Respiration Physiology 115 (1999) 215–227

K+ and Ca2+ channel activity and cytosolic [Ca2+] inoxygen-sensing tissues

J. Lopez-Barneo *, R. Pardal, R.J. Montoro, T. Smani, J. Garcıa-Hirschfeld,J. Urena

Departamento de Fisiologıa Medica y Biofısica, Uni6ersidad de Se6illa, Facultad de Medicina, A6enida Sanchez Pizjuan 4,E-41009 Se6illa, Spain

Accepted 1 February 1999

Abstract

Ion channels are known to participate in the secretory or mechanical responses of chemoreceptor cells to changesin oxygen tension (PO2

). We review here the modifications of K+ and Ca2+ channel activity and the resulting changesin cytosolic [Ca2+] induced by low PO2

in glomus cells and arterial smooth muscle which are well known examplesof O2-sensitive cells. Glomus cells of the carotid body behave as presynaptic-like elements where hypoxia produces areduction of K+ conductance leading to enhanced membrane excitability, Ca2+ entry and release of dopamine andother neurotransmitters. In arterial myocytes, hypoxia can inhibit or potentiate Ca2+ channel activity, thus regulatingcytosolic [Ca2+] and contraction. Ca2+ channel inhibition is observed in systemic myocytes and most conduitpulmonary myocytes, whereas potentiation is seen in a population of resistance pulmonary myocytes. The mechanismwhereby O2 modulates ion channel activity could depend on either the direct allosteric modulation by O2-sensingmolecules or redox modification by reactive chemical species. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Carotid body, ion channels; Control of breathing, carotid body; Ion channels, K+, Ca2+; Smooth muscle,vascular, ion channels

1. Introduction

Oxygen-sensing is a general phenomenon thatallows cells to adapt to stressing hypoxic environ-ments. In most cell types, protracted hypoxialeads to alteration of metabolic function and the

* Corresponding author. Tel.: +34-954-389299; Fax: +34-954-551769.

E-mail address: lbarneo@cica.es (J. Lopez-Barneo)

0034-5687/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

PII: S 0034 -5687 (99 )00016 -X

J. Lopez-Barneo et al. / Respiration Physiology 115 (1999) 215–227216

expression of genes encoding enzymes, hormonesand growth factors (for a review see Bunn andPoyton, 1996). However, there are cells able torespond to decrements in O2 tension (PO2

) in thecourse of seconds or minutes with changes in theirexcitability, contractility or secretory activity.These fast-responsive, O2-sensitive cells are nor-mally located in chemoreceptor organs (i.e. thecarotid, aortic and neuroepithelial cell bodies,neonatal adrenal medulla or vascular smoothmuscle) that participate in cardiovascular andventilatory control (for reviews see Lopez-Barneoand Weir, 1998). Besides their physiological sig-nificance, O2-sensitive chemoreceptors have also agrowing clinical interest because they appear to beinvolved in the pathogenesis of several diseasessuch as sleep apnea, sudden infant death syn-drome or pulmonary hypertension (Cutz et al.,1997; Weir et al., 1998). Over the past decade ithas become well established that modulation ofion channel activity by changes in PO2

participatesin the secretory or mechanical responses ofchemoreceptor cells to low PO2

(see Lopez-Barneo, 1996; Lopez-Barneo et al., 1998). O2-reg-ulated K+ channels, initially observed in glomuscells of the carotid body, are present in a varietyof tissues including pulmonary vascular smoothmuscle, neuroepithelial cells in the airways, chro-maffin cells and central neurons. Recent work hasshown that in vascular myocytes or neurons Ca2+

channel activity can be under control of PO2, and

there are reports indicating that in some prepara-tions Na+ and Cl− channels are also altered byhypoxia. In this article we briefly describe theproperties of K+ and Ca2+ channels in O2-sens-ing tissues, emphasizing the work done in ourlaboratory on carotid body and vascular smoothmuscle cells as representative examples of O2-sen-sitive neurosecretory and contractile systems,respectively.

2. Modulation of K+ channels by O2 tension

The first clues suggesting the existence of ionchannels regulated by O2 tension came from workin dispersed glomus cells of the carotid bodyshowing that, at least in rabbits, they are electri-

cally excitable (Duchen et al., 1988; Lopez-Barneoet al., 1988) and have a K+ current inhibited byhypoxia (Lopez-Barneo et al., 1988; Delpiano andHescheler, 1989; Peers, 1990; Stea and Nurse,1991; Chou and Shirahata, 1996; for additionalreferences see Gonzalez et al., 1994; Lopez-Barneo et al., 1998). An example of O2-sensitiveK+ current is illustrated in Fig. 1A with record-ings from a patch-clamped rabbit glomus cellduring depolarizing pulses to +20 mV and ex-posed to normoxic (PO2

:150 Torr) and hypoxic(PO2:20 Torr) solutions. In this experiment the

inward current (mainly carried by Ca2+ ions) wasunaffected by hypoxia but the outward K+ cur-rent amplitude was reduced by about 25% onexposure to low PO2

. These observations werefollowed by the biophysical characterization ofthe various types of voltage-gated K+ channelsexisting in rabbit glomus cells and the identifica-tion of O2-sensitive K+ channels, expressed witha density of :1000–2000 per cell, as the majorcontributor to the whole-cell K+ current (Gan-fornina and Lopez-Barneo, 1991, 1992). As shown

Fig. 1. Major O2-dependent electrophysiological properties ofrabbit glomus cells. (A) Macroscopic inward and outwardcurrents of a glomus cell and reversible inhibition of theoutward current by hypoxia (PO2

:20 Torr). Control andrecovery traces in normoxia (PO2

:150 Torr) are shown super-imposed. Tetrodotoxin was added to the external solution toblock Na+ conductance. (B) Single-channel recordings froman excised membrane patch containing at most one openO2-sensitive K+ channel. Depolarizing pulses applied from−80 to +20 mV. Ensemble averages indicating the single-channel open probability in normoxia and hypoxia are from15 and 22 successive recordings in the two experimental condi-tions. (Modified from Ganfornina and Lopez-Barneo, 1992;Montoro et al., 1996.)

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in Fig. 1B, hypoxia decreases single-channel openprobability (between 20 and 40%) but leaves unal-tered the single-channel conductance (about 20 pSin physiological extra and intracellular K+ con-centrations). Although inhibition of K+ conduc-tance by hypoxia is a common feature of glomuscells, the type of K+ channel regulated by O2

varies in the different mammalian species andexperimental conditions. In cat glomus cells theO2-sensitive K+ current has some characteristicssimilar to the current in the rabbit (Chou andShirahata, 1996) but in neonatal and adult ratsCa2+-dependent, maxi-K+ channels are the onesregulated by low PO2

(Peers, 1990; Lopez-Lopez etal., 1997). It seems, however, that the type of ratK+ channel influenced by hypoxia changes ifanimals are reared in low PO2

environments (Wy-att et al., 1995). The O2-sensitive channel retainssimilar activation and inactivation characteristicsbut looses its sensitivity to charybdotoxin and nolonger contributes to resting potential. Macro-scopic K+ currents similar to those of the carotidbody are reduced by hypoxia in neuroepithelialcells of the lung airways (Youngson et al., 1993)as well as in adrenomedullary (Thompson et al.,1997) and PC12 (Zhu et al., 1996) cells. In this cellline the expression of Kv1.2 channels is up-regu-lated by low PO2

and these channels appear to bethose O2-sensitive (Conforti and Millhorn, 1997).Besides the voltage-dependent O2-sensitive K+

channels, it has been reported in rat glomus cellsa leak K+ conductance which seems to mediatethe depolarizing receptor potential triggered bylow PO2

(Buckler, 1997). We have observed insome rabbit glomus cells exposed to hypoxiachanges in the holding current compatible withinhibition of an O2-sensitive leak K+ currentsimilar to that in the rat. However in other cellsthe holding current is either unaffected by hy-poxia or varies in the opposite direction. Interest-ingly, in some of the cells exhibiting O2-dependentchanges in the holding current the voltage-depen-dent K+ currents are unaltered by low PO2

. Hy-poxic inhibition of voltage-dependent K+

channels favours the increase of action potentialfiring frequency in spontaneously active rabbitglomus cells (Montoro et al., 1996), but in quies-

cent cells (such as those in the rat carotid bodylacking Na+ channels and with reduced ability ofspontaneous pacemaking activity) the leak O2-sensitive channels may have an important role inthe initiation of the response to low PO2

.Regardless of the K+ channel type modulated

by PO2, the consequence of exposure to hypoxia in

glomus and chromaffin-like cells is the reductionof K+ conductance and an increase of cellularexcitability leading to the opening of Ca2+ chan-nels, Ca2+ influx and elevation of cytosolic [Ca2+](Lopez-Barneo et al., 1993; Buckler and Vaughan-Jones, 1994; Urena et al., 1994; Zhu et al., 1996;Zhong et al., 1997). The relationship betweencytosolic [Ca2+] and ambient PO2

in rabbit glo-mus cells is illustrated in Fig. 2. Application ofgraded hypoxia to the cells results in parallelelevations of cytosolic [Ca2+] (Fig. 2A). Fig. 2Bshows that the relation between cytosolic Ca2+

and PO2follows an hyperbolic function (see be-

low). Fig. 2C and D demonstrate that suppressionof Ca2+ currents by removal of external Ca2+ orblockade of the channels with Cd2+, reversiblyabolishes the rise of cytosolic Ca2+ evoked bylow PO2

. Since glomus cells were known to con-tain large amounts of dopamine and other trans-mitters (see Fidone and Gonzalez, 1986) we havecharacterized the neurosecretory response to hy-poxia in single cells by the amperometric detec-tion of the released oxidizable substances (Urenaet al., 1994). Application of a polarized carbonfiber near the surface of a glomus cells permits theidentification of secretory spikes representing dis-crete events due to release of single vesicles (Fig.3, top). Typical exocytotic responses to hypoxiaare shown in Fig. 3 (bottom), where it is alsoillustrated that the low PO2

-induced transmitterrelease requires extracellular Ca2+. Thus, glomuscells behave as O2-sensitive presynaptic-like ele-ments with a secretory response to hypoxia that isalmost absolutely dependent on the influx of ex-tracellular Ca2+. The resemblance between theO2-sensitive responses at the cellular and organlevels indicates that the neurosecretory propertiesof single glomus cells are major contributors tothe chemosensory function of the carotid body.For example, both in single cells and in wholecarotid bodies, the hypoxic response is blocked by

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Fig. 2. Rise of cytosolic Ca2+ in glomus cells in response to low PO2. (A) Gradual elevations of cytosolic [Ca2+] in a fura-2-loaded

cell in response to various levels of hypoxia. The signal from an O2-sensitive electrode is shown at the top. (B) Relationship betweencytosolic [Ca2+] and PO2

in several rabbit glomus cells. (C, D) Abolishment of the hypoxia-induced rise of cytosolic Ca2+ afterremoval of extracellular Ca2+ or blockade of Ca2+ channels with 0.2 mM Cd2+. (Modified from Urena et al., 1994; Montoro etal., 1996.)

Ca2+ channel antagonists. In addition, the hyper-bolic relationship between cytosolic Ca2+ orsecretory rate and PO2

in single cells is almostsuperimposable to the curve relating the changesin afferent sensory discharges as a function of O2

tension in either isolated or in situ carotid bodies.However, whether or not dopamine release isabsolutely required for the chemosensory functionof the carotid body is still open to discussion sincethe nature of the transmitter that activates theafferent fibers of the sinus nerve is unknown. Theactual role of dopamine might be autocrine, asfeedback regulator of glomus cell secretory activ-ity, rather than that of a direct transmittermolecule. It is known that exogenous dopamineinhibits the hypoxic chemosensory discharges inafferent nerve fibers (Donnelly et al., 1981) andwe have shown that in dispersed glomus cellsapplication of dopamine can selectively reduce theamplitude of Ca2+ currents (Benot and Lopez-Barneo, 1990).

O2-sensitive K+ channels have been also stud-ied in some central neurons (Jiang and Haddad,1994) and, in more detail, in smooth muscle cellsof the pulmonary arterial tree (Post et al., 1992;Yuan et al., 1993; Osipenko et al., 1997). Inmyocytes dispersed from resistance pulmonaryvessels inhibition of voltage-dependent K+ chan-nels by low PO2

causes membrane depolarization,opening of L-type Ca2+ channels and contrac-tion. These phenomena contribute to explain hy-poxic pulmonary vasoconstriction, a responsespecific of vessels in the pulmonary circulationwhich helps to maintain high resistance in fetallung and in the adult contributes to the matchingof ventilation and perfusion by diverting blood tothe better ventilated alveoli (see Weir et al., 1998).As it occurs in the neurosecretory systems, thetype of O2-sensitive K+ channel in the resistancevessels of the pulmonary circulation might changewith age or animal species. In sheep fetuses theO2-sensitive channel has characteristics of the

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Ca2+-dependent maxi-K+ channels (Cornfieldet al., 1996) but in young and adult animals thechannel modulated by hypoxia is of the delayedrectifier type (Yuan et al., 1993; Archer et al.,1996). The molecular nature of the O2-regulatedK+ channel in vascular myocytes is unknownalthough it has been shown that chronic hy-poxia inhibits selectively the expression of Kv1.2and Kv1.5 channels in pulmonary vascularsmooth muscle cells (Wang et al., 1997). Inmost O2-sensitive cells studied so far the K+

conductance is reduced by hypoxia but the op-posite effect (potentiation of K+ current by lowPO2

) has been also described. In sheep ductusarteriosus there are K+ channels potentiated bylow PO2

which are inhibited in normoxic condi-tions. Reduction of the K+ current after birthmay contribute to the closure of the ductus thuspreventing in the newborn the mixture of oxy-genated and deoxygenated blood (Tristani-Firouzi et al., 1996).

3. Ca2+ channel activity and cellular responses tohypoxia

As discussed in the previous section, it is well-established that changes in transmembrane Ca2+

influx are ultimately necessary for the secretory ormechanical responses to hypoxia in O2-sensitivetissues. These modifications appear to result fromprimary alterations in the function of O2-sensitiveK+ channels which regulate membrane potentialand, thus, the activity of voltage-dependent Ca2+

channels. However, there are recent indicationssuggesting that Ca2+ channels may be also di-rectly influenced by changes in O2 tension(Franco-Obregon et al., 1995; Franco-Obregonand Lopez-Barneo, 1996; Soloviev et al., 1996;Miranov and Richter, 1998). Our initial experi-mental observations suggesting the modulation ofCa2+ channels by O2 tension were done in dis-persed myocytes from systemic (femoral, celiacand mesenteric) and coronary arteries. These

Fig. 3. Secretory response of a single glomus cell to low PO2. As indicated by the diagram at the top, dopamine release was

monitored by amperometry with an 8 mm polarized (to +750 mV) carbon fiber electrode placed near the cell and quantal secretoryevents appeared as spike-like activity representing the fusion of individual secretory vesicles. The trace at the bottom indicates thatthe hypoxia-induced secretory activity was abolished by removal and chelation of extracellular Ca2+.

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Fig. 4. Alteration of Ca2+ homeostasis in vascular myocytes by hypoxia. (A) Recording of cytosolic [Ca2+] in a fura-2-loadeddispersed rabbit celiac myocyte illustrating the reversible inhibition of Ca2+ oscillations in response to hypoxia. (B) Reversiblereduction of cytosolic [Ca2+] in a pig coronary myocyte in which membrane voltage-dependent Ca2+ channels were activated bydepolarization with high extracellular K+. (C) Calcium currents recorded from a rabbit celiac myocyte during 15-msec stepdepolarizations to +10 mV from a holding potential of −80 mV. After exposure to hypoxia (PO2

:20 Torr) there is a progressiveinhibition of current amplitude. Reversibility is illustrated by the recovery trace. (Modified from Franco-Obregon et al., 1995, 1998).

preparations are known to dilate in response tolocal decrements in PO2

by means of several mech-anisms, some of them independent of endothelialintegrity (see Franco-Obregon et al., 1998). Dis-persed systemic myocytes loaded with fura-2 canexhibit spontaneous oscillations of cytosolic[Ca2+] (Ca2+ spikes) due to release of the cationfrom internal stores. Hypoxia produces a re-versible decrease in basal Ca2+ and reduction infrequency, or even suppression, of the Ca2+ oscil-lations (Fig. 4A). Similar low PO2

-dependent re-ductions in cytosolic Ca2+ are also observed inmyocytes that do not have spontaneous Ca2+

spikes but where cytosolic [Ca2+] is increased bydepolarization with high external K+ (Fig. 4B).ATP regulated K+ (KATP) channels in myocytesare known to be major regulators of arterial tonein conditions of strong and protracted exposureto low PO2

since their opening leads to membranehyperpolarization and smooth muscle relaxation

(Daut et al., 1990). However, there are severalfacts indicating that activation of these channelscannot fully account for the responses to hypoxiadescribed above. Low PO2

-induced reductions incytosolic [Ca2+] are seen with mild hypoxia with-out compromise of cell’s respiration and in thepresence of glibenclamide, a blocker of KATP

channels (Franco-Obregon et al., 1998). More-over, hypoxia can also relax arterial rings afterK+-evoked contractures (Marriott and Marshall,1990) and decreases cytosolic Ca2+ in cells bathedin high external K+ (Fig. 4B and lower left panelin Fig. 6; see also Vadula et al., 1993), a conditionat which opening of KATP channels would favourcell depolarization to a voltage near the K+

equilibrium potential and Ca2+ influx. Modula-tion of Ca2+ channels by changes in O2 tensioncan be evidenced in patch-clamped myocyteswhere exposure to hypoxia leads to a gradual andreversible reduction in the amplitude of macro-

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scopic Ca2+ currents (Fig. 4C). Besides in arterialmyocytes, inhibition of Ca2+ currents by hypoxiahas been described in other types of smooth mus-cle cells, in glomus cells at potentials nearthreshold and in central neurones ( for review seeLopez-Barneo et al., 1998). Interestingly, L-typeCa2+ channels resulting from the stable expres-sion of the cardiac a1C subunit in HEK cells arealso reversibly inhibited by lowering PO2

(Fearonet al., 1997).

Given that hypoxic pulmonary vasoconstrictionis mainly observed in fine branches of the pul-monary arterial tree whereas vasodilation is nor-mally produced in the main pulmonary artery, wehave tested whether low PO2

has differential ef-fects on Ca2+ currents and cytosolic [Ca2+]homeostasis in conduit and resistance myocytes.In patch-clamped smooth muscle cells dispersedfrom the main pulmonary arterial trunk (conduitmyocytes) hypoxia produces a reduction of Ca2+

Fig. 5. Dual modulation of Ca2+ channel activity by low PO2in pulmonary arterial myocytes. (A) Currents recorded from

patch-clamped cells dispersed from proximal (conduit) and distal (resistance) pulmonary arteries during depolarizations to −10 mVfrom a holding potential of −80 mV. The onset and end of the depolarizing pulses are indicated by the arrows. The control andrecovery traces recorded in the normoxic solution are shown superimposed. (B) Families of Ca2+ currents recorded fromrepresentative conduit and resistance pulmonary myocytes during depolarizations to the indicated membrane potentials from aholding potential of −80 mV. (C) Top: mean calcium current–voltage relationships in conduit (open circles; n=41) and resistance(filled circles; n=21) myocytes of the pulmonary arterial tree. The plot shows the average peak inward current (ordinate,mean9standard error) elicited with voltage pulses to the indicated membrane potentials (abscissa) from the holding potential of−80 mV. Despite the differences in magnitude, the shapes of the current–voltage curves are the same in the two classes of smoothmuscle cells. (C) Bottom: current density in conduit and resistance myocytes. (Modified from Franco-Obregon and Lopez-Barneo,1996.)

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current amplitude similar to the effect describedbefore in systemic and coronary arteries (Fig. 5A,left). However, in a high percentage of cells dis-persed from tertiary arterial branches (resistancemyocytes) hypoxia produces a potentiation of thecalcium current which is more apparent in cur-rents elicited by depolarizations near threshold(Fig. 5A, right). Apart from the differential mod-ulation by low PO2

, Ca2+ currents appear to besimilar in the two classes of myocytes as indicatedby their time course and current-voltage relation-ship (Fig. 5B and C). Interestingly, Ca2+ currentdensity in resistance myocytes is about 2-fold thevalue in conduit myocytes (Fig. 5C), which mighthelp to maintain a high vasomotor tone in theperipheral branches of the pulmonary arterialtree. The opposite effects of low PO2

on Ca2+

channel activity in conduit and resistance my-ocytes are paralleled by differential responses ofcytosolic Ca2+ to hypoxia in the two types ofcells (Fig. 6). Measurements of cytosolic Ca2+ inboth conduit and resistance pulmonary myocytesreveal the existence of oscillations of cytosolicCa2+, or Ca2+ spikes, much like those previouslydescribed in cells of the systemic vessels (Urena etal., 1996). The Ca2+ spikes in some conduit andresistance pulmonary myocytes are modulated bychanges in bathing PO2

, although in diametricallyopposed manners. In the majority of conduit my-ocytes a reduction in bathing PO2

induces a dropin basal cytosolic Ca2+ concentration and a de-crease, or complete supression, in the Ca2+ spikefrequency. These changes are generally associatedwith an increase in Ca2+ spike amplitude (Fig. 6,left, top). Although this response to hypoxia isalso seen in some myocytes isolated from finearterial branches, in more than 50% of the resis-tance myocytes low PO2

induces the opposite ef-fect: increase in basal cytosolic [Ca2+]accompanied by a decrement in Ca2+ spike am-plitude (Fig. 6, right, top). In conduit myocytesthe drop in PO2

mimics the effects of removal ofextracellular Ca2+ (Fig. 6, left, center) and cancounteract the increment of basal cytosolic [Ca2+]and reduction of Ca2+ spike amplitude observedwith elevated extracellular K+ (Fig. 6, left, bot-tom). In contrast, the response to hypoxia ofresistance myocytes resembles the effect of high

external K+ (Fig. 6, right, center) and can bereversibly counteracted by selectively blocking theactivity of L-type Ca2+ channels with nifedipine(0.5 mM) (Fig. 6, right, bottom). These resultsstrongly suggest that low PO2

modulates the activ-ity of Ca2+ channels in opposed manners indifferent regions of the pulmonary arterial tree. Inmyocytes of conduit arteries hypoxia leads toinhibition of the Ca2+ channels, whereas it canpotentiate or inhibit Ca2+ channel activity indifferent subpopulations of resistance myocytes.These results also agree with previous work show-ing that the development of hypoxic pulmonaryvasoconstriction in the distal regions of the pul-monary artery can be either inhibited or enhancedwith L-type Ca2+ channel antagonist or agonist,respectively (see Franco-Obregon et al., 1998).Potentiation of L-type Ca2+ channel activity bylow PO2

may not be specific of resistance pul-monary myocytes since a similar observation hasbeen recently reported in inspiratory neurons (Mi-ranov and Richter, 1998).

4. Oxygen-sensitivity of ion channels

The progress in research in recent years hasproduced numerous observations describing newO2-regulated channels and/or cellular phenomenawhere they may play a role. However, the molecu-lar nature of the mechanisms underlying O2-sens-ing by ion channels remain essentially unknown.Our own experience with carotid body and vascu-lar smooth muscle cells indicates that the O2-sen-sitivity of ion channels is a rather labile process,easily altered by cell dissociation procedures,which may depend on subtle physico-chemicalchanges difficult to detect and characterize. It isalso unknown whether the O2-dependent mecha-nisms that mediate ion channel regulation in fastO2-responsive cells are similar to those operatingin a broad variety of cells types where protractedhypoxia regulates gene expression (see Ratcliffe etal., 1998). In some preparations regulation of ionchannels by PO2

appears to be a membrane delim-ited mechanism since it is resistant to intracellulardialysis and can be observed in excised membranepatches (Ganfornina and Lopez-Barneo, 1991; Ji-

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Fig. 6. Contrasting effects of hypoxia on the cytosolic [Ca2+] and Ca2+ oscillations (Ca2+ spikes) in conduit (left column) andresistance (right column) pulmonary myocytes. The shaded regions represent the times during which the bath solution wasexchanged for the indicated experimental conditions. Left column: top, reduction of basal Ca2+ levels (dotted lines) and thefrequency of the oscillations by low PO2

in a representative conduit myocyte; center, removing extracellular Ca2+ from the bathingsolution reversibly decreases basal cytosolic [Ca2+] and decreases the frequency of the Ca2+ spikes; bottom, increase of basal Ca2+

and the frequency of the oscillations by high external K+. The effect of K+ is counteracted by hypoxia. Right column: top, increaseof basal Ca2+ (dotted lines) and reduction of the Ca2+ spikes amplitude during exposure to hypoxia in a resistance myocyte; center,depolarizing the myocytes with 30 mM extracellular K+ increases basal cytosolic [Ca2+], spikes frequency and reduces spikeamplitude; bottom, the effect of hypoxia is counteracted by blockade of L-type Ca2+ channels with nifedipine (0.5 mM). The PO2

of the solutions were :150 (normoxia) and 20–30 (hypoxia) Torr. All calibration bars indicate 1 min. (Modified from Urena et al.,1996.)

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Fig. 7. Redox regulation of the O2-sensitive K+ channels of rabbit glomus cells. (A) Currents recorded from an inside-outmacropatch during depolarizing pulses to +20 mV in the standard solution (control) and after addition of reduced glutathione(GSH, 0.5 mM) to the bath. GSH reversibly reduced current amplitude. (B) Modulation of a single O2-regulated K+ channel bythe application of reduced glutathione (GSH, 0.5 mM) to the internal face of the membrane. The recordings were obtained from aninside-out membrane patch with a functional channel during depolarization to +20 mV. The concentrations of K+ were 2.7 and140 mM in the external and internal solutions, respectively. (Modified from Benot et al., 1993; Lopez-Barneo et al., 1998.)

ang and Haddad, 1994). Therefore, it is conceiv-able that there are O2 sensors associated with theion channels capable of undergoing conforma-tional changes during oxygenation and deoxy-genation and, thus, modifying allosterically thechannel’s kinetic properties. The best candidatesfor this kind of regulation are membrane boundheme proteins such as the cloned O2 sensor ofRhizobium meliloti (Fix L) that can change activ-ity according to its oxygenated state (see Bunnand Poyton, 1996). Interestingly, the oxygenation

state of hemoglobin, associated with the internalface of the membrane, is also known to alter theion transport properties of erythrocytes (Motais etal., 1987; Lauf, 1998). Another possibility to con-sider is the existence of structural domains insome of the main (a or b) channel subunits thatcan bind reversibly molecular oxygen and modifytheir function. In this respect, it is important tomention that there are several reports describingthe modulation by PO2

of recombinant K+ andCa2+ channels expressed in heterologous systems,

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although the precise molecular mechanisms in-volved have remained unexplained so far (Ortega-Saenz et al., 1996; Fearon et al., 1997;Lopez-Barneo et al., 1997; Patel et al., 1997;Perez-Garcıa et al., 1998).

Besides the direct effect of O2 tension on ionchannels, another possibility is the participation ofredox based mechanisms in O2-sensing. An attrac-tive hypothesis is that the modifications of O2

tension are linked to channel activity throughvariations in the concentration of oxidants orreductants which modify the redox state of thiolgroups in the channel molecule (Acker et al., 1989;Archer et al., 1993; Benot et al., 1993). It has beenshown in recombinant K+ channels that the redoxstate of cysteine residues in the amino terminal ofthe a subunits or in some auxiliary b subunitsregulates inactivation. Moreover, it is known thatreductants, such as reduced glutathione (GSH) ordithiothreitol (DTT), can mimic the effect of hy-poxia on the O2-regulated K+ channels (Archer etal., 1993; Benot et al., 1993; Yuan et al., 1994;Lopez-Barneo et al., 1998). The redox regulationof K+ conductance in rabbit glomus cells is illus-trated in Fig. 7. The traces show the currentsrecorded from an inside-out macropatch with ap-proximately 20 channels (A) and from a patchwith a single functional channel (B). In both casesapplication of GSH to the internal face of themembrane produced reversible inhibition of chan-nel activity. As it occurs during exposure to lowPO2

, GSH reduces channel open probability with-out affecting the single-channel conductance(Lopez-Barneo et al., 1998). Hence, it is plausiblethat some of the PO2

-dependent effects observed inthe various O2-sensitive K+ channels studied aredue to changes in the production of oxygen radi-cals which modify the redox state of the channelprotein. In the cell types that exhibit a specialsensitivity to PO2

changes (for instance glomus andneuroepithelial cells or pulmonary arterial smoothmuscle) it has been postulated the existence ofspecific membrane bound oxidases associated withthe channel molecule and capable of generatingthe reactive species in the vicinity of the targetresidues. Among the proposed candidates are theoxidases similar to the NADPH oxidase of neu-trophils or the family of cytochrome P-450 oxi-

dases (Acker et al., 1989; Youngson et al., 1993;Yuan et al., 1995). The putative oxidase acting asO2 sensor could be co-expressed with the mainpore-forming a subunit, thus conferring to thechannels O2-sensitivity.

In conclusion, O2-regulated K+ and Ca2+ ionchannels are involved in the cellular responses tohypoxia. These channels, affected by changes inO2 tension in the range of seconds or minutes, arepresent in fast O2-responsive cells of chemorecep-tor tissues or organs. Although the nature of themechanisms underlying the interaction of O2 andthe channels is still unknown, several possibleforms, which may well act in parallel, are dis-cussed. O2-sensing by ion channels and the O2-de-pendent regulation of gene expression are possiblyrelated phenomena, acting in different time ranges,that may share similar basic principles and mecha-nisms.

Acknowledgements

The work in our laboratory is supported by theDireccion General de Investigacion Cientıfica yTecnica (DGICYT) of the Spanish Ministry ofScience and Education, Fundacion La Caixa andthe Andalusian Government.

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