Oxygen-sensitive ion channels

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Oxygen-sensitive ion channels Chris Peers It is well known that ion channels are regulated by altered cellular metabolism, and therefore indirectly by O2 levels of the local environment. For example, by decreasing intracellular ATP levels in cardiac tissue, brief periods of severe hypoxia or ischaemia can cause opening of ATPsensitive K+ channels1. This can reduce contractility and be arrhythmogenic. In the CNS, hypoxia or ischaemia alters neuronal excitability, contributing to the manifestation of stroke2. Owing to such detrimental consequences, and to the potential for pharmacological intervention, much effort has been directed towards studying the cellular mechanisms that couple hypoxia/ischaemia to altered ion channel activity. In recent years, a body of evidence has emerged indicating that O2 levels per se can regulate ion channel activity in a seemingly direct and sensitive manner. This modulation of channel activity, which can be very rapid, does not involve a fall in ATP levels via reduced oxidative phosphorylation, or other consequences of compromised metabolism such as acidosis, and might result from O2-sensing by the channel itself. Two tissue types, the carotid body and pulmonary vascular smooth muscle (PVSM), exhibit responses to hypoxia that are unique in certain aspects. Perhaps not surprisingly, many of these studies were initiated in these tissues. The carotid body is the principal arterial chemoreceptor3, responding to falls in arterial PO2 by increasing afferent sensory information to the central respiratory centres. This leads to a reflex increase in ventilation, so that arterial PO2 levels are restored. The O2 sensors within the carotid body are the type I or glomus cells3,4, which release transmitters onto afferent sensory nerve endings to alter their activity. PVSM is unique within the circulation, since it constricts rather than relaxes in

hypoxia; this has the beneficial effect of directing blood to better-ventilated regions of the lung, optimizing uptake of O2 into the bloodstream5. This property may also be fundamental to the development of pulmonary hypertension in a number of hypoxic diseases.

Oxygen-sensitive K+ channels Some nine years ago, O2-sensitive K+ channels were first reported in rabbit carotid body type I cells6. Subsequent studies indicated that hypoxia selectively inhibited a specific class of K+ channels, although this appears to be species dependent and different O2-sensitive K+ channel types may co-exist (see Table 1; Refs 7–24); the degree of diversity, however, may in some cases be due to differing experimental conditions. The remarkable features of the hypoxic inhibition of K+ channels in rabbit type I cells were: (1) the high sensitivity to changes in O2 levels7; (2) the rapidity of the response (quicker than block of Na+ channels by tetrodotoxin25); and (3) the fact that hypoxia also inhibited K+ channels in excised membrane patches8. This latter observation was crucial in dissociating channel inhibition from changes in cellular metabolic status. Similarly, in PVSM, O2-sensitive K+ channels were identified5. Whilst the majority of workers have shown that hypoxia inhibits K+ channels in pulmonary myocytes, there is also evidence for hypoxic enhancement of K+ channels in myocytes isolated from large (conduit) pulmonary vessels26 and cerebral arteries27. Such O2-sensitive channels are not however unique to the carotid body or PVSM but have been observed in a variety of cell types (Table 1). Oxygensensitive K+ currents have been recorded in different cell types (Fig. 1). Importantly, K+ channel inhibition has also been demonstrated in excised membrane patches of central neurones, but the time

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course of such inhibition was far slower than was seen in the carotid body15,16. In each of these cell types, the inhibition of K+ channels is likely to be of major physiological importance, since channel closure leads to membrane depolarization and therefore Ca2+ influx via voltage-gated Ca2+ channels28. Consequently, cells may respond rapidly to hypoxia either by releasing neurotransmitters (as for type I carotid body cells3,4) or by contracting (as for PVSM cells5; see also Ref. 28). In arterial myocytes that display hypoxic enhancement of K+ channels26,27, hyperpolarization and vasorelaxation may occur. The mechanism by which hypoxia alters channel activity is currently a topic of major interest. Differing ideas are apparent, but redox modulation of channels is a popular theory: the ratio of cytosolic redox couples such as glutathione (GSHGSSG) or nicotinamide adenine dinucleotide (NADH-NAD) may well favour the reduced forms (GSH or NADH) in hypoxia. These reduced forms have been shown to inhibit O2sensitive K+ channels in type I cells9 and PVSM cells5,29. Such redox control of K+ channels may well be important, but this does not identify these agents as being O2 sensors; rather, their levels are the consequence of a response to hypoxia earlier in the pathway. Acker and colleagues have indicated a cytosolic cytochrome b-linked NAD(P)H oxidase that, in hypoxia, fails to maintain superoxide (and hence H2O2) production and so cannot keep redox couples preferentially in the oxidized state30. In contrast (or perhaps in addition) to redox modulation, there is evidence for membrane-bound O2 sensors. Thus for example, in the rabbit carotid body (where hypoxia inhibits K+ channels in excised patches), the effects of hypoxia can be rapidly reversed by carbon monoxide, arguing strongly for the involvement of a plasmalemmal haem protein as a regulator of these K+ channels25. However, by contrast, hypoxic regulation of Ca2+-sensitive K+ channels in rat type I cells does not appear to be membrane limited10.

Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0165 – 6147/97/$17.00 PII: S0165-6147(97)01120-6

TiPS – November 1997 (Vol. 18)

C. Peers, British Heart Foundation Lecturer, Institute for Cardiovascular Research, University of Leeds, Leeds, UK LS2 9JT.

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Table 1. Features of native O2-sensitive K+ and Ca2+ channels Tissue/cell type

O2-sensitive channel

Channel features

Refs

Carotid body

K+

Ca2+

40 pS, voltage gated, inactivating, blocked by 4-AP 190 pS, Ca2+ sensitive, blocked by CTX and TEA Not voltage gated, low conductance, resistant to TEA and 4-AP Voltage gated, possibly L-type (found in rabbit but not rat cells)

7–9 10 11 12, 13

Neuroepithelial cell body

K+

Voltage gated, blocked by TEA

14

Central neurones

K+

200 pS, voltage gated, Ca2+ activated, inhibited by ATPi

15, 16

Neonatal chromaffin cells

K+

Voltage gated, possibly also Ca2+ sensitive

17

PC12 phaeochromocytoma cells

K+

20 pS, voltage gated, blocked by TEA, inactivating, possibly Kv1.2?

Ca2+

Pulmonary smooth muscle

K+

Ca2+

Ca2+ sensitive, blocked by Leiurus quinquestriatus venom Voltage gated, blocked by 4-AP Low threshold, non-inactivating DHP sensitive, voltage gated L-type

20 21 22 23

Ca2+

DHP sensitive, voltage gated L-type

24

Systemic smooth muscle

insensitive, slowly

18, 19

4-AP, 4-aminopyridine; ATPi, intracellular adenosine 5′-triphosphate; CTX, charybdotoxin; DHP, dihydropyridine; TEA, tetraethylammonium.

In central neurones, two possible mechanisms account for slow hypoxic inhibition of K+ channels in excised patches: first, exogenous

application of reduced moieties suggests the redox mechanism described above. Second, there is evidence for the involvement of an

Fig. 1. Examples of inhibition of K+ currents by hypoxia (PO2 17–40 mmHg) in a variety of tissue types as indicated. In each case, currents were recorded using conventional whole-cell patch clamp recordings. In a–c, currents were evoked by step depolarizations a: from –70 mV to +20 mV, b: from –60 mV to +50 mV and c: from –60 mV to +20 mV. d: Currents were evoked by ramp depolarizations (0.042 mV ms–1). Reproduced with permission from Refs 40, 14, 17 and 20, respectively.

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iron-containing but nonhaem protein. This is based on the observation that the effects of hypoxia could be mimicked by agents that chelate metal in metal-containing proteins16. Using canine pulmonary myocytes, Post et al.31 demonstrated that hypoxia inhibited delayed rectifierlike K+ channels via release of Ca2+ from caffeine- sensitive intracellular Ca2+ stores. Thus, different mechanisms, and evidence to support these mechanisms, exist to account for hypoxic inhibition of K+ channels. However, what remains unresolved (and is of paramount importance to determine) is whether the channels can themselves directly act as O2 sensors32. That is, can they directly respond to low PO2, or does hypoxia require an intermediate O2 sensor that couples to the channel? Direct redox modulation, for example, could be mediated via thiol-containing cysteine residues in the channel protein. Certainly, K+ channels (both a- and b-subunits) possess cysteine residues on their cytosolic aspect which can undergo oxidation and reduction and so modulate channel inactivation. This has been shown in a recombinant voltage-gated K+ channel33, but in response to reducing agents, and not so far to hypoxia

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itself. Nevertheless, direct O2 sensitivity of recombinant K+ channels may soon be demonstrated, since the molecular identity of K+ channels present in phaeochromocytoma (PC12) cells has recently been reported, and one of these (possibly Kv1.2) is sensitive to PO2 changes18. Furthermore, numerous recombinant K+ channels (Kv1.3, 1.4, 1.5, 3.4 and IRK3, but interestingly not Kv1.2) have been shown to be modulated by reactive oxygen species that are implicated in myocardial and cerebral ischaemia34.

Oxygen-sensitive Ca2+ channels Some years after the report of O2sensitive K+ currents in type I carotid body cells, it was found that voltagegated Ca2+ channels could also be inhibited by hypoxia in these cells12. This effect was voltage dependent, with inhibition only being seen in hypoxia at lower activating test potentials. Subsequently, LopezBarneo and colleagues also found dihydropyridine-sensitive L Ca2+ channels in cells isolated from vascular (both systemic and pulmonary) smooth muscle to be O2 sensitive23,24 (e.g. Fig. 2a). In the systemic circulation, Ca2+ channel inhibition could account for hypoxia’s well known action as a vasodilator, but in the pulmonary circulation the effects of hypoxia were different between cells from resistance and conduit vessels23. In conduit vessels, inhibitory effects such as those found in systemic cells were found, but in resistance vessels hypoxia enhanced Ca2+ channel currents. Both of these effects could be accounted for in part by opposite actions on current activation, such that current–voltage relationships were shifted in a positive direction for conduit vessels and in a negative direction for resistance vessels. These exciting findings have no mechanism to account for them. However, as for K+ channels, this may soon change since a recent study has shown that the poreforming a1-subunit (a1C) of the human cardiac L channel (which has >95% sequence homology with the smooth muscle a1-subunit35), when

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expressed in HEK293 cells, also shows O2 sensitivity36 (Fig. 2b). This recombinant subunit could be the most O2-sensitive channel discovered to date: hypoxic levels as mild as 90 mmHg caused inhibition36. As in vascular smooth muscle, hypoxic inhibition was only seen at lower activating test potentials, and the inhibition was accompanied by a slowing of activation kinetics. Thus with a recombinant expression system, the way is open to investigate hypoxic inhibition of Ca2+ channels at the molecular level. Of primary importance is the investigation of the possible role of cysteine residues that might be modulated by oxidation or reduction. Interestingly, a recent report has indicated the presence of such redox-modulated residues, and these appear to be located on the extracellular surface of the native L channels in ferret ventricular myocytes37, supporting the possibility of direct O2 sensing. As for K+ channels, it remains then to be determined whether hypoxia can directly inhibit Ca2+ channels, or whether an intermediary O2 sensor is required. In summary, this brief article intends to increase awareness of the existence of O2-sensitive ion channels, and their potential importance in diverse cellular activities. Whilst the focus of attention has, to date, been largely on K+ and Ca2+ channels, it is likely that other channel types will be found to be O2 sensitive: there is recent evidence for O2sensitive Cl– channels in skeletal muscle sarcoplasmic reticulum38, and the vast family of ligand-gated ion channels have the potential (through possessing cysteine residues39) to be regulated directly by hypoxia. Furthermore, it should be apparent that there are marked differences in the effects of hypoxia: not only may low PO2 affect numerous types of channels (Table 1), but the rate at which channel modulation is affected by hypoxia can vary widely. Moreover, the severity of hypoxia required to observe channel modulation also varies enormously. Together, these factors serve to suggest that there is unlikely to be

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Fig. 2. Inhibition of L-type Ca2+ channels by hypoxia. a: Inhibition of currents evoked in a pulmonary myocyte by lowering PO2 levels to approximately 20 mmHg. A similar level of hypoxia inhibits recombinant human cardiac L channel a1C-subunits stably expressed in HEK293 cells (b). In each case, whole cell patch-clamp techniques were used, and currents were evoked by step depolarizations a: from –80 mV to –10 mV or b: from –80 mV to +10 mV. Modified, from Refs 23 and 36, respectively, with permission .

a single unifying mechanism to account for the modulation of ion channels by O2. However, direct O2 sensing may be a vital feature of some channels. Future studies will doubtlessly be aimed at identifying these mechanisms. Such studies must also consider structural modifications caused by hypoxia: ion channels comprise a large family of proteins that are important therapeutic targets. Drug affinity or efficacy has the potential to be altered by conformational changes induced by hypoxia, perhaps at times when therapeutic intervention is most needed. References 1 Benndorf, K., Thierfelder, S., Doepner, B., Gebhardt, C. and Hirche, H. (1997) News Physiol. Sci. 12, 78–83 2 Martin, R. L., Lloyd, H. G. and Cowan, A. I. (1994) Trends Neurosci. 17, 251–257 3 Gonzalez, C., Almaraz, L., Obeso, A. and Rigual, R. (1994) Physiol. Rev. 74, 829–898 4 Peers, C. and Buckler, K. J. (1995) J. Membr. Biol. 144, 1–9

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Acknowledgements The author’s own studies in this field are supported by the Wellcome Trust and the British Heart Foundation. I am grateful to S. G. Ball and E. Carpenter for helpful comments, and all my colleagues for allowing me to reproduce their work.

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5 Weir, E. K. and Archer, S. L. (1995) FASEB J. 9, 183–189 6 Lopez-Barneo, J., Lopez-Lopez, J. R., Urena, J. and Gonzalez, C. (1988) Science 241, 580–582 7 Lopez-Lopez, J. R., Gonzalez, C., Urena, J. and Lopez-Barneo, J. (1989) J. Gen. Physiol. 93, 1001–1015 8 Ganfornina, M. D. and Lopez-Barneo, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2927–2930 9 Benot, A. R., Ganfornina, M. D. and Lopez-Barneo, J. (1993) in Ion Flux in Pulmonary Vascular Control (Weir, E. K., Hume, J. R. and Reeves, J. T., eds), pp. 177–183, Plenum 10 Wyatt, C. N. and Peers, C. (1995) J. Physiol. 483, 559–565 11 Buckler, K. J. (1997) J. Physiol. 498, 649–662 12 Montoro, R. J., Urena, J., FernandezChacon, R., Alvarez de Toledo, G. and Lopez-Barneo, J. (1996) J. Gen. Physiol. 107, 133–143 13 Lopez-Lopez, J. R., Gonzalez, C. and PerezGarcia, M. T. (1997) J. Physiol. 499, 429–411 14 Youngson, C., Nurse, C., Yeger, H. and Cutz, E. (1993) Nature 365, 153–155 15 Jiang, C. and Haddad, G. G. (1994) J. Physiol. 481, 15–26

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16 Jiang, C. and Haddad, G. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7198–7201 17 Thompson, R. J., Jackson, A. and Nurse, C. A. (1997) J. Physiol. 498, 503–510 18 Conforti, L. and Millhorn, D. E. (1997) J. Physiol. 502, 293–605 19 Zhu, W. H., Conforti, L., CzyzykKrzeska, M. F. and Millhorn, D. E. (1996) Am. J. Physiol. 271, C658–C665 20 Post, J. H., Hume, J. R., Archer, S. L. and Weir, E. K. (1992) Am. J. Physiol. 262, C882–C890 21 Yuan, X-J., Goldman, W. F., Tod, M. L., Rubin, L. J. and Blaustein, M. P. (1993) Am. J. Physiol. 264, L116–L123 22 Osipenko, O. N., Evans, A. M. and Gurney, A. M. (1997) Br. J. Pharmacol. 120, 1461–1470 23 Franco-Obregon, A. and Lopez-Barneo, J. (1996) J. Physiol. 491, 511–518 24 Franco-Obregon, A. Urena, J. and Lopez-Barneo, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4715–4719 25 Lopez-Lopez, J. R. and Gonzalez, C. (1992) FEBS Lett. 299, 251–254 26 Bonnet, P., Vandier, C., Cheliakine, C. and Garnier, D. (1994) Exp. Physiol. 79, 597–600 27 Gebremedhin, D. et al. (1994) Pflügers Arch.

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Stephen A. Douglas

S. A. Douglas, Investigator, Department of Cardiovascular Pharmacology (UW2510), SmithKline Beecham Pharmaceuticals, PO Box 1539, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA.

*3rd International Symposium on Endothelin Inhibitors: Advances in Therapeutic Application and Development, 26–27 June 1997, Philadelphia, USA. 408

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428, 621–630 28 Lopez-Barneo, J. (1996) Trends Neurosci. 19, 435–440 29 Lee, S., Park, M., So, I. and Earm, Y. E. (1994) Pflügers Arch. 427, 378–380 30 Acker, H. and Xue, D. (1995) News Physiol. Sci. 10, 211–216 31 Post, J. M., Gelband, C. H. and Hume, J. R. (1995) Circ. Res. 77, 131–139 32 Lopez-Barneo, J. (1997) J. Physiol. 500, 549 33 Ruppersberg, J. P. et al. (1991) Nature 352, 711–714 34 Duprat, F. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11796–11800 35 Hofmann, F., Biel, M. and Flockerzi, V. (1994) Annu. Rev. Neurosci. 17, 399–418 36 Fearon, I. M. et al. (1997) J. Physiol. 500, 551–556 37 Campbell, D. L., Stamler, J. S. and Strauss, H. C. (1996) J. Gen. Physiol. 108, 277–293 38 Kourie, J. I. (1997) Am. J. Physiol. 272, C324–C332 39 Lipton, S. A., Choi, Y. B., Sucher, N. J., Pan, Z. H. and Stamler, J. S. (1996) Trends Pharmacol. Sci. 17, 186–187 40 Hatton, C. J., Carpenter, E., Pepper, D. R., Kumar, P. and Peers, C. (1997) J. Physiol. 501, 49–58

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Clinical development of endothelin receptor antagonists The rapid preclinical development of diverse endothelin (ET) inhibitors bears testimony to the perceived pathological significance of this endothelium-derived peptide within the medical community. The development of agents with distinct pharmacological profiles has facilitated the identification of ET-1 as a pathogenic mediator in preclinical models, something complimented by the recent successful application of alternative molecular approaches (e.g. ET-related knockout mice). Most encouraging, however, has been the successful progression of ET receptor antagonists into clinical development. Phase I/II trials have established clinical utility for these therapeutic modalities. The most significant advances within the field were discussed at a recent meeting in Philadelphia*.

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Advances in the development of ET knockout mice M. Yanagisawa (University of Texas Southwestern, USA) has pioneered the development of ET knockout mice strains. ET-1 (Ref. 1), ET-3 (Ref. 2) and ETB (Ref. 3) receptor knockouts have delineated biological roles for this neurohumoral system including embryonic craniofacial/ cardiovascular (first brachial arch) and neural crest development/ maturation (enteric neurone and epidermal melanocyte loss leading to Hirschsprung’s disease, etc.). Endothelin converting enzyme-1 (ECE-1) knockout mice have been bred successfully. However, although ECE-1(–/–) homozygotes exhibit no ECE activity ex vivo, plasma ET-1 levels remain ≥70% of those seen in ECE-1(+/+) wild types, i.e. bigET-1 is

subject to alternate processing by proteases distinct from ECE-1 [e.g. neutral endopeptidase (NEP), angiotensin converting enzyme (ACE), distinct ECE isoforms] thus questioning the therapeutic utility of selective ECE-1 inhibitors (see below). Attempts are in progress to breed ECE-1/2 ‘double homozygous’ knockout mice to address this question. Targeted disruption of the ETB (or ET-3) gene is associated with a lethal phenotype (aganglionic megacolon)3 and ETB(–/–) mice rarely survive three weeks post partum. As such, homozygotes can only be studied as immature individuals. Although a milder phenotype exists in heterozygous ETB(+/–) mice or within a strain of piebald (s′) mice (spontaneous ETB mutation), mature, adult ETB(–/–) mice are being raised using a transgene approach whereby a human dopamine-b-hydroxylase promoter/ ‘rescue’ construct preserves the development of enteric neurones even in the presence of an ETB(–/–) background. Such animals survive to

Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0165 – 6147/97/$17.00 PII: S0165-6147(97)01121-8