Lipin-1-derived diacylglycerol activates intracellular TRPC3 which is critical for inflammatory signaling.

Exposure to Gram-negative bacterial LPS exacerbates host immune responses and may lead to sepsis, a life-threatening condition. Despite its high mortality and morbidity, no drugs specifically directed to treating sepsis are currently available. Using human cell genetic depletion, pharmacological inhibition, live-cell microscopy and organelle-targeted molecular sensors we present evidence that the channel TRPC3 is activated intracellularly during macrophage exposure to LPS and is essential for Ca2+ release from internal stores. In this manner, TRPC3 participates in cytosolic Ca2+ elevations, activation of the transcription factor NF-κB and cytokine upregulation. We also report that TRPC3 is activated by diacylglycerol generated by the phosphatidic acid phosphatase lipin-1. In accord with this, lipin-1-deficient cells exhibit reduced Ca2+ responses to LPS challenge. Finally, pharmacological inhibition of TRPC3 reduces systemic inflammation induced by LPS in mice. Collectively, our study unveils a central component of LPS-triggered Ca2+ signaling that involves intracellular sensing of lipin-1-derived DAG by TRPC3, and opens new opportunities for the development of strategies to treat LPS-driven inflammation.

Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries.

The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC50, of 14 and 100 µM). The low EC50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by ß-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries.

Differences in TRPC3 and TRPC6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension.

KEY POINTS: Canonical transient receptor potential (TRPC)3 and TRPC6 channels of vascular smooth muscle cells (VSMCs) mediate stretch- or agonist-induced cationic fluxes, contributing to membrane potential and vascular tone. Native TRPC3/C6 channels can form homo- or heterotetrameric complexes, which can hinder individual TRPC channel properties. The possibility that the differences in their association pattern may change their contribution to vascular tone in hypertension is unexplored. Functional characterization of heterologously expressed channels showed that TRPC6-containing complexes exhibited Pyr3/Pyr10-sensitive currents, whereas TRPC3-mediated currents were blocked by anti-TRPC3 antibodies. VSMCs from hypertensive (blood pressure high; BPH) mice have larger cationic basal currents insensitive to Pyr10 and sensitive to anti-TRPC3 antibodies. Consistently, myography studies showed a larger Pyr3/10-induced vasodilatation in BPN (blood pressure normal) mesenteric arteries. We conclude that the increased TRPC3 channel expression in BPH VSMCs leads to changes in TRPC3/C6 heteromultimeric assembly, with a higher TRPC3 channel contribution favouring depolarization of hypertensive VSMCs. ABSTRACT: Increased vascular tone in essential hypertension involves a sustained rise in total peripheral resistance. A model has been proposed in which the combination of membrane depolarization and higher L-type Ca2+ channel activity generates augmented Ca2+ influx into vascular smooth muscle cells (VSMCs), contraction and vasoconstriction. The search for culprit ion channels responsible for membrane depolarization has provided several candidates, including members of the canonical transient receptor potential (TRPC) family. TRPC3 and TRPC6 are diacylglycerol-activated, non-selective cationic channels contributing to stretch- or agonist-induced depolarization. Conflicting information exists regarding changes in TRPC3/TRPC6 functional expression in hypertension. However, although TRPC3-TRPC6 channels can heteromultimerize, the possibility that differences in their association pattern may change their functional contribution to vascular tone is largely unexplored. We probe this hypothesis using a model of essential hypertension (BPH mice; blood pressure high) and its normotensive control (BPN mice; blood pressure normal). First, non-selective cationic currents through homo- and heterotetramers recorded from transfected Chinese hamster ovary cells indicated that TRPC currents were sensitive to the selective antagonist Pyr10 only when TRPC6 was present, whereas intracellular anti-TRPC3 antibody selectively blocked TRPC3-mediated currents. In mesenteric VSMCs, basal and agonist-induced currents were more sensitive to Pyr3 and Pyr10 in BPN cells. Consistently, myography studies showed a larger Pyr3/10-induced vasodilatation in BPN mesenteric arteries. mRNA and protein expression data supported changes in TRPC3 and TRPC6 proportions and assembly, with a higher TRPC3 channel contribution in BPH VSMCs that could favour cell depolarization. These differences in functional and pharmacological properties of TRPC3 and TRPC6 channels, depending on their assembly, could represent novel therapeutical opportunities.

Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation.

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K(+) fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.

Kv1.3 channels modulate human vascular smooth muscle cells proliferation independently of mTOR signaling pathway.

Phenotypic modulation (PM) of vascular smooth muscle cells (VSMCs) is central to the process of intimal hyperplasia which constitutes a common pathological lesion in occlusive vascular diseases. Changes in the functional expression of Kv1.5 and Kv1.3 currents upon PM in mice VSMCs have been found to contribute to cell migration and proliferation. Using human VSMCs from vessels in which unwanted remodeling is a relevant clinical complication, we explored the contribution of the Kv1.5 to Kv1.3 switch to PM. Changes in the expression and the functional contribution of Kv1.3 and Kv1.5 channels were studied in contractile and proliferating VSMCs obtained from human donors. Both a Kv1.5 to Kv1.3 switch upon PM and an anti-proliferative effect of Kv1.3 blockers on PDGF-induced proliferation were observed in all vascular beds studied. When investigating the signaling pathways modulated by the blockade of Kv1.3 channels, we found that anti-proliferative effects of Kv1.3 blockers on human coronary artery VSMCs were occluded by selective inhibition of MEK/ERK and PLCγ signaling pathways, but were unaffected upon blockade of PI3K/mTOR pathway. The temporal course of the anti-proliferative effects of Kv1.3 blockers indicates that they have a role in the late signaling events essential for the mitogenic response to growth factors. These findings establish the involvement of Kv1.3 channels in the PM of human VSMCs. Moreover, as current therapies to prevent restenosis rely on mTOR blockers, our results provide the basis for the development of novel, more specific therapies.

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells.

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

Vascular smooth muscle ion channels in essential hypertension.

Hypertension is a highly prevalent chronic disease and the major risk factor for cardiovascular diseases, the leading cause of death worldwide. Hypertension is characterized by an increased vascular tone determined by the contractile state of vascular smooth muscle cells that depends on intracellular calcium levels. The interplay of ion channels determine VSMCs membrane potential and thus intracellular calcium that controls the degree of contraction, vascular tone and blood pressure. Changes in ion channels expression and function have been linked to hypertension, but the mechanisms and molecular entities involved are not completely clear. Furthermore, the literature shows discrepancies regarding the contribution of different ion channels to hypertension probably due to differences both in the vascular preparation and in the model of hypertension employed. Animal models are essential to study this multifactorial disease but it is also critical to know their characteristics to interpret properly the results obtained. In this review we summarize previous studies, using the hypertensive mouse (BPH) and its normotensive control (BPN), focused on the identified changes in the expression and function of different families of ion channels. We will focus on L-type voltage-dependent Ca2+ channels (Cav1.2), canonical transient receptor potential channels and four different classes of K+ channels: voltage-activated (Kv), large conductance Ca2+-activated (BK), inward rectifiers (Kir) and ATP-sensitive (KATP) K+ channels. We will describe the role of these channels in hypertension and we will discuss the importance of integrating individual changes in a global context to understand the complex interplay of ion channels in hypertension.

Elastin-like recombinamer-based devices releasing Kv1.3 blockers for the prevention of intimal hyperplasia: An in vitro and in vivo study.

Coronary artery disease (CAD) is the most common cardiovascular disorder. Vascular surgery strategies for coronary revascularization (either percutaneous or open) show a high rate of failure because of restenosis of the vessel, due to phenotypic switch of vascular smooth muscle cells (VSMCs) leading to proliferation and migration. We have previously reported that the inhibition of Kv1.3 channel function with selective blockers represents an effective strategy for the prevention of restenosis in human vessels used for coronary angioplasty procedures. However, delivery systems for controlled release of these drugs have not been investigated. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models. The dose and time course of PAP-1 release from ELRs click hydrogels was able to inhibit human VSMC proliferation in vitro as well as remodeling of human vessels in organ culture and restenosis in in vivo models. We conclude that this combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients. STATEMENT OF SIGNIFICANCE: Vascular surgery strategies for coronary revascularization show a high rate of failure, because of occlusion (restenosis) of the vessel, due to vascular smooth muscle cells proliferation and migration. We have previously reported that blockers of Kv1.3 channels represent an effective anti-restenosis therapy, but delivery systems for their controlled release have not being explored. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models, both in vivo and in vitro, and also in human vessels. We demonstrated that combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients.

Kv1.3 blockade inhibits proliferation of vascular smooth muscle cells in vitro and intimal hyperplasia in vivo.

The modulation of voltage-gated K+ (Kv) channels, involved in cell proliferation, arises as a potential therapeutic approach for the prevention of intimal hyperplasia present in in-stent restenosis (ISR) and allograft vasculopathy (AV). We studied the effect of PAP-1, a selective blocker of Kv1.3 channels, on development of intimal hyperplasia in vitro and in vivo in 2 porcine models of vascular injury. In vitro phenotypic modulation of VSMCs was associated to an increased functional expression of Kv1.3 channels, and only selective Kv1.3 channel blockers were able to inhibit porcine VSMC proliferation. The therapeutic potential of PAP-1 was then evaluated in vivo in swine models of ISR and AV. At 15-days follow-up, morphometric analysis demonstrated a substantial reduction of luminal stenosis in the allografts treated with PAP-1 (autograft 2.72 ± 1.79 vs allograft 10.32 ± 1.92 vs allograft + polymer 13.54 ± 8.59 vs allograft + polymer + PAP-1 3.06 ± 1.08 % of luminal stenosis; P = 0.006) in the swine model of femoral artery transplant. In the pig model of coronary ISR, using a prototype of PAP-1-eluting stent, no differences were observed regarding % of stenosis compared to control stents (31 ± 13 % vs 37 ± 18%, respectively; P = 0.372) at 28-days follow-up. PAP-1 treatment was safe and did not impair vascular healing in terms of delayed endothelialization, inflammation or thrombosis. However, an incomplete release of PAP-1 from stents was documented. We conclude that the use of selective Kv1.3 blockers represents a promising therapeutic approach for the prevention of intimal hyperplasia in AV, although further studies to improve their delivery method are needed to elucidate its potential in ISR.

MaxiK potassium channels in the function of chemoreceptor cells of the rat carotid body.

Hypoxia activates chemoreceptor cells of the carotid body (CB) promoting an increase in their normoxic release of neurotransmitters. Catecholamine (CA) release rate parallels the intensity of hypoxia. Coupling of hypoxia to CA release requires cell depolarization, produced by inhibition of O(2)-regulated K(+) channels, and Ca(2+) entering the cells via voltage-operated channels. In rat chemoreceptor cells hypoxia inhibits large-conductance, calcium-sensitive K channels (maxiK) and a two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) channel (TASK)-like channel, but the significance of maxiK is controversial. A proposal envisions maxiK contributing to set the membrane potential (E(m)) and the hypoxic response, but the proposal is denied by authors finding that maxiK inhibition does not depolarize chemoreceptor cells or alters intracellular Ca(2+) concentration or CA release in normoxia or hypoxia. We found that maxiK channel blockers (tetraethylammonium and iberiotoxin) did not modify CA release in rat chemoreceptor cells, in either normoxia or hypoxia, and iberiotoxin did not alter the Ca(2+) transients elicited by hypoxia. On the contrary, both maxiK blockers increased the responses elicited by dinitrophenol, a stimulus we demonstrate does not affect maxiK channels in isolated patches of rat chemoreceptor cells. We conclude that in rat chemoreceptor cells maxiK channels do not contribute to the genesis of the E(m), and that their full inhibition by hypoxia, preclude further inhibition by maxiK channel blockers. We suggest that full inhibition of this channel is required to generate the spiking behavior of the cells in acute hypoxia.

Contribution of Kv channels to phenotypic remodeling of human uterine artery smooth muscle cells.

Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K+ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kvalpha subunits in both preparations as well as their functional contribution to the K+ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K+ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling.

Oxygen sensitive Kv channels in the carotid body.

Hypoxic inhibition of K(+) channels has been documented in many native chemoreceptor cells, and is crucial to initiate reflexes directed to improve tissue O(2) supply. In the carotid body (CB) chemoreceptors, there is a general consensus regarding the facts that a decrease in P(O2) leads to membrane depolarization, increase of Ca(2+) entry trough voltage-dependent Ca(2+) channels and Ca(2+)-dependent release of neurotransmitters. Central to this pathway is the modulation by hypoxia of K(+) channels that triggers depolarization. However, the details of this process are still controversial, and even the molecular nature of these oxygen-sensitive K(+) (K(O2)) channels in the CB is hotly debated. Clearly there are inter-species differences, and even in the same preparation more that one K(O2) may be present. Here we recapitulate our present knowledge of the role of voltage dependent K(+) channels as K(O2) in the CB from different species, and their functional contribution to cell excitability in response to acute and chronic exposure to hypoxia.

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins.

Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

Oxygen-sensitive potassium channels in chemoreceptor cell physiology: making a virtue of necessity.

The characterization of the molecular mechanisms involved in low-oxygen chemotransduction has been an active field of research since the first description of an oxygen-sensitive K(+) channel in rabbit carotid body (CB) chemoreceptor cells. As a result, a large number of components of the transduction cascade, from O(2) sensors to O(2)-sensitive ion channels, have been found. Although the endpoints of the process are analogous, the heterogeneity of the elements involved in the different chemoreceptor tissues precludes a unifying theory of hypoxic signaling, and it has been a source of controversy. However, when these molecular constituents of the hypoxic cascade are brought back to their physiological context, it becomes clear that the diversity of mechanisms is necessary to build up an integrated cellular response that demands the concerted action of several O(2) sensors and several effectors.

Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes.

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.

Down regulation of Kv3.4 channels by chronic hypoxia increases acute oxygen sensitivity in rabbit carotid body.

The carotid body (CB) chemoreceptors participate in the ventilatory responses to acute and chronic hypoxia (CH). Arterial hypoxaemia increases breathing within seconds, and CB chemoreceptors are the principal contributors to this reflex hyperventilatory response. Acute hypoxia induces depolarization of CB chemoreceptors by inhibiting certain K+ channels, but the role of these channels in CH, as in high-altitude acclimatization, is less known. Here we explored the effects of prolonged (24-48 h) hypoxic exposure of rabbit CB chemoreceptor cells in primary cultures on the voltage-dependent K+ currents and on their response to acute hypoxia. We found that CH induces a decrease in the amplitude of outward K+ currents due to a reduction in a fast-inactivating BDS- and highly TEA-sensitive component of the current. In spite of this effect, acute hypoxic inhibition of K+ currents is increased in CH cultures, as well as hypoxia-induced depolarization. These data suggest that downregulation of this component (that does not contribute to the oxygen-sensitive K+ current (IKO2) participates in the hypoxic sensitization. Pharmacological, immunocytochemical and quantitative PCR (qPCR) experiments demonstrate that CH-induced decrease in outward K+ currents is due to a downregulation of the expression of Kv3.4 channels. Taken together, our results suggest that CH sensitization in rabbit CB could be achieved by an increase in the relative contribution of IKO2 to the outward K+ current as a consequence of the decreased expression of the oxygen-insensitive component of the current. We conclude that acute and chronic hypoxia can exert their effects acting on different molecular targets.

Characterization of the Kv channels of mouse carotid body chemoreceptor cells and their role in oxygen sensing.

As there are wide interspecies variations in the molecular nature of the O(2)-sensitive Kv channels in arterial chemoreceptors, we have characterized the expression of these channels and their hypoxic sensitivity in the mouse carotid body (CB). CB chemoreceptor cells were obtained from a transgenic mouse expressing green fluorescent protein (GFP) under the control of tyrosine hydroxylase (TH) promoter. Immunocytochemical identification of TH in CB cell cultures reveals a good match with GFP-positive cells. Furthermore, these cells show an increase in [Ca(2+)](i) in response to low P(O(2)), demonstrating their ability to engender a physiological response. Whole-cell experiments demonstrated slow-inactivating K(+) currents with activation threshold around -30 mV and a bi-exponential kinetic of deactivation (tau of 6.24 +/- 0.52 and 32.85 +/- 4.14 ms). TEA sensitivity of the currents identified also two different components (IC(50) of 17.8 +/- 2.8 and 940.0 +/- 14.7 microm). Current amplitude decreased reversibly in response to hypoxia, which selectively affected the fast deactivating component. Hypoxic inhibition was also abolished in the presence of low (10-50 microm) concentrations of TEA, suggesting that O(2) interacts with the component of the current most sensitive to TEA. The kinetic and pharmacological profile of the currents suggested the presence of Kv2 and Kv3 channels as their molecular correlates, and we have identified several members of these two subfamilies by single-cell PCR and immunocytochemistry. This report represents the first functional and molecular characterization of Kv channels in mouse CB chemoreceptor cells, and strongly suggests that O(2)-sensitive Kv channels in this preparation belong to the Kv3 subfamily.