The carotid body oxygen sensor

The carotid body oxygen sensor Lin Gao 1,2,3 , Alejandro Moreno-Domínguez 1,2,3 , Patricia Ortega-Sáenz 1,2,3 and José López-Barneo 1,2,3 Carotid body (CB) chemoreceptor glomus cells sense hypoxia through the inhibition of plasmalemmal K + channels, which leads to the opening of Ca 2+ channels, Ca 2+ influx, and neurotransmitter release. The mechanism of O 2 sensing and the regulation of membrane ion channels by O 2 have remained undefined and a subject of debate. Here, we summarize the molecular pathway that underlies acute O 2 sensing in the CB. This process does not rely on a single-molecule O 2 sensor expressed in glomus cells but rather on HIF2 a -dependent genetically specialized mitochondria that can detect changes in O 2 tension, within physiological ranges, and generate biochemical signals that regulate membrane ion channels. The acute O 2 -sensing pathway in glomus cells could provide new targets for respiratory and cardiovascular pharmacology. Addresses 1 Institute of Biomedicine of Seville (IBiS), University Hospital “Virgen del Rocío”/CSIC/University of Seville, Seville, Spain 2 Department of Medical Physiology and Biophysics. School of Medicine, University of Seville, Seville, Spain 3 CIBERNED, Madrid, Spain Corresponding author: López-Barneo, José (lbar[email protected]) Current Opinion in Neurobiology 2025, 92:103022 This review comes from a themed issue on Interoception 2025 Edited by Stephen Liberles and Zachary Knight For complete overview of the section, please refer the article collection - Interoception 2025 Available online 22 April 2025 https://doi.org/10.1016/j.conb.2025.103022 0959-4388/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). Introduction The carotid body (CB) is the prototypical acute oxygen (O 2 )-sensing organ in mammals, playing an essential role in initiating rapid adaptive cardiorespiratory reflexes, such as hyperventilation and increased cardiac output, triggered by low O 2 tension (hypoxia). O 2 sensing is an intrinsic property of neural crestderived chemoreceptor glomus cells, which are the most abundant cell type in the CB. These cells are in close contact with a dense network of fenestrated capillaries and are innervated by sensory fibers of the petrosal ganglion, which connects to brainstem respiratory and autonomic centers [1]. Glomus cells are excitable, presynaptic-like elements that contain O 2 -sensitive K þ channels. The inhibition of these channels during hypoxia leads to depolarization, Ca 2þ influx, and the exocytotic release of neurotransmitters [2e4]. Glomus cells are highly dopaminergic but also contain a variety of other transmitters that play a modulatory autocrine and paracrine role. ATP is the main excitatory transmitter, acting on ionotropic purinergic receptors located in the afferent sensory terminals [2]. While the basic electrophysiological and neurosecretory characteristics supporting the sensory properties of glomus cells are well established, the molecular mechanisms underlying O 2 sensing and the modulation of membrane ion channels by O 2 have remained elusive. As a result, these mechanisms were represented by question marks in a summary of glomus cell function published in this journal in 2003 [5*]. The identity of the O 2 sensor in the CB has been a subject of debate [6,7], with several promising candidates, including enzymes and receptors directly or indirectly regulated by O 2 tension, discussed in the literature [5,7e12]. However, studies using knockout mouse models, which lack the genes encoding the putative O 2 -sensing molecules (e.g. heme oxygenase 2, cystathionineg -lyase, AMP kinase, or olfactory receptor 78), have shown that although these molecules may have relevant functional roles, none are essential for acute O 2 sensing in the CB [5*,13e17]. For the past few years, the combined use of genetically modified mice, comparative gene expression analyses, and robust single-cell monitoring of responsiveness to hypoxia has enabled us to complete the description of the molecular pathway that underlies, and is indispensable for, CB acute O 2 -sensing. This process does not depend on a single-molecule O 2 sensor expressed in glomus cells, but rather on genetically specialized mitochondria that can detect hypoxia within the physiological range and generate biochemical signals that regulate membrane ion channels [18**,19*,20**,21**, 22*]. Available online at www.sciencedirect.com ScienceDirect Current Opinion in Neurobiology www.sciencedirect.com Current Opinion in Neurobiology 2025, 92:103022 Mitochondrial complex I-dependent generation of hypoxic signals and modulation of ion channel function It has long been known that mitochondrial inhibitors are potent activators of the CB [23,24*] and that some mitochondrial parameters in glomus cells are modulated by O 2 tension [25*,26e28*]. We demonstrated that, similar to hypoxia [1], activation of CB glomus cells by mitochondrial inhibitors requires extracellular Ca 2þ influx and that rotenone, a selective inhibitor of mitochondrial complex I (MCI), not only mimics hypoxia but in its presence the cells become insensitive to hypoxia [24*]. These observations suggested that mitochondria may modulate membrane electrical activity in glomus cells and that acute O 2 sensing could depend on MCI function. To test these hypotheses, we used a mouse model with conditional genetic disruption of MCI, created by ablation of the Ndufs2 gene, which encodes a subunit of the catalytic core of MCI necessary for coenzyme Q (CoQ) binding and rotenone blockade [18**,29]. MCI is a multimeric NADH dehydrogenase that transfers electrons from NADH to CoQ, generating CoQH 2 , which in turn serves as the substrate for mitochondrial complex III (MCIII). This reaction is coupled to proton pumping, where MCI transports H þ from the mitochondrial matrix to the intermembrane space (IMS). NDUFS2-deficient mice, in which glomus cells lack MCI activity, can survive for several months, likely due to upregulation of glycolysis [18**,19*,22*]. These cells maintain normal morphology, electrical properties, and neurosecretory activity, with normal dopamine content in their secretory vesicles, suggesting that they retain fundamental physiological functions. However, hypoxia-induced exocytotic catecholamine release is completely abolished in MCIdeficient glomus cells, although their secretory response to hypercapnia and other stimuli remains intact (Figure 1aed). In parallel, hypoxia-induced modulation of membrane ionic currents and increases in cytosolic Ca 2þ were also eliminated in MCI-deficient glomus cells [18**,19*]. These findings led us to propose a dynamic mitochondrial-tomembrane signaling (MMS) model of acute O 2 sensing, which is based on the role of O 2 as an essential substrate for cytochrome coxidase (CCO) in mitochondrial complex IV (MCIV) and the reversibility of preceding reactions in the mitochondrial electron transport chain (ETC) (Figure 2a). In the MMS model, a decrease in O 2 tension during hypoxia leads, via mass action, to a backlog of substrates in the reduced state, favoring the production of superoxide anion (converted to H 2 O 2 by superoxide dismutase) in the mitochondrial IMS. The increase in CoQH 2 results in a slowdown of MCI activity and the accumulation of NADH. Following equilibration with the cytosol, NADH and H 2 O 2 interact with membrane ion channels, triggering cell depolarization and transmitter release [18**,30**]. Support for the MMS model has come from microfluorimetric analyses of single glomus cells, which showed graded increases in NADH and H 2 O 2 levels in response to various levels of hypoxia. Moreover, these signals were abolished in MCI-deficient glomus cells (Figure 2b) [19*]. Both NADH and H 2 O 2 are wellknown ion channel modulators [31,32], and in glomus cells, they mimic and attenuate the effects of hypoxia on background and voltage-dependent K þ currents [18**]. However, hypoxia-induced membrane depolarization in glomus cells does not appear to depend on a specific class of O 2 -regulated K þ channels but rather on the interaction of NADH and H 2 O 2 with several channel types. For example, TASK1 and TASK3 K þ channels, which are highly expressed in glomus cells [20**,33**], have been suggested to mediate the background K þ current whose inhibition by hypoxia triggers membrane depolarization [28,34]. Nevertheless, glomus cells from mice with genetic ablation of Task1 and Task3 still exhibit normal secretory responses to hypoxia. These findings suggest that other ion channels, regulated by NADH and/or H 2 O 2 dpossibly voltage-gated K þ channels, which also contribute to setting the resting potentialdcompensate for the absence of TASK1 and TASK3 channels [35*]. The MMS model (Figure 2a) assumes that the essential role of MCI in acute O 2 sensing is to generate the signals that regulate ion channel function, rather than directly sensing changes in O 2 tension. In agreement with this concept, transgenic expression of NDI1, a singlemolecule alternative yeast NADH/CoQ oxidoreductase that is structurally unrelated to the 45-subunit mammalian MCI, completely rescues the O 2 -sensing function of MCI-deficient glomus cells [22*]. NDI1 expression restores hypoxia-induced NADH and H 2 O 2 signals (Figure 2b) and the secretory response of glomus cells to hypoxia. Mice-expressing NDI1 also recover the hypoxic ventilatory response (HVR), a systemic reflex dependent on CB function [1], which is selectively lost in MCI-deficient mice (Figure 2c) [22*]. Since NDI1 cannot pump protons, these observations indicate that the dynamic changes in NADH dehydrogenase activity, induced by the decrease in O 2 availability during hypoxia (Figure 2a), are essential for the acute O 2 -sensing pathway in CB glomus cells. Additional support to the MMS model of acute O 2 -sensing was recently provided by experiments in mice with genetic ablation of MCIII in glomus cells. These MCIII-deficient mice exhibit complete absence of the HVR, while they have normal ventilatory responses to hypercapnia. Moreover, MCIIIdeficient glomus cells, with an interrupted ETC, are insensitive to hypoxia and cyanidedan MCIV 2Interoception 2025 Current Opinion in Neurobiology 2025, 92:103022 www.sciencedirect.com blockerdbut have normal secretory responses to hypercapnia and other stimuli [36]. Genetically specialized mitochondria for physiological acute O 2 -sensing CB glomus cells are extraordinarily sensitive to hypoxia as they can detect changes in O 2 tension in their surrounding environment at levels slightly below 80e90 mmHg [13,35]. In all tissues studied, the reported Km values of CCO for O 2 are w1 mmHg or lower. Therefore, even assuming a marked O 2 gradient between the extracellular medium and mitochondria, the O 2 levels that activate glomus cells are clearly above saturation of MCIV. These facts suggest that glomus cells possess an atypical mitochondrial ETC capable of responding to fluctuations in high levels of O 2 tension. In support of this idea, transcriptomic studies [20**,33**] have identified a “signature genetic profile” characteristic of O 2 -sensitive CB cells, distinguishing them from other neural crest-derived cellsde.g. sympathetic neurons in the superior cervical gangliond, which are O 2 -insensitive [37**]. Among the transcripts highly expressed in CB cells are those coding for pyruvate carboxylaseda mitochondrial anaplerotic enzyme that maintains the supply of substrates for mitochondrial ETCd[38], two MCIV core subunit isoforms (COX4I2 and COX8B), and two additional ETC subunits believed to be associated with MCIV (NDUFA4L2 and HIGD1C) [20**,33**,39**]. Notably, the expression of Cox4i2, Cox8b, and Ndufa4l2 depends on the constitutive transcriptional activity of HIF2 a [21**,40*], which is highly expressed in CB Figure 1 Recording of secretory activity in single carotid body glomus cells by amperometry (a) and examples of single secretory events (b).(c,d) Selective abolition of the secretory response to hypoxia in glomus cells from mitochondrial complex I (MCI)-deficient mice. Activation of the cells by hypercapnia (CO 2 ) remains unaltered. Acute oxygen sensing Gao et al. 3 www.sciencedirect.com Current Opinion in Neurobiology 2025, 92:103022 glomus cells under normal (normoxic) conditions [20**,33**,41]. Moreover, Higd1c contains several putative hypoxia-responsive elements in its promoter region, and its expression in the CB also seems to depend on HIF2 a [40*]. Expression of Epas1dthe HIF2 a -encoding genedis required for CB embryonic development [42*] and disruption of Epas1 in adulthood strongly inhibits glomus cell responsiveness to hypoxia [21**] and the HVR [21**,43*]. Similar results are obtained by ablation of either the Cox4i2 gene [21*]or, with less potency, the Higd1c gene [39**]. However, Ndufa4l2 deficiency does not alter glomus cell sensitivity to hypoxia or the HVR [21**]. These findings suggest that much of the O 2 -sensing function of glomus cell MCIV depends on the HIF2 a -dependent expression of COX4I2 and HIGD1C. COX4, a nuclear-encoded protein, is one of the 13 subunits forming the catalytic core of CCO [44]. It is located at the periphery of MCIVand consists of a helix spanning the mitochondrial inner membrane, with N-terminus (matrix) and C-terminus (IMS) domains. In mammals, COX4 has two isoforms: COX4I1, which is widely expressed in all tissues, and COX4I2, which is tissuespecific and mainly expressed in the lung, vascular smooth muscle, and the CB [45*]. Interestingly, tissuespecific expression of Cox4i2 mRNA is induced by hypoxia through the upregulation of hypoxia inducible factors Figure 2 b 50 a.u. Hx Hx IMS H2O2 Hx Hx Hx Hx 2 min 0.005 Hx Hx 400/484 Hx Hx 2 min Hx Hx NADH hypoxia-induced mitochondrial signals wild-type MCI-KO MCI-KO/Ndi1 c hypoxic ventilatory response 5 min 100 150 200 250 300 breaths/min 21% O 2 tension wild-type MCI-deficient (MCI-KO) MCI-deficient/transgenic Ndi1 (MCI-KO/Ndi1) a MCIV MCI CoQH 2 MCIII MCII O 2 H 2 O (Hx) dynamic changes in ETC induced by hypoxia IMM CytC NADH H 2 O 2 OMM IMS Matrix H 2 O 2 10% Current Opinion in Neurobiology (a) Scheme of the dynamic changes in mitochondrial electron transport chain (ETC) induced by hypoxia (red arrows and symbols). OMM: outer mitochondrial membrane; IMS: intermembrane space; IMM: inner mitochondrial membrane. (b) Changes in the intracellular levels of NADH and IMS H 2 O 2 during repeated exposure to hypoxia monitored by microfluorimetry in single glomus cell from wild type mice, mitochondrial complex I (MCI)-deficient mice and MCI-deficient mice expressing transgenic yeast NDI1. (c) Hypoxic ventilatory response of the three mice strains indicated in (b). 4Interoception 2025 Current Opinion in Neurobiology 2025, 92:103022 www.sciencedirect.com (HIFs) and other transcription factors [21**,46*,47]. Although the differential functional characteristics of COX4 isoforms are not completely understood, it is known that the presence of COX4I2 instead of COX4I1 markedly accelerates CCO activity, possibly because this subunit is less susceptible to allosteric negative modulation by ATP [45*]. In permeabilized HEK cells, expression of COX4I2, instead of COX4I1, decreases the affinity of CCO for O 2 . However, the reported Km valuesdO 2 tensions of 0.5 and 1 mmHg in mitochondria exclusively expressing COX4I1 or COX4I2, respectivelyd[48], are much lower than the O 2 tension levels that modulate glomus cell function. Similar effects have been reported for HIGD1C expressed in HEK cells [39**], which suggests that, besides the potential effects of COX4I2 and HIGD1C on the binding affinity of CCO for O 2 ,other local factorsdsuch as the rate of electron transport or the interactions of COX4I2 and HIGD1C with NDUFA4L2 and/or COX8Bdinfluence the O 2 sensitivity of mitochondrial CCO in glomus cells. The presence of glomus cell-specific MCIV subunit isoforms may hinder the accessibility of O 2 to the catalytic center (CuB/heme a 3 ) and in this manner decrease the apparent affinity of the enzyme [21**,48,49]. In mitochondria with an accelerated ETC and high CCO turnover [20**,44], a decrease in O 2 availability during hypoxia could cause a mismatch between substrates (electrons and O 2 ), leading to an accumulation of reduced intermediates in the ETC and the generation of hypoxia-induced mitochondrial signals. The acute O 2 -sensing pathway in glomus cells is schematically summarized in Figure 3.Itdependson HIF2 a -dependent genetically specialized mitochondria, with high metabolic activity and high sensitivity to decreases in O 2 tension. Under hypoxic conditions, the relative lack of O 2 causes a decrease in CCO activity (step 1), leading to an accumulation of reduced cytochrome c and a backlog of reduced intermediates in the ETC (step 2). The hypoxia-induced dynamic changes in the ETC promote the formation of H 2 O 2 in the IMS andaccumulationofNADHinthematrix(step3). After equilibration with the cytosol, these mitochondrial signals inhibit K þ channels (step 4), leading to cell depolarization, opening of Ca 2þ channels (step 5), and release of transmitters that activate afferent fibers (step6).Itcouldbespeculatedthatinglomuscells, mitochondria specialized for acute O 2 -sensing are located near the membrane, close to ion channels, forming “O 2 -sensing microdomains”. Supporting this idea, electron microscopy studies reveal that the distance between mitochondria and the plasma membraneinglomuscellsisshorterthaninothercell types [50]. Figure 3 Current Opinion in Neurobiology Acute oxygen-sensing pathway in carotid body glomus cells. IMS, mitochondrial intermembrane space. See text for explanation. Acute oxygen sensing Gao et al. 5 www.sciencedirect.com Current Opinion in Neurobiology 2025, 92:103022 Conclusions CB glomus cells are the primary acute O 2 -sensing elements in the body and are essential for triggering rapid adaptive responses, such as hyperventilation and increased cardiac output, to hypoxia. Understanding of the mechanisms underlying O 2 sensing by glomus cells has significantly progressed in the last 20 years. Recent data indicate that the CB O 2 sensor is not a singlemolecule structure but rather an intricate signaling pathway that relies on specialized mitochondria, largely dependent on the constitutive high expression of HIF2 a [21**,40*,51**]. These mitochondria can detect changes in O 2 tension within physiological ranges and signal membrane ion channels to modulate glomus cell transmitter release. Similar O 2 -sensing mechanisms appear to operate in cells of the adrenal medulla and in systemic and pulmonary vascular smooth muscle, which also respond acutely to hypoxia [18**,52,53*,54**]. Beyond its pathophysiological significance, the acute O 2 - sensing pathway based on mitochondria-to-membrane signaling provides new possibilities in respiratory and cardiovascular pharmacology, including the potential therapeutic application of NADH mimetics, mitochondrial metabolism modulators, and HIF inhibitors. Authors’contribution All the authors contributed to the design of the article and the writing of the draft. J.L.-B. coordinated the writing of the paper. Declaration of competing interest There are no competing interests to disclose. Acknowledgements This research was supported by the Spanish Ministries of Science and Innovation and Health (Grants PID2022138131OB-I00, and PID2023-146862OB-100 funded by MCIN/AEI/10.13039/501100011033 to J.L.-B., L.G. 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Additionally, the combined heterologous expression of the two MCIV subunit isoforms, COX4I2 and HIGD1C, is reported to decrease the affinity of cytochrome coxidase for oxygen. 40 * .Colinas O, Moreno-Dominguez A, Ortega-Saenz P, LopezBarneo J: Constitutive expression of Hif2alpha confers acute O 2 sensitivity to carotid body glomus cells.Adv Exp Med Biol 2023, 1427:153–162. Demonstration that the expression of several genes encoding mitochondrial complex IV subunits, including Higd1c, in glomus cells depends on HIF2 a . 41. Tian H, Hammer RE, Matsumoto AM, Russell DW, McKnight SL: The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development.Genes Dev 1998, 12:3320–3324. 42 * .Macías D, Cowburn AS, Torres-Torrelo H, Ortega-Saenz P, Lopez-Barneo J, Johnson RS: HIF-2alpha is essential for carotid body development and function.Elife 2018, 7, e34681. Embryonic ablation of the gene encoding HIF2 a leads to atrophy of the carotid body (CB). Mice with CB atrophy exhibit an almost complete Acute oxygen sensing Gao et al. 7 www.sciencedirect.com Current Opinion in Neurobiology 2025, 92:103022 loss of the hypoxic ventilatory response, along with alterations in other relevant physiological and metabolic parameters. 43 * .Hodson EJ, Nicholls LG, Turner PJ, Llyr R, Fielding JW, Douglas G, Ratnayaka I, Robbins PA, Pugh CW, Buckler KJ, et al.: Regulation of ventilatory sensitivity and carotid body proliferation in hypoxia by the PHD2/HIF-2 pathway.J Physiol 2016, 594:1179–1195. Downregulation of the gene encoding HIF2 a results in a decrease in the hypoxic ventilatory response while overexpression of HIF2 a induces carotid body hypertrophy. 44. 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Rakoczy RJ, Schiebrel CM, Wyatt CN: Acute oxygen-sensing via mitochondria-generated temperature transients in rat carotid body type I cells.Front Physiol 2022, 13, 874039. 51 ** .Bishop T, Ratcliffe PJ: Genetic basis of oxygen sensing in the carotid body: HIF2alpha and an isoform switch in cytochrome c oxidase subunit 4.Sci Signal 2020, 13. An authoritative discussion of data reported in reference 21 within the broader context of HIF2 a -dependent regulation of the development and function of the carotid body (CB) and other catecholaminergic tissues. The discussion emphasizes the critical role of HIF2 a in regulating the expression of CB-specific mitochondrial complex IV subunit isoforms essential for acute oxygen sensing in the CB. 52. Dunham-Snary KJ, Wu D, Potus F, Sykes EA, Mewburn JD, Charles RL, Eaton P, Sultanian RA, Archer SL: Ndufs2, a core subunit of mitochondrial complex I, is essential for acute oxygen-sensing and hypoxic pulmonary vasoconstriction. Circ Res 2019, 124:1727–1746. 53 * .Sommer N, Huttemann M, Pak O, Scheibe S, Knoepp F, Sinkler C, Malczyk M, Gierhardt M, Esfandiary A, Kraut S, et al.: Mitochondrial complex IV subunit 4 isoform 2 is essential for acute pulmonary oxygen sensing.Circ Res 2017, 121: 424–438. This paper describes how the ablation of the gene encoding the mitochondrial complex IV subunit isoform COX4I2 abolished pulmonary hypoxic vasoconstriction. 54 ** .Moreno-Dominguez A, Colinas O, Arias-Mayenco I, Cabeza JM, Lopez-Ogayar JL, Chandel NS, Weissmann N, Sommer N, Pascual A, Lopez-Barneo J: Hif1alpha-dependent mitochondrial acute O 2 sensing and signaling to myocyte Ca 2+ channels mediate arterial hypoxic vasodilation.Nat Commun 2024, 15:6649. A detailed description of the mechanism underlying inhibition of L-type calcium channel activity in systemic arterial smooth muscle by acute hypoxia. The hypoxic signaling pathway described here is essentially similar to the mitochondrial-to-membrane signaling model proposed for carotid body glomus cells. 8Interoception 2025 Current Opinion in Neurobiology 2025, 92:103022 www.sciencedirect.com