Keith J. Buckler

An oxygen‐, acid‐ and anaesthetic‐sensitive TASK‐like background potassium channel in rat arterial chemoreceptor cells

1 The biophysical and pharmacological properties of an oxygen‐sensitive background K+ current in rat carotid body type‐I cells were investigated and compared with those of recently cloned two pore domain K+ channels. 2 Under symmetrical K+ conditions the oxygen‐sensitive whole cell K+ current had a linear dependence on voltage indicating a lack of intrinsic voltage sensitivity. 3 Single channel recordings identified a K+ channel, open at resting membrane potentials, that was inhibited by hypoxia. This channel had a single channel conductance of 14 pS, flickery kinetics and showed little voltage sensitivity except at extreme positive potentials. 4 Oxygen‐sensitive current was inhibited by 10 mM barium (57 % inhibition), 200 μM zinc (53 % inhibition), 200 μM bupivacaine (55 % inhibition) and 1 mM quinidine (105 % inhibition). 5 The general anaesthetic halothane (1.5 %) increased the oxygen‐sensitive K+ current (by 176 %). Halothane (3 mM) also stimulated single channel activity in inside‐out patches (by 240 %). Chloroform had no effect on background K+ channel activity. 6 Acidosis (pH 6.4) inhibited the oxygen‐sensitive background K+ current (by 56 %) and depolarised type‐I cells. 7 The pharmacological and biophysical properties of the background K+ channel are, therefore, analogous to those of the cloned channel TASK‐1. Using in situ hybridisation TASK‐1 mRNA was found to be expressed in type‐I cells. We conclude that the oxygen‐ and acid‐sensitive background K+ channel of carotid body type‐I cells is likely to be an endogenous TASK‐1‐like channel.

A novel oxygen‐sensitive potassium current in rat carotid body type I cells.

1. Hypoxic stimuli depolarize carotid body type I cells causing voltage‐gated calcium influx. This study investigates the cause of this membrane depolarization. Isolated type I cells from neonatal (11‐16 day) rat carotid bodies were used in the experiments. 2. Tetraethylammonium (TEA; 10 mM), 1 and 5 mM 4‐aminopyridine (4‐AP) and 20 nM charybdotoxin all failed to evoke a significant rise in [Ca2+]i. Similarly, in perforated patch whole‐cell recordings, a combination of 10 mM TEA and 5 mM 4‐AP failed to depolarize type I cells. 3. In type I cells voltage clamped at ‐70 mV, anoxia evoked a small inward current under control conditions, but had no effect in the absence of pipette and extracellular K+. 4. Anoxia decreased resting membrane conductance from 322 to 131 pS. The anoxia‐sensitive current (measured using voltage ramps from ‐100 to ‐40 mV) had a reversal potential of ‐89 mV in 4.5 mM Ko+ and ‐66 mV in 20 mM Ko+, indicating that this current was carried principally by potassium ions. In contrast, 10 mM TEA + 5 mM 4‐AP had little effect on the current‐voltage relationship of the cells over the same range. 5. This O2‐sensitive K+ conductance showed only mild outward rectification over the range ‐90 to +30 mV, which could be approximated by the Goldman‐Hodgkin‐Katz current equation. In addition, there was no time‐dependent activation or inactivation of membrane currents elicited by voltage steps in the range ‐100 to ‐30 mV. 6. The O2‐sensitive K+ conductance was inhibited by graded reductions in PO2 to 40 Torr and below, with a K1/2 of about 12 Torr. 7. The data suggest that hypoxia depolarizes type I cells principally through the inhibition of a small voltage‐insensitive resting (or background) K+ conductance, and not through the inhibition of voltage‐gated TEA and 4‐AP‐sensitive K+ channels (e.g. maxi‐K or KO2 channels), as has been previously suggested.

Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells.

1 Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4‐dinitrophenol (DNP) and carbonyl cyanide p‐trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mm Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response. 2 In perforated‐patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to −70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current. 3 Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response. 4 FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3–4 s. 5 We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage‐gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation.

Effects of hypercapnia on membrane potential and intracellular calcium in rat carotid body type I cells.

1. An acid‐induced rise in the intracellular calcium concentration ([Ca2+]i) of type I cells is thought to play a vital role in pH/PCO2 chemoreception by the carotid body. In this present study we have investigated the cause of this rise in [Ca2+]i in enzymatically isolated, neonatal rat type I cells. 2. The rise in [Ca2+]i induced by a hypercapnic acidosis was inhibited in Ca(2+)‐free media, and by 2 mM Ni2+. Acidosis also increased Mn2+ permeability. The rise in [Ca2+]i is dependent, therefore, upon a Ca2+ influx from the external medium. 3. The acid‐induced rise in [Ca2+]i was attenuated by both nicardipine and methoxyverapamil (D600), suggesting a role for L‐type Ca2+ channels. 4. Acidosis depolarized type I cells and often (approximately 50% of cells) induced action potentials. These effects coincided with a rise in [Ca2+]i. When membrane depolarization was prevented by a voltage clamp, acidosis failed to evoke a rise in [Ca2+]i. The acid‐induced rise in [Ca2+]i is a consequence, therefore, of membrane depolarization. 5. Acidosis decreased the resting membrane conductance of type I cells. The reversal potential of the acid‐sensitive current was about ‐75 mV. 6. A depolarization (30 mM [K+]o)‐induced rise in [Ca2+]i was blocked by either the removal of extracellular Ca2+ or the presence of 2 mM Ni2+, and was also substantially inhibited by nicardipine. Under voltage‐clamp conditions, [Ca2+]i displayed a bell‐shaped dependence on membrane potential. Depolarization raises [Ca2+]i, therefore, through voltage‐operated Ca2+ channels. 7. Caffeine (10 mM) induced only a small rise in [Ca2+]i (< 10% of that induced by 30 mM extracellular K+). Ca(2+)‐induced Ca2+ release is unlikely, therefore, to contribute greatly to the rise in [Ca2+]i induced by depolarization. 8. Although the replacement of extracellular Na+ with N‐methyl‐D‐glucamine (NMG), but not Li+, inhibited the acid‐induced rise in [Ca2+]i, this was due to membrane hyperpolarization and not to the inhibition of Na(+)‐Ca2+ exchange or Na(+)‐dependent action potentials. 9. The removal of extracellular Na+ (NMG substituted) did not have a significant effect upon the resting [Ca2+]i, and only slowed [Ca2+]i recovery slightly following repolarization from 0 to ‐60 mV. Therefore, if present, Na(+)‐Ca2+ exchange plays only a minor role in [Ca2+]i homeostasis. 10. In summary, in the neonatal rat type I cell, hypercapnic acidosis raises [Ca2+]i through membrane depolarization and voltage‐gated Ca2+ entry.

The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells.

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN−) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca2+, which was inhibited on removal of extracellular Ca2+. Under current clamp conditions rotenone and CN− caused a rapid membrane depolarization and elevation of [Ca2+]i. Voltage clamping cells to −70 mV substantially attenuated this rise in [Ca2+]i. Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K+ currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K+ current leading to membrane depolarization and voltage‐gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN− or rotenone or the mitochondrial uncoupler p‐trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.

MUSCARINIC AND NICOTINIC RECEPTORS RAISE INTRACELLULAR CA2+ LEVELS IN RAT CAROTID BODY TYPE I CELLS

1. The effects of cholinergic agonists upon intracellular free Ca2+ levels ([Ca2+]i) have been studied in enzymically isolated rat carotid body single type I cells, using indo‐1. 2. Acetylcholine (ACh) dose‐dependently increased [Ca2+]i in 55% of cells studied (EC50 = 13 microM). These [Ca2+]i rises were partially inhibited by atropine or mecamylamine. 3. Specific nicotinic and muscarinic agonists also elevated [Ca2+]i in a dose‐dependent manner (nicotine, EC50 = 15 microM; methacholine, EC50 = 20 microM). 4. While the majority of the ACh‐sensitive cells responded to both classes of cholinergic agonist, 29% responded exclusively to nicotinic stimulation and 9% responded exclusively to muscarinic stimulation. 5. In the presence of nicotinic agonists, Ca2+i responses were transient. In the presence of muscarinic agonists, Ca2+i responses consisted of an initial rise, which then declined to a lower plateau level. 6. Nicotinic responses were rapidly abolished in Ca(2+)‐free medium, suggesting that they are dependent on Ca2+ influx. 7. The plateau component of the muscarinic‐activated response was also abolished in Ca(2+)‐free conditions. The rapid initial [Ca2+]i rise, however, could still be evoked after several minutes in Ca(2+)‐free medium. Muscarine also increased Mn2+ quenching of intracellular fura‐2 fluorescence. These data suggest that the full muscarinic response depends on both Ca2+ release from intracellular stores and Ca2+o influx. 8. The results indicate that, in rat carotid body type I cells, both nicotinic and muscarinic acetylcholine receptors increase [Ca2+]i, but achieve this via different mechanisms. ACh may therefore play a role in carotid body function by modulating Ca2+i in the chemosensory type I cells.

Modulation of TASK‐like background potassium channels in rat arterial chemoreceptor cells by intracellular ATP and other nucleotides

The carotid bodys physiological role is to sense arterial oxygen, CO2 and pH. It is however, also powerfully excited by inhibitors of oxidative phosphorylation. This latter observation is the cornerstone of the mitochondrial hypothesis which proposes that oxygen is sensed through changes in energy metabolism. All of these stimuli act in a similar manner, i.e. by inhibiting a background TASK‐like potassium channel (KB) they induce membrane depolarization and thus neurosecretion. In this study we have evaluated the role of ATP in modulating KB channels. We find that KB channels are strongly activated by MgATP (but not ATP4−) within the physiological range (K1/2= 2.3 mm). This effect was mimicked by other Mg‐nucleotides including GTP, UTP, AMP‐PCP and ATP‐γ‐S, but not by PPi or AMP, suggesting that channel activity is regulated by a Mg‐nucleotide sensor. Channel activation by MgATP was not antagonized by either 1 mm AMP or 500 μm ADP. Thus MgATP is probably the principal nucleotide regulating channel activity in the intact cell. We therefore investigated the effects of metabolic inhibition upon both [Mg2+]i, as an index of MgATP depletion, and channel activity in cell‐attached patches. The extent of increase in [Mg2+]i (and thus MgATP depletion) in response to inhibition of oxidative phosphorylation were consistent with a decline in [MgATP]i playing a prominent role in mediating inhibition of KB channel activity, and the response of arterial chemoreceptors to metabolic compromise.

The lipid-activated two-pore domain K+ channel TREK-1 is resistant to hypoxia: implication for ischaemic neuroprotection

TREK‐1 is a member of the two‐pore domain potassium (K2P) channel family that is mechano‐, heat, pH, voltage and lipid sensitive. It is highly expressed in the central nervous system and probably encodes one of the previously described arachidonic acid‐activated K+ channels. Polyunsaturated fatty acids and lysophospholipids protect the brain against global ischaemia. Since both lipids are openers of TREK‐1, it has been suggested that this K2P channel is directly involved in neuroprotection. Recently, however, this view has been challenged by a report claiming that TREK‐1 and its activation by arachidonic acid is inhibited by hypoxia. In the present study, we demonstrate that the bubbling of saline with gases results in the loss of arachidonic acid from solution. Using experimental conditions which obviate this experimental artefact we demonstrate that TREK‐1 is resistant to hypoxia and is strongly activated by arachidonic acid even at low PO2 (< 4 Torr). Furthermore, hypoxia fails to affect basal as well as 2,4,6‐trinitrophenol‐ and acid‐stimulated TREK‐1 currents. These data are supportive for a possible role of TREK‐1 in ischaemic neuroprotection and in cell signalling via arachidonic acid.

Background leak K+-currents and oxygen sensing in carotid body type 1 cells

One model of oxygen sensing by the carotid body is that hypoxia depolarises type 1 cells leading to voltage-gated calcium entry and the secretion of neurotransmitters which then excite afferent nerves. This paper revues the mechanisms responsible for the membrane depolarisation in response to hypoxia. It concludes that depolarisation is caused not through the inhibition of calcium activated or delayed rectifier K+-channels but through the inhibition of an entirely new type of background K+-channel. This channel lacks sensitivity to the classical K+-channel inhibitors TEA and 4-AP. New evidence does however reveal that background K+-channels in the type 1 cell can be inhibited by Ba2+ and that Ba2+ depolarises isolated type 1 cells. Intriguingly, Ba2+ is the only K+-channel inhibitor thus far reported to stimulate the carotid body. These studies therefore support the hypothesis that depolarisation of the type 1 cell is an integral part of the oxygen sensing pathway in the carotid body.

Oxygen sensitivity of mitochondrial function in rat arterial chemoreceptor cells

•  Arterial chemoreceptors measure blood oxygen and are involved in the control of both breathing and the cardiovascular system. •  Oxygen is mostly used by cells in their mitochondria to generate energy. •  In this study we have investigated the effects of oxygen starvation (hypoxia) on the mitochondria of specialised oxygen sensing cells from arterial chemoreceptors. •  Our data confirm that the mitochondria of these oxygen sensing cells are unusually sensitive to modest hypoxia. This effect seems to stem from a reduced affinity of the oxygen utilising enzyme cytochrome oxidase for oxygen. •  These results are consistent with a functional adaptation of the mitochondria of oxygen sensing cells that may enable them to play a direct role in the oxygen sensing process itself.