, 25) identified voltage-gated Na+, Ca2+, and K+ channels and demonstrated a selective, reversible inhibition of K+ currents by acute hypoxia. These findings provide evidence that NEB cells might respond to acute hypoxia in a manner comparable to that seen in carotid body glomus cells (20). However, there has been a paucity of further information due to the extreme difficulty in preparing isolated NEB cells for such studies: in the fetal lung, NEBs represent 0.2% of perinatal lung tissue mass, and this declines to 0.04% in adult tissue (11).

O'Kelly et al. (18) recently suggested that the small cell lung carcinoma cell line H-146, which is believed to be derived from the same precursor pool as NEB cells (8), represents a suitable model in which to study human airway O2 sensing, principally due to the significant electrophysiological similarities between NEB cells and H-146 cells (24, 25). H-146 cells contain three main voltage-activated current types: 1) a Cd2+-blockable Ca+ current (~20% of the inward current), 2) a tetrodotoxin-sensitive Na+ current (~80% of the inward current), and 3) an outward K+ current. The K+ current is ~85% blocked by a maximally effective concentration of tetraethylammonium (TEA; 10 mM) and contains both Cd2+-sensitive (~40% of total) and -insensitive (~60% of total) components. Furthermore, the Cd2+-resistant K+ current is acutely sensitive to a reduction in PO2 from 150 to 20 mmHg. The inward currents were unaffected by acute hypoxia (18).

With the aim of identifying the O2-sensitive K+ channel in airway NEB cells, we studied the effects of two additional K+-channel blockers on the ability of hypoxia to suppress outward currents in H-146 cells. Furthermore, we investigated further the potential physiological importance of these currents to maintenance of the resting membrane potential. Finally, based on pharmacological profiles of the currents, we screened for candidate K+ channels and demonstrated that mRNA of one member of the recently cloned mammalian twin P domain K+ channels (1, 5-7), with novel functional characteristics, is differentially expressed in this cell line.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell Culture

The small cell lung carcinoma cell line H-146 was purchased from American Type Culture Collection (Manassas, VA) and was of unknown passage number. On delivery, the cells were thawed rapidly at 37°C, diluted 1:12 with RPMI 1640 culture medium (containing L-glutamine) supplemented with 10% fetal calf serum, 2% sodium pyruvate, and 2% penicillin-streptomycin (all from GIBCO, Paisley, UK), and incubated at 37°C for 18 h in a humidified atmosphere of 5% CO2-95% air. After this period, the cells in the suspension culture were removed from the flask, centrifuged at 150 g for 5 min, resuspended in fresh medium, and reseeded in flasks at low density. This point was designated passage 1. Subsequently, the medium was changed every 2 days, and the cells were passaged every 6-7 days by splitting in a 1:5 ratio. Cells were used between passages 1 and 6.

Electrophysiology

Solutions and chemicals. Unless otherwise stated, all chemicals were of the highest grade available and were purchased from Sigma (Poole, UK). The standard pipette solution was K+ rich and contained (in mM) 10 NaCl, 117 KCl, 2 MgSO4, 10 HEPES, 11 EGTA, 1 CaCl2, and 2 Na2ATP, pH 7.2 with KOH; the free Ca2+ concentration was 27 nM. The standard bath solution was Na+-rich and contained (in mM) 135 NaCl, 5 KCl, 1.2 MgCl2, 5 HEPES, 2.5 CaCl2, and 10 D-glucose, pH 7.4 with NaOH. 4-Aminopyridine (4-AP), quinidine, and CdCl2 were added to the bath solution where indicated, and where the final concentration exceeded 1 mM, the osmolarity was maintained by isosmotic substitution of NaCl. A reduction in extracellular pH (pHo) in a CdCl2-containing extracellular solution (from 7.4 to 6.5) was achieved by back-titration with 1 M HCl.

All tubing was gas impermeant (Tygon tubing, BDH, Atherstone, UK) and kept as short as possible. Normoxic solutions were equilibrated with room air. Solutions were made hypoxic, where appropriate, by bubbling with N2 for at least 30 min before perfusion of the cells. This procedure produced no shift in pH. The solution flow rate was ~5 ml/min. In some experiments, PO2 was measured (at the cell) with a polarized (-800-mV), calibrated carbon-fiber electrode (16); PO2 under hypoxic conditions ranged from 15 to 20 mmHg for the experiments reported.

Whole cell recording. After trituration (10 passes through a 1-ml automatic pipette tip), the cells were allowed to adhere to poly-L-lysine-coated glass coverslips for at least 1 h at 37°C before being placed in a temperature-controlled perfusion chamber (Brook Industries, Lake Villa, IL) mounted on the stage of a Nikon TMS inverted microscope. All experiments were carried out at 21 ± 1°C. Patch-clamp pipettes were manufactured from standard-walled borosilicate glass capillary tubing on a two-stage Narishige PP-83 pipette puller (Narishige Scientific Instrument Laboratory, Tokyo, Japan), were routinely heat polished on a Narishige microforge, and had measured tip resistances of 3-8 MOmega (when filled with K+-rich pipette solution).

Resistive-feedback voltage clamp was achieved with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Voltage protocols were generated and currents were recorded with pClamp 6.0.3 software with a Digidata 1200 analog-to-digital converter (Axon Instruments). Data were filtered (4-pole Bessel) at 2 kHz and digitized at 5 kHz. After successful transition to the whole cell recording mode (12), capacitance transients were compensated for and measured. To evoke ionic currents in H-146 cells, two voltage protocols were used: 1) ramp protocol: holding potential -70 mV, -100 mV to +60mV, ramp duration = 1 s, and frequency = 0.1 Hz; and 2) time series: holding potential = -70 mV, single increment to 0 mV, step duration = 50 ms, and frequency = 0.1 Hz.

Currents resulting from the time-series protocols were recorded at two analog channels: one unsubtracted and one on-line subtracted with a P/N protocol (8 prepulses), where P is the pulse amplitude and N is the number of prepulses, each 1/N of P. Where necessary, series resistance compensation was used at 100%.

Fast current clamp was achieved with the same amplifier, and the solutions were the same as those used in the voltage-clamp experiments. The cells were clamped at current (I) = 0 pA, and the recorded voltage was filtered at 1 kHz and digitized at 2 kHz.

RT-PCR. Total RNA was extracted from pelleted cells with the RNeasy Micro Kit (Qiagen, Crawley, UK). The extracted RNA was then divided, with 60% being treated (cleaned) with RQ-1 RNase-free DNase (1 U/µg RNA; Promega, Southampton, UK) to remove genomic DNA contamination before reextraction with the RNeasy Micro Kit as before. The remaining 40% was kept in an untreated (uncleaned) state. The yield, purity, and integrity of the RNA were verified by spectrophotometry at 260/280 nm, followed by electrophoresis on 1% agarose, and was then stored in aqueous solution at -80°C. RT was performed on 1-µg aliquots of both cleaned and uncleaned RNAs with the reverse transcription system A3500 (Promega) composed of avian myeloblastosis virus reverse transcriptase and oligo(dT)15 primers.

The resulting cDNA was amplified by PCR and screened for K+-channel expression with a panel of paired oligonucleotide primers (21-23 mers) specific for the 3' regions of the human delayed rectifier K+ channel Kv2.1 (GenBank accession no. AF026005), human TWIK-1 (GenBank accession no. U33632), human TASK-1 (GenBank accession no. AF006823), and murine TREK-1 (GenBank accession no. U73488), human TREK-1 not being available. All primers were designed against the complete mRNA sequences as published on GenBank, synthesized by Genosys Biotechnologies (Pampisford, UK), and purified to an HPLC standard. PCRs with the cleaned and uncleaned (as positive control) cDNA were run in parallel with reactions using RNA that had not been reverse transcribed (no-RT controls) and negative controls containing nuclease-free water. Reactions were performed with a Hybaid (Ashford, UK) OMN-E thermal cycler in a volume of 50 µl containing 0.8-1.0 units of Taq DNA polymerase (Promega) under conditions optimized for each set of primers (Table 1). All PCR reactions were run as follows: denaturing at 94°C for 2 min, annealing at X°C (see Table 1) for 2 min, and extension at 72°C for 2.5 min for 32 cycles, followed by a final cycle that had a 5-min extension.

                              
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Table 1.   Primers and conditions used

The sequence of TASK-1 is highly repetitive, particularly in the coding region, making primer design and amplification difficult. Thus the first set of primers, used for the initial screening, were located in the 3'-untranslated region. It was then possible to design an upstream primer with one degenerate base (TASK-1C); this disrupted a highly stable primer dimer resulting from a sequence repeat and allowed further PCR in conjunction with the original downstream primer; this PCR was also troublesome because of the high GC content of TASK-1. Therefore, the reaction conditions were optimized by increasing the primer concentration to 400 nM, increasing the MgCl2 concentration to 2 mM, increasing Taq DNA polymerase to 1.5 units/reaction, and adding 1.9 M betaine monohydrate (which acts as an isostabilizing agent for GC and AT base pairs; Sigma). In addition, this PCR was performed with a "hot start" whereby MgCl2 was added when the reaction temperature had reached 94°C (there was a 4-min hold at 94°C, which also helped denature the template). This avoided formation of spurious short products due to nonselective primer-primer or primer-template interactions between 30 and 60°C, where Taq polymerase retains partial activity and which would be preferentially amplified at the expense of the longer template in later cycles. Sequencing was done by dye-terminator PCR with a Perkin-Elmer ABI PRISM automated sequencer by Oswel Sequencing Services (Southampton, UK).

Data Handling and Calculations

The magnitude of the steady-state outward currents (time-series protocol) was measured as the mean current between 46 and 49 ms of the voltage pulse. With the ramp protocol, current was measured at 0 mV. Where indicated, the currents were normalized to a nominal cell area by dividing by measured cell capacitance (to yield current density). In the time-series plots, the currents from each separate experiment were normalized by dividing the current at each point by the mean of the first five currents in that series; in Figs. 1-6, the values are means ± SE; n is the number of cells. Statistical comparisons were made with paired Student's t-test, with P < 0.05 being considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Pharmacological Dissection of the Outward K+ Currents

Outward K+ currents were reversibly inhibited by the K+-channel blockers 4-AP (Fig. 1A) and quinidine (Fig. 1B). Both inhibitors caused a concentration-dependent suppression of the K+ currents (Fig. 1C). Quinidine produced complete blockade at concentrations >=  1 mM, and the concentration-response curve shown in Fig. 1C allowed calculation of the quinidine IC50 as 16.6 ± 4.2 µM (degrees of freedom = 31; Hill coefficient not significantly different from unity; n = 8). In contrast, maximally effective concentrations of 4-AP (>= 10 mM) were unable to block >60% of the current; the IC50 value was calculated as 1.52 ± 0.39 mM (degrees of freedom = 39; Hill coefficient not different from unity; ratio of I to maximal I = 0.40 ± 0.04; n = 12). As expected, the 4-AP-resistant K+ current was completely and reversibly abolished by the addition of 1 mM quinidine (Fig. 2), indicating that a significant component of the K+ current was 4-AP resistant but quinidine sensitive.


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Fig. 1.   Effects of quinidine (Quin) and 4-aminopyridine (4-AP) on outward K+ currents of H-146 cells. A: representative currents evoked by 50-ms step depolarizations from a holding potential of -70 to 0 mV (time-series protocol) before (control), during, and after (wash) treatment with 4-AP as indicated. B: representative currents evoked by ramp protocol before, during, and after treatment with Quin. C: concentration dependence of effects of 4-AP and Quin. [Inhibitor], inhibitor concentration; I/Imax, ratio of current to maximal current. Values are means ± SE. IC50 values were calculated by iterative fitting with Hill equation. Quin IC50 = 16.6 ± 4.2 µM (degrees of freedom = 31; Hill coefficient not significantly different from unity; n = 8 cells). 4-AP IC50 = 1.52 ± 0.39 mM (degrees of freedom = 39; Hill coefficient not different from unity; I/Imax = 0.40 ± 0.04; n = 12 cells).


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Fig. 2.   Effect of cumulative addition of 4-AP and Quin on outward K+ currents. A: exemplar currents evoked (employing time-course protocol) in a cell before, during, and after sequential and cumulative bath application of 4-AP and 4-AP+Quin as indicated. B: time-series plot of cumulative blockade of sustained outward K+ currents evoked in a cell by repeated step depolarizations from -70 to 0 mV (time-series protocol). Solid bars, periods of application of blockers. Currents were normalized for each cell by dividing each current by mean of 1st 5 currents recorded before population mean was calculated.

Effect of Hypoxia

O'Kelly et al. (18) previously demonstrated that reducing the bath PO2 from 150 to 20 mmHg causes a reversible suppression of outward K+ currents in H-146 cells of ~30%. To investigate the possibility that this hypoxia-sensitive current is 4-AP sensitive, we perfused the cells with a maximal inhibitory concentration of the inhibitor and then the reduced bath PO2 in its continued presence. As shown in Fig. 3, a maximally effective concentration of 4-AP failed to abrogate significantly the rapid inhibitory effect of hypoxia on the K+ currents. Ten millimolar 4-AP caused a significant 56.4 ± 4.9% reduction in the total current density (from 58.8 ± 8.5 to 27.3 ± 5.8 pA/pF, measured at 0 mV; P < 0.001; n = 8). Hypoxia caused a further reduction in current density to 13.2 ± 2.4 pA/pF (P < 0.005). Because this reduction in absolute current density is similar to the value that O'Kelly et al. previously reported, it appears that the hypoxia-sensitive current in H-146 cells is quinidine sensitive but 4-AP resistant. It follows, therefore, that the effect of hypoxia in the presence of 4-AP is proportionally larger than the hypoxic inhibition in its absence. Hypoxia was unable to cause any further reduction in the presence 1 mM quinidine (data not shown).


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Fig. 3.   Effect of 4-AP on response of K+ currents to hypoxia. A: representative currents evoked (employing time-course protocol) in a cell after reduction of PO2 to 20 mmHg in presence of 4-AP. Currents evoked before and after washout of 4-AP are also shown. B: time-series plot of cumulative blockade of sustained outward K+ currents by 4-AP and hypoxia (n = 8 cells). Solid bars, periods of application. Currents were normalized as in Fig. 2.

Effects on Membrane Potential

The role of the hypoxia-sensitive current in H-146 cells was recently demonstrated (18). Because hypoxia causes a significant membrane depolarization (even in the presence of TEA), it is clear that this current is active at, and contributes to, the resting membrane potential. Figure 4 reinforces this idea. Under current-clamp conditions, 10 mM 4-AP significantly depolarized the cells from -57.8 ± 4.0 to -40.3 ± 5.1 mV (P < 0.0001; n = 8). Reduction of the PO2 to 20 mmHg in the continued presence of 4-AP caused a further significant depolarization of 4.1 ± 1.0 mV (P < 0.01; n = 8). On return to normoxia, in the continued presence of 4-AP, the membrane potential was restored to -39.7 ± 5.0 mV (e.g., Fig. 4A). This hypoxia-induced mild depolarization was not significantly different from the value that O'Kelly et al. (18) previously reported in the absence of pharmacological blockade (i.e., 5.8 ± 0.53 mV; P > 0.13 by unpaired t-test). Figure 4, B and C, demonstrates the effect of quinidine addition on membrane potential in H-146 cells. One millimolar quinidine almost completely collapsed the membrane potential (from -49.5 ± 4.8 to -4.8 ± 4.3 mV; P < 0.0001; n = 7), and its effect was concentration dependent (Fig. 4B). Furthermore, consistent with the voltage-clamp records described above, 1 mM quinidine abolished the hypoxic response (membrane potential -4.8 ± 3.3 mV; Fig. 4C).


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Fig. 4.   Effect of 4-AP, Quin, and hypoxia on membrane potential. A: representative current-clamp (I = 0 pA) recording of membrane potential during exposure to hypoxia in presence of 4-AP. Solid bars, periods of application. B: cumulative concentration-dependent membrane depolarization by Quin. Arrows, dose application; [Quinidine], Quin concentration. C: abbrogation of hypoxia-evoked membrane depolarization by pretreatment with Quin. Solid bars, periods of application.

RT-PCR Screening of Candidate Hypoxia-Sensitive K+ Channels

The pharmacological and electrophysiological evidence presented above suggested that the hypoxia-sensitive K+ channel in these cells may belong to the family of twin P domain leak K+ channels recently cloned from mammalian tissues. We employed RT-PCR to screen rapidly for the expression of mRNA encoding three members of this gene family: TASK (5), TREK (1), and TWIK (6). In addition, we also probed for expression of Kv2.1 because it has been reported that this channel is sensitive to a chronic reduction in PO2 in pulmonary vascular myocytes (19). Figure 5A shows a representative agarose gel of PCR products amplified from primer pairs designed to TWIK and TASK. Products of the predicted size of both TWIK and TASK could be amplified from uncleaned cDNA samples (used as a positive control). However, in samples that contained no genomic DNA contamination, only TASK could be amplified, suggesting that this channel is differentially expressed in H-146 cells. Sequence analysis showed that there was 100% base identity between our amplified fragment and the published sequence of TASK (Fig. 5D). Neither TREK nor Kv2.1 could be amplified from cleaned H-146 cDNA samples, although Kv2.1 could be amplified from cDNA untreated with DNase (Fig. 5C). In addition, we have used a further set of primers, designed to amplify a much larger portion of TASK (extending from the 3'-untranslated region to 385 bp in the open reading frame). This PCR amplified two products: one was of the expected 1,546-bp length, but the other was ~300 bp shorter (Fig. 5B). The shorter product could not be eliminated or reduced by increasing the annealing temperature, suggesting strongly that it represents a splice variant of TASK.


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Fig. 5.   RT-PCR screening for candidate O2-sensitive K+ channels. A: 2% agarose gel electrophoresis stained with ethidium bromide after PCR with TWIK and TASK primer sets with cDNA reverse transcribed from cleaned and uncleaned H-146 cell RNA. No RT, control with RNA that was not reverse transcribed; H2O, nuclease-free water control. Sizes of these PCR products corresponded with those predicted from published mRNA sequences of TWIK-1 and TASK (178 and 157 bp, respectively; arrows). TASK mRNA is differentially expressed in these cells. Nos. at left, DNA standards in bp. B: TASK-1C primer set with cDNA reverse transcribed from cleaned H-146 cell mRNA diluted as indicated (before PCR) and reamplified by turbo PCR with the same primer set. Amplification was seen only at 50-fold template dilution, with discrete bands corresponding to a product of the predicted size (1,546 bp) and to another ~300 bp shorter (arrows). C: Kv2.1 primer set with cDNA reverse transcribed from cleaned and uncleaned H-146 cell mRNAs. PCR product was of correct size (403 bp; arrow) predicted from location of priming sites within published mRNA sequence but could not be amplified in cleaned sample. D: alignment of forward and reverse sequencing products, with the region of published mRNA sequence corresponding to predicted TASK PCR product. Overlap of the 2 products gives 100% agreement between expected and observed sequences.

Effect of Change in pHo on Outward K+ Currents

Having established that the acid-sensitive TASK was differentially expressed, we studied the effects of a change in pHo on the outward K+ currents (Fig. 6). To increase the signal-to-noise ratio, currents were recorded after blockade of Ca2+ influx (and, therefore, Ca2+-activated K+-channel activity) with 200 µM CdCl2. Cd2+ caused a rapid inhibition of K+ currents by 35.1 ± 5.3% [from 74.5 ± 12.2 to 48.0 ± 8.4 pA/pF; P < 0.005; n = 6; consistent with previous data of O'Kelly et al. (18)]. The reduction in pHo from 7.4 to 6.5 did not significantly suppress the currents further. Mean current density at pHo 6.5 was 48.2 ± 9.1 pA/pF (P > 0.9). Furthermore, raising the pHo back to 7.4 caused no significant change in current density (51.7 ± 8.81 pA/pF; P > 0.15), but an almost complete recovery to 68.7 ± 10.41 pA/pF was achieved on removal of Cd2+ (P < 0.02). These data demonstrate that the Cd2+-resistant K+ channels in H-146 cells are not pHo sensitive.


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Fig. 6.   Effect of changing extracellular pH (pHo) on Cd2+-resistant outward K+ currents. A: representative Cd2+-resistant K+ currents evoked (employing time-course protocol) in a cell after reduction of pHo from 7.4 to 6.5. Currents evoked before and after washout of Cd2+ are also shown. B: time-series plot showing that reduction in pHo from 7.4 to 6.5 had no effect on Cd2+-resistant K+ currents. Solid bars, periods of application. Currents were normalized as in Fig. 2.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The previous study by O'Kelly et al. (18) established H-146 small cell lung carcinoma cells as a suitable model system for studying O2 sensing by airway chemoreceptor NEB cells. They demonstrated that H-146 cells possess outward K+ currents and that a component of these currents was sensitive to inhibition by acute hypoxia. Furthermore, the O2-sensitive K+ channels were found to contribute to the resting membrane potential, and their inhibition by hypoxia led to membrane depolarization. The establishment of such a model system for NEB cells is of particular importance because NEBs represent only a tiny fraction [0.04% in adult lung (11)] of lung mass and are therefore extremely difficult to isolate to conduct the type of investigations reported herein.

In this present study, we have further characterized the O2-sensitive component of the whole cell K+ current of H-146 cells, and we provide some evidence as to its molecular identity. Our electrophysiological studies revealed that whole cell K+ currents could be fully inhibited by quinidine but that a component of the K+ current was resistant to 4-AP, even at extremely high concentrations (10-100 mM; Fig. 1). Importantly, hypoxic inhibition of K+ currents was still apparent in the presence of 10 mM 4-AP (Fig. 3), and hypoxia was able to depolarize H-146 cells in the presence of this concentration of 4-AP (Fig. 4A). In contrast, quinidine was able to evoke a far greater depolarization in these cells (Fig. 4B), and in the presence of a near-maximal concentration of quinidine (1 mM), hypoxia was unable to depolarize the cells further (Fig. 4C). These findings led to the conclusion that the O2-sensitive component of the whole cell K+ current in H-146 cells is quinidine sensitive but 4-AP resistant. Furthermore, although O2- and quinidine-sensitive K+ channels contribute to the resting membrane potential of H-146 cells, they are not the only channels to do so because 4-AP could also cause membrane depolarization (although it should be noted that even at a concentration of 10 mM, the degree of depolarization was far less than that caused by quinidine; Fig. 4). Thus, together with the previous finding by O'Kelly et al. (18) that the O2-sensitive K+ current in H-146 cells is weakly sensitive to blockade by TEA, we have now established a pharmacological profile of this important K+ current in H-146 cells.

In a series of elegant studies, Arrighi et al. (1), Duprat et al. (5), and Fink and colleagues (6, 7) recently described a novel family of K+ channels that are widely distributed in mammalian tissues. Although the members of this family show marked variations in their amino acid sequences, they share common novel features. The most distinctive feature of these channels is that they possess two P domains (which are thought to form the ion-conducting pore lining of the channel) within a single subunit, and the subunits are likely to form functional channels by dimerization (15). This distinct structural feature has led to these channels being termed TWIK (tandem of P domains in a weak inward rectifying K+ channel) or TWIK-related channels. These channels are time and voltage insensitive and only weakly (if at all) rectifying. Their lack of voltage sensitivity and wide distribution suggests that these channels may exert important influences on the resting membrane potential of a wide variety of cell types [including the carotid body glomus cell (2)]. Because O'Kelly et al. (18) previously showed that the O2-sensitive K+ current in H-146 cells influences the resting membrane potential, we compared the pharmacological profile of this current (i.e., quinidine sensitivity, weak TEA sensitivity, and insensitivity to 4-AP) with those reported for the various members of the twin P domain class of K+ channel. Clearly, the effects of the three blocking agents tested pointed to the possibility that this channel resembled TWIK, the first member of this family of channels to be described.

To investigate the possible identity of the O2-sensitive K+ current in H-146 cells further, we used RT-PCR with primers to the 3'-untranslated regions of TWIK-1 and TASK (TWIK-related acid-sensitive K+ channel), another TWIK-related channel that uniquely shows sensitivity to changes in pHo (5); these members of the TWIK-related family of channels are the only ones from human tissue with published sequences. RT-PCR of genomic DNA amplified products of the correct size with both primer sets, but RT-PCR of purified mRNA amplified only the TASK product (Fig. 5A). Sequencing of this product confirmed its identity as TASK (Fig. 5D). These findings pointed to the possibility that the O2-sensitive K+ current in H-146 cells was closely related, or even identical, to the TASK channel. If this were the case, then it would be anticipated that a reduction in pHo would lead to inhibition of this current. However, as indicated in Fig. 6, this was clearly not the case. The experimental protocol used in this part of the study was designed to avoid potential artifacts; ideally, we would have examined the effects of reduced pHo in the presence of 4-AP (thereby isolating the O2-sensitive K+ current). However, 4-AP is a weak base, and its ability to block K+ channels is therefore strongly dependent on pH (22) because it enters the cells in the uncharged form to block K+ channels from the cytosolic face. Indeed, as predicted, we found that currents recorded in the presence of 10 mM 4-AP were actually enhanced by a reduction in pHo (data not shown). To avoid the influence of pHo on the ability of 4-AP to block a component of the whole cell K+ current in H-146 cells, we instead chose to investigate the effects of external acidification in the presence of Cd2+. The previous study by O'Kelly et al. (18) demonstrated that by blocking Ca2+ influx into H-146 cells, Cd2+ reduced a Ca2+-dependent component of the whole cell K+ current. This component was not O2 sensitive, and so, by recording K+ currents in the presence of Cd2+, we were able to enhance the fractional inhibition of residual (Cd2+-insensitive) K+ currents by hypoxia. Thus, if the O2-sensitive K+ current in H-146 cells was a TASK current, the effects of external acidification would be expected to be enhanced in the presence of Cd2+. Clearly, this was not the case (Fig. 6).

The results of the present study, therefore, suggest that the O2-sensitive K+ current in H-146 cells resembles TWIK-1 in terms of its pharmacological profile and ability to influence membrane potential. Our finding that this current in H-146 cells was highly sensitive to blockade by quinidine argues against it being TASK (5). However, our PCR studies showed that, although we could amplify both TWIK-1 and TASK from H-146 cell genomic DNA, we could only amplify TASK from purified mRNA, suggesting that H-146 cells express TASK but not TWIK-1 channels. We cannot at present account for this apparent discrepancy, but it is noteworthy that knowledge of this family of TWIK-related channels is in its infancy, and, indeed, a most recent report (7) has identified another member (TRAAK), which is found in two splice variant forms. It is therefore possible that we have identified an O2-sensitive K+ channel that is structurally very similar to TASK but does not display the sensitivity to pHo reported for this cloned channel when expressed in Xenopus oocytes or COS cells. If the acid sensitivity of TASK is conferred by regulatory subunits, it is possible that these are not expressed in H-146 cells. Another possible explanation for our findings may be that acid sensitivity is dependent on the level of channel expression. Alternatively, the channel may indeed be coded for by the TWIK-1 gene, but its steady-state mRNA levels are too low to detect with our PCR conditions because, perhaps, of low stability. In support of the notion that the O2-sensitive channel may be related closely to TASK, Fig. 5B shows that H-146 cells express the mRNA of a potential splice variant. This information presents the intriguing possibility that H-146 cells differentially express this shorter splice variant and that this posttranscriptional event confers novel functional characteristics on the expressed protein.

    ACKNOWLEDGEMENTS

We thank The Anonymous Trust for partial financial support of this work. We are indebted to Christine Jones and Malcolm Hunter for invaluable advice on the molecular component of this study and Susan Shaw for technical assistance.

    FOOTNOTES

I. O'Kelly holds a University of Leeds Research Scholarship. C. Peers is a British Heart Foundation Senior Lecturer.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: P. J. Kemp, School of Biomedical Sciences, Worsley Medical and Dental Bldg., University of Leeds, Leeds LS2 9JT, UK.

Received 6 August 1998; accepted in final form 5 October 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Arrighi, I., F. Lesage, J. C. Scimeca, G. F. Carle, and J. Barhanin. Structure, chromosome localization, and tissue distribution of the mouse TWIK K+ channel gene. FEBS Lett. 425: 310-316, 1998[Medline].

2.   Buckler, K. J. A novel oxygen-sensitive potassium current in rat carotid body type I cells. J. Physiol. (Lond.) 498: 649-662, 1997[Abstract].

3.   Cutz, E. Introduction to pulmonary neuroendocrine cell system, structure-function correlations. Microsc. Res. Tech. 37: 1-3, 1997[Medline].

4.   Cutz, E., J. E. Gillan, and N. S. Track. Pulmonary endocrine cells in the developing human lung and during neonatal adaptation. In: The Endocrine Lung in Health and Disease, edited by K. L. Becker, and A. F. Gazdar. Philadelphia, PA: Saunders, 1984, p. 210-231.

5.   Duprat, F., F. Lesage, M. Fink, R. Reyes, C. Heurteaux, and M. Lazdunski. TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J. 16: 5464-5471, 1997[Abstract/Free Full Text].

6.   Fink, M., F. Duprat, F. Lesage, R. Reyes, G. Romey, C. Heurteaux, and M. Lazdunski. Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel. EMBO J. 15: 6854-6862, 1996[Abstract].

7.   Fink, M., F. Lesage, F. Duprat, C. Heurteaux, R. Reyes, M. Fosset, and M. Lazdunski. A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsatuarated fatty acids. EMBO J. 17: 3297-3308, 1998[Abstract/Free Full Text].

8.   Gazdar, A. F., L. J. Helman, M. A. Israel, E. K. Russel, R. I. Linnoila, J. L. Mulshine, H. M. Sculler, and J. G. Park. Expression of neuro-endocrine cell markers L-Dopa decarboxylase, chromogranin-A, and dense core granules in human tumors of endocrine and nonendocrine origin. Cancer Res. 48: 4078-4082, 1988[Abstract].

9.   Gonzalez, C., L. Almarez, A. Obeso, and R. Rigual. Oxygen and acid chemoreception in the carotid body chemoreceptors. Trends Neurosci. 15: 146-153, 1992[Medline].

10.   Gonzalez, C., L. Almarez, A. Obeso, and R. Rigual. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol. Rev. 74: 829-898, 1994[Free Full Text].

11.   Gosney, J. R. Pulmonary neuroendocrine cell system in pediatric and adult lung disease. Microsc. Res. Tech. 37: 107-113, 1997[Medline].

12.   Hamill, O. P., A. Marty, E. Neher, B. Sackmann, and F. J. Sigworth. Improved patch-clamp techniques for high resolution recordings from cells and cell-free membrane patches. Pflügers Arch. 391: 85-100, 1981[Medline].

13.   Lauweryns, J. M., and M. Cokeleare. Hypoxia sensitive neuroepithelial bodies intrapulmonary secretory neuroreceptors, modulated by CNS. Z. Zellforsch. Mikrosk. Anat. 145: 521-540, 1973[Medline].

14.   Leach, R. M., C. H. C. Twort, I. R. Cameron, and J. P. T. Ward. A comparison of the pharmacological and mechanical properties in vitro of large and small pulmonary arteries of the rat. Clin. Sci. (Colch.) 82: 55-62, 1992[Medline].

15.   Lesage, F., R. Reyes, M. Fink, F. Duprat, E. Guillemare, and M. Lazdunski. Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge. EMBO J. 15: 6400-6407, 1996[Medline].

16.   Mojet, M. H., E. Mills, and M. R. Duchen. Hypoxia-induced catecholamine secretion in isolated newborn rat adrenal chromaffin cells is mimicked by inhibition of mitochondrial respiration. J. Physiol. (Lond.) 504: 175-189, 1997[Abstract].

17.   Newman, C., D. Wang, and E. Cutz. Serotonin (5-hydroxytryptamine) expression in pulmonary neuroendocrine cells (NE) and NE tumor cell line. In: Neurobiology and Cell Physiology of Chemoreception, edited by P. G. Data. New York: Plenum, 1993, p. 73-77.

18.   O'Kelly, I., C. Peers, and P. J. Kemp. O2-sensitive K+ channels in neuroepithelial body-derived small cell carcinoma cells of the human lung. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L709-L716, 1998[Abstract/Free Full Text].

19.   Patel, A. J., M. Lazdunski, and E. Honore. Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes. EMBO J. 16: 6615-6625, 1997[Abstract/Free Full Text].

20.   Peers, C., and K. J. Buckler. Transduction of chemostimuli by the type I carotid body cell. J. Membr. Biol. 144: 1-9, 1995[Medline].

21.   Speirs, V., E. Bienkowski, V. Wong, and E. Cutz. Paracrine effects of bombesin/gastrin-releasing peptide and other growth factors on pulmonary neuroendocrine cells in vitro. Anat. Rec. 236: 53-61, 1993[Medline].

22.   Stephens, G. J., J. C. Garratt, B. Robertson, and D. G. Owen. On the mechanism of 4-aminopyridine action on the cloned mouse-brain potassium channel mKv1.1. J. Physiol. (Lond.) 477: 187-196, 1994[Abstract].

23.   Weir, E. K., and S. L. Archer. The mechanism of acute hypoxic pulmonary vasoconstriction---the tale of 2 channels. FASEB J. 9: 183-189, 1995[Abstract/Free Full Text].

24.   Youngson, C., C. Nurse, H. Yeger, and E. Cutz. Oxygen sensing in airway chemoreceptors. Nature 365: 153-155, 1993[Medline].

25.   Youngson, C., C. Nurse, H. Yeger, and E. Cutz. Characterization of membrane currents in pulmonary neuroepithelial bodies: hypoxia-sensitive airway chemoreceptors. In: Arterial Chemoreceptors. Cell to System, edited by R. G. O'Regan, P. Nolan, D. S. McQueen, and D. J. Paterson. New York: Plenum, 1994, p. 179-182.


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