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Mutations in the actively expressed, maternal allele of the imprinted KCNK9 gene cause Birk-Barel intellectual disability syndrome (BBIDS). Using a BBIDS mouse model, we identify here a partial rescue of the BBIDS-like behavioral and neuronal phenotypes mediated via residual expression from the paternal Kcnk9 (Kcnk9 pat ) allele. We further demonstrate that the second-generation HDAC inhibitor CI-994 induces enhanced expression from the paternally silenced Kcnk9 allele and leads to a full rescue of the behavioral phenotype suggesting CI-994 as a promising molecule for BBIDS therapy. Thus, these findings suggest a potential approach to improve cognitive dysfunction in a mouse model of an imprinting disorder.

External acidity induces catecholamine secretion by inhibiting TASK1-like channels in rat adrenal medullary (AM) cells. TASK channels can function as a heteromer or homomer in the TASK subfamily. In this study, we elucidate the molecular identity of TASK1-like channels in mouse AM cells using gene knockout. Genetic deletion of TASK1, but not TASK3, abolished the depolarizing inward current and catecholamine secretion in response to acidity, whereas it did not affect the resting current level. Immunocytochemistry revealed that AM cells exhibited predominantly TASK1-like and little TASK3-like immunoreactivity. A proximity ligation assay showed that TASK1/3 heteromeric channels were not formed in AM cells or PC12 cells. However, the exogenous expression of p11 in PC12 cells resulted in the heteromeric formation of TASK isoforms, which were mainly located in the cytoplasm, and p11 was not expressed in rat adrenal medullae or PC12 cells. In AM cells, genetic deletion of TASK1 resulted in enhancement of the immunoreactivity of the TALK2 channel, but not TASK3. The results indicate that TASK1 homomeric channels function as acidity sensors in AM cells, and that function is facilitated by the lack of p11 expression.-Inoue, M., Matsuoka, H., Lesage, F., Harada, K. Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

We previously proposed a role for the two-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy among the three TREK isoforms (TREK-1, TREK-2, and TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all three TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient [triple knockout (KO)] mice to either room air, 72-h HO, MV [high and low tidal volume (TV)], or a combination of HO + MV and measured quasistatic lung compliance, bronchoalveolar lavage (BAL) protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression and attempted to prevent HO-induced lung injury by prophylactically administering an exogenous surfactant (Curosurf). HO treatment increased lung injury in triple KO but not WT mice, including an elevated LIS, BAL protein concentration, and markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO + MV (low TV) further decreased lung compliance in triple KO but not WT mice, and HO + MV (high TV) was lethal for triple KO mice. In triple KO mice, the HO-induced lung injury was associated with decreased surfactant protein (SP) A and SPC but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors nuclear factor-1 (NF-1), NKX2.1/thyroid transcription factor-1 (TTF-1) or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple KO mice., (Copyright © 2017 the American Physiological Society.)

Despite intensive research, the exact function of TASK potassium channels in central and peripheral chemoreception is still under debate. In this study, we investigated the respiration of unrestrained TASK-3 (TASK-3 -/- ) and TASK-1/TASK-3 double knockout (TASK-1/3 -/- ) adult male mice in vivo using a plethysmographic device. Ventilation parameters of TASK-3 -/- mice were normal under control condition (21% O 2 ) and upon hypoxia and hypercapnia they displayed the physiological increase of ventilation. TASK-1/3 -/- mice showed increased ventilation under control conditions. This increase of ventilation was caused by increased tidal volumes (V T ), a phenomenon similarly observed in TASK-1 -/- mice. Under acute hypoxia, TASK-1/3 -/- mice displayed the physiological increase of the minute volume. Interestingly, this increase was not related to an increase of the respiratory frequency (f R ), as observed in wild-type mice, but was caused by a strong increase of V T . This particular respiratory phenotype is reminiscent of the respiratory phenotype of carotid body-denervated rodents in the compensated state. Acute hypercapnia (5% CO 2 ) stimulated ventilation in TASK-1/3 -/- and wild-type mice to a similar extent; however, at higher CO 2 concentrations (>5% CO 2 ) the stimulation of ventilation was more pronounced in TASK-1/3 -/- mice. At hyperoxia (100% O 2 ), TASK-1 -/- , TASK-3 -/- and wild-type mice showed the physiological small decrease of ventilation. In sharp contrast, TASK-1/3 -/- mice exhibited an abnormal increase of ventilation under hyperoxia. In summary, these measurements showed a grossly normal respiration of TASK-3 -/- mice and a respiratory phenotype of TASK-1/3 -/- mice that was characterized by a markedly enhanced tidal volume, similar to the one observed in TASK-1 -/- mice. The abnormal hyperoxia response, exclusively found in TASK-1/3 -/- double mutant mice, indicates that both TASK-1 and TASK-3 are essential for the hyperoxia-induced hypoventilation. The peculiar respiratory phenotype of TASK-1/3 knockout mice is reminiscent of the respiration of animals with long-term carotid body dysfunction. Taken together, TASK-1 and TASK-3 appear to serve specific and distinct roles in the complex processes underlying chemoreception and respiratory control., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Task3 K(+) channels are highly expressed in the adrenal cortex and contribute to the angiotensin II and K(+) sensitivity of aldosterone-producing glomerulosa cells. Adult Task3(-/-) mice display a partially autonomous aldosterone secretion, subclinical hyperaldosteronism, and salt-sensitive hypertension. Here, we investigated the age dependence of the adrenal phenotype of Task3(-/-) mice. Compared with adults, newborn Task3(-/-) mice displayed a severe adrenal phenotype with strongly increased plasma levels of aldosterone, corticosterone, and progesterone. This adrenocortical dysfunction was accompanied by a modified gene expression profile. The most strongly up-regulated gene was the protease renin. Real-time PCR corroborated the strong increase in adrenal renin expression, and immunofluorescence revealed renin-expressing cells in the zona fasciculata. Together with additional factors, activation of the local adrenal renin system is probably causative for the severely disturbed steroid hormone secretion of neonatal Task3(-/-) mice. The changes in gene expression patterns of neonatal Task3(-/-) mice could also be relevant for other forms of hyperaldosteronism.

Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake.

TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.

K+ channels with two-pore domain (K2p) form a large family of hyperpolarizing channels. They produce background currents that oppose membrane depolarization and cell excitability. They are involved in cellular mechanisms of apoptosis, vasodilatation, anesthesia, pain, neuroprotection and depression. This review focuses on TREK-1, TREK-2 and TRAAK channels subfamily and on the mechanisms that contribute to their molecular heterogeneity and functional regulations. Their molecular diversity is determined not only by the number of genes but also by alternative splicing and alternative initiation of translation. These channels are sensitive to a wide array of biophysical parameters that affect their activity such as unsaturated fatty acids, intra- and extracellular pH, membrane stretch, temperature, and intracellular signaling pathways. They interact with partner proteins that influence their activity and their plasma membrane expression. Molecular heterogeneity, regulatory mechanisms and protein partners are all expected to contribute to cell specific functions of TREK currents in many tissues.

Sensing of sugar by specialized 'glucose-inhibited' cells helps organisms to counteract swings in their internal energy levels. Evidence from several cell types in both vertebrates and invertebrates suggests that this process involves sugar-induced stimulation of plasma membrane K(+) currents. However, the molecular composition and the mechanism of activation of the underlying channel(s) remain controversial. In mouse hypothalamic neurones and neurosecretory cells of the crab Cancer borealis, glucose stimulates K(+) currents displaying leak-like properties. Yet knockout of some of the candidate 'leak' channel subunits encoded by the KCNK gene family so far failed to abolish glucose inhibition of hypothalamic cells. Moreover, in other tissues, such as the carotid body, glucose-stimulated K(+) channels appear to be not leak-like but voltage-gated, suggesting that glucose-induced inhibition may engage multiple types of K(+) channels. Other mechanisms of glucose-induced inhibition, such as hyperpolarization mediated by opening of Cl(-) channels and depolarization block caused by closure of K(ATP) channels have also been proposed. Here we review known ionic and pharmacological features of glucose-induced inhibition in different cell types, which may help to identify its molecular correlates.

Introduction: K(+) channels allow the passive and selective transport of K(+) ions through the membranes. They control K(+) homeostasis, neuronal and muscular excitabilities, and neurotransmitter and hormone release., Exegesis: K(+) channels are composed of pore-forming subunits associated with regulatory subunits. Many different K(+) channels have been identified., Conclusion: This diversity is stressed by the growing number of genetic and acquired diseases associated with these channels.

TWIK1 belongs to a family of K(+) channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.

The distribution of two-pore-domain (2P-domain) K(+) channels of the TREK subfamily was studied using immunocytochemistry in the peripheral vestibular system of mouse and rat. Using RT-PCR, the mRNA for TREK-1, but not for TREK-2 or TRAAK, were detected in mouse vestibular endorgans and ganglia. The TREK-1 channel protein was immunodetected in both nerve fibers and nerve cell bodies in the vestibular ganglion, both afferent fibers and nerve calyces innervating type I hair cells in the utricle and cristae. The post-synaptic localization in afferent calyces may suggest a neuroprotective role in glutamatergic excitotoxicity during ischemic conditions. In non-neuronal cells, TREK-1 was immunodetected in the apical membrane of dark cells and transitional cells, both of which are involved in endolymph K(+) secretion and recycling. TREK-1 may subserve some neuroprotective function in afferent nerve fibers as well as play a role in endolymph potassium homeostasis.

The acid- and volume-sensitive TASK2 K+ channel is strongly expressed in renal proximal tubules and papillary collecting ducts. This study was aimed at investigating the role of TASK2 in renal bicarbonate reabsorption by using the task2 -/- mouse as a model. After backcross to C57BL6, task2 -/- mice showed an increased perinatal mortality and, in adulthood, a reduced body weight and arterial blood pressure. Patch-clamp experiments on proximal tubular cells indicated that TASK2 was activated during HCO3- transport. In control inulin clearance measurements, task2 -/- mice showed normal NaCl and water excretion. During i.v. NaHCO3 perfusion, however, renal Na+ and water reabsorption capacity was reduced in -/- animals. In conscious task2 -/- mice, blood pH, HCO3- concentration, and systemic base excess were reduced but urinary pH and HCO3- were increased. These data suggest that task2 -/- mice exhibit metabolic acidosis caused by renal loss of HCO3-. Both in vitro and in vivo results demonstrate the specific coupling of TASK2 activity to HCO3- transport through external alkalinization. The consequences of the task2 gene inactivation in mice are reminiscent of the clinical manifestations seen in human proximal renal tubular acidosis syndrome.

Background K+ conductances are a major determinant of membrane resting potential and input resistance, two key components of neuronal excitability. Background channels have been cloned and form a K+ channel family structurally different from Kv, KCa and Kir channels. These channels with 2P domains (K2P channels) are voltage- and time-independent. They are relatively insensitive to classical potassium channels blockers such as TEA, 4-AP, Ba2+ and Cs+. TASK and TREK subunits are widely expressed in the nervous system. Open at rest, these channels mainly contribute to the resting potential of somatic motoneurons, brainstem respiratory and chemoreceptor neurones, and cerebellar granule cells. K2P channels are regulated by numerous physical and chemical stimuli including extracellular and intracellular pH, temperature, hypoxia, pressure, bioactive lipids, and neurotransmitters. The regulation of these background K+ channels profoundly alters the neuronal excitability. For example, in Aplysia, regulation of a background potassium conductance by neurotransmitters is involved in synaptic modulation, a simple and primitive form of learning. The recent discovery that clinical compounds such as volatile anaesthetics and other neuroprotective agents including riluzole and unsaturated fatty acids activate K2P channels suggest that neuronal background K+ channels are attractive targets for the development of new drugs.

Group I metabotropic glutamate receptors (mGluRs) are implicated in diverse processes such as learning, memory, epilepsy, pain and neuronal death. By inhibiting background K(+) channels, group I mGluRs mediate slow and long-lasting excitation. The main neuronal representatives of this K(+) channel family (K(2P) or KCNK) are TASK and TREK. Here, we show that in cerebellar granule cells and in heterologous expression systems, activation of group I mGluRs inhibits TASK and TREK channels. D-myo-inositol-1,4,5-triphosphate and phosphatidyl-4,5-inositol-biphosphate depletion are involved in TASK channel inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels. Mechanisms described here with group I mGluRs will also probably stand for many other receptors of hormones and neurotransmitters.

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.

During tooth development, odontoblasts are the cells that form dentin and possibly mediate early stages of sensory processing in teeth. It is suggested that ion channels assist in these events. Indeed, mechanosensitive potassium currents, transducing mechanical stimuli into electrical cell signals, have been previously recorded in the human odontoblast cell membrane. Here, we show by RT-PCR that the mechanosensitive potassium channel TREK-1 (a member of the two-pore-domain potassium channel family) is overexpressed in these cultured cells compared with pulp cells in vitro. In situ hybridization showed that transcripts are detected in the odontoblast layer in vivo. The use of antibodies shows that TREK-1 is strongly expressed in the membrane of coronal odontoblasts and absent in the root. This distribution is related to the spatial distribution of nerve endings identified by labeling of the low-affinity nerve growth factor (NGF) receptor (p75(NTR)). These results demonstrate the expression of TREK-1 in human odontoblasts in vitro and in vivo.

Background or leak conductances are a major determinant of membrane resting potential and input resistance, two key components of neuronal excitability. The primary structure of the background K(+) channels has been elucidated. They form a family of channels that are molecularly and functionally divergent from the voltage-gated K(+) channels and inward rectifier K(+) channels. In the nervous system, the main representatives of this family are the TASK and TREK channels. They are relatively insensitive to the broad-spectrum K(+) channel blockers tetraethylammonium (TEA), 4-aminopyridine (4-AP), Cs(+), and Ba(2+). They display very little time- or voltage-dependence. Open at rest, they are involved in the maintenance of the resting membrane potential in somatic motoneurones, brainstem respiratory and chemoreceptor neurones, and cerebellar granule cells. TASK and TREK channels are also the targets of many physiological stimuli, including intracellular and extracellular pH and temperature variations, hypoxia, bioactive lipids, and neurotransmitter modulation. Integration of these different signals has major effects on neuronal excitability. Activation of some of these channels by volatile anaesthetics and by other neuroprotective agents, such as riluzole and unsaturated fatty acids, illustrates how the neuronal background K(+) conductances are attractive targets for the development of new drugs.

Many members of the two-pore-domain potassium (K(+)) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether I(KAA), a background K(+) current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that I(KAA), like TREK-1, was outwardly rectifying in physiological K(+) conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, I(KAA) was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, I(KAA) was inhibited by chlorophenylthio-cAMP (500 micromol/L) and also by stimulation of beta-adrenergic receptors with isoproterenol (1 micromol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by beta-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.

We isolated three novel 2P domain K(+) channel subunits from human. The first two subunits, TALK-1 and TALK-2, are distantly related to TASK-2. Their genes form a tight cluster of 25 kb on chromosome 6p21.1-p21.2. The corresponding channels produce quasi-instantaneous and non-inactivating currents that are activated at alkaline pHs. These currents are sensitive to Ba(2+), quinine, quinidine, chloroform, halothane, and isoflurane but are not affected by TEA, 4-AP, Cs(+), arachidonic acid, hypertonic solutions, agents activating protein kinases C and A, changes of internal Ca(2+) concentrations, and by activation of G(i) and G(q) proteins. TALK-1 is exclusively expressed in the pancreas. TALK-2 is mainly expressed in the pancreas, but is also expressed at a lower level in liver, placenta, heart, and lung. We also cloned a third subunit, named hTHIK-2 which is present in many tissues with high levels again in the pancreas but which could not be functionally expressed., (Copyright 2001 Academic Press.)

The two-pore-domain K(+) channels, or K(2P) channels, constitute a novel class of K(+) channel subunits. They have four transmembrane segments and are active as dimers. The tissue distribution of these channels is widespread, and they are found in both excitable and nonexcitable cells. K(2P) channels produce currents with unusual characteristics. They are quasi-instantaneous and noninactivating, and they are active at all membrane potentials and insensitive to the classic K(+) channel blockers. These properties designate them as background K(+) channels. They are expected to play a major role in setting the resting membrane potential in many cell types. Another salient feature of K(2P) channels is the diversity of their regulatory mechanisms. The weak inward rectifiers TWIK-1 and TWIK-2 are stimulated by activators of protein kinase C and decreased by internal acidification, the baseline TWIK-related acid-sensitive K(+) (TASK)-1 and TASK-2 channels are sensitive to external pH changes in a narrow range near physiological pH, and the TWIK-related (TREK)-1 and TWIK-related arachidonic acid-stimulated K(+) (TRAAK) channels are the first cloned polyunsaturated fatty acids-activated and mechanogated K(+) channels. The recent demonstration that TASK-1 and TREK-1 channels are activated by inhalational general anesthetics, and that TRAAK is activated by the neuroprotective agent riluzole, indicates that this novel class of K(+) channels is an interesting target for new therapeutic developments.

Mechano-sensitive and fatty acid-activated K(+) belong to the structural class of K(+) channel with two pore domains. Here, we report the isolation and the characterization of a novel member of this family. This channel, called TREK2, is closely related to TREK1 (78% of homology). Its gene is located on chromosome 14q31. TREK2 is abundantly expressed in pancreas and kidney and to a lower level in brain, testis, colon, and small intestine. In the central nervous system, TREK2 has a widespread distribution with the highest levels of expression in cerebellum, occipital lobe, putamen, and thalamus. In transfected cells, TREK2 produces rapidly activating and non-inactivating outward rectifier K(+) currents. The single-channel conductance is 100 picosiemens at +40 mV in 150 mm K(+). The currents can be strongly stimulated by polyunsaturated fatty acid such as arachidonic, docosahexaenoic, and linoleic acids and by lysophosphatidylcholine. The channel is also activated by acidification of the intracellular medium. TREK2 is blocked by application of intracellular cAMP. As with TREK1, TREK2 is activated by the volatile general anesthetics chloroform, halothane, and isoflurane and by the neuroprotective agent riluzole. TREK2 can be positively or negatively regulated by a variety of neurotransmitter receptors. Stimulation of the G(s)-coupled receptor 5HT4sR or the G(q)-coupled receptor mGluR1 inhibits channel activity, whereas activation of the G(i)-coupled receptor mGluR2 increases TREK2 currents. These multiple types of regulations suggest that TREK2 plays an important role as a target of neurotransmitter action.

The present study was conducted to explore the possible contribution of a recently described leak K+ channel, TASK (TWIK-related acid-sensitive K+ channel), to the high resting K+ conductance of adrenal glomerulosa cells. Northern blot analysis showed the strongest TASK message in adrenal glomerulosa (capsular) tissue among the examined tissues including heart and brain. Single-cell PCR demonstrated TASK expression in glomerulosa cells. In patch-clamp experiments performed on isolated glomerulosa cells the inward current at -100 mV in 30 mM [K+] (reflecting mainly potassium conductance) was pH sensitive (17+/-2% reduction when the pH changed from 7.4 to 6.7). In Xenopus oocytes injected with mRNA prepared from adrenal glomerulosa tissue the expressed K+ current at -100 mV was virtually insensitive to tetraethylammonium (3 mM) and 4-aminopyridine (3 mM). Ba2+ (300 microM) and Cs+ (3 mM) induced voltage-dependent block. Lidocaine (1 mM) and extracellular acidification from pH 7.5 to 6.7 inhibited the current (by 28% and 16%, respectively). This inhibitory profile is similar (although it is not identical) to that of TASK expressed by injecting its cRNA. In oocytes injected with adrenal glomerulosa mRNA, TASK antisense oligonucleotide reduced significantly the expression of K+ current at -100 mV, while the sense oligonucleotide failed to have inhibitory effect. Application of angiotensin II (10 nM) both in isolated glomerulosa cells and in oocytes injected with adrenal glomerulosa mRNA inhibited the K+ current at -100 mV. Similarly, in oocytes coexpressing TASK and ATla angiotensin II receptor, angiotensin II inhibited the TASK current. These data together indicate that TASK contributes to the generation of high resting potassium permeability of glomerulosa cells, and this background K+ channel may be a target of hormonal regulation.

Peripheral and central thermoreceptors are involved in sensing ambient and body temperature, respectively. Specialized cold and warm receptors are present in dorsal root ganglion sensory fibres as well as in the anterior/preoptic hypothalamus. The two-pore domain mechano-gated K(+) channel TREK-1 is highly expressed within these areas. Moreover, TREK-1 is opened gradually and reversibly by heat. A 10 degrees C rise enhances TREK-1 current amplitude by approximately 7-fold. Prostaglandin E2 and cAMP, which are strong sensitizers of peripheral and central thermoreceptors, reverse the thermal opening of TREK-1 via protein kinase A-mediated phosphorylation of Ser333. Expression of TREK-1 in peripheral sensory neurons as well as in central hypothalamic neurons makes this K(+) channel an ideal candidate as a physiological thermoreceptor.

Riluzole (RP 54274) is a potent neuroprotective agent with anticonvulsant, sedative, and anti-ischemic properties. It is currently used in the treatment of amyotrophic lateral sclerosis. This article reports that riluzole is an activator of TREK-1 and TRAAK, two important members of a new structural family of mammalian background K(+) channels with four transmembrane domains and two pore regions. Whereas riluzole activation of TRAAK is sustained, activation of TREK-1 is transient and is followed by an inhibition. The inhibitory process is attributable to an increase of the intracellular cAMP concentration by riluzole that produces a protein kinase A-dependent inhibition of TREK-1. Mutants of TREK-1 lacking the Ser residue where the kinase A phosphorylation takes place are activated in a sustained manner by riluzole. TRAAK is permanently activated by riluzole because, unlike TREK-1, it lacks the negative regulation by cAMP.

The two P domain hTRAAK K(+) channel has been cloned from human brain. hTRAAK cDNA encodes a 393 amino acid polypeptide with 88% of homology with its mouse counterpart. The hTRAAK gene has been mapped to chromosome 11q13 and the study of its organization indicates that the hTRAAK open reading frame is contained in six exons. hTRAAK is expressed abundantly in brain and placenta. In COS cells, hTRAAK currents are K(+)-selective, instantaneous and non-inactivating. These currents are insensitive to the classical K(+) channels blockers 4-aminopyridine, tetraethylammonium, barium and quinidine, but are strongly stimulated by application of arachidonic acid as well as other polyunsaturated fatty acids. hTRAAK can also be activated by a stretch of the membrane.

The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honoré, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honoré, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honoré, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell-attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation.

The recent cloning, functional expression and brain localization of two new potassium channels, TREK and TRAAK, led us to examine whether both channels are present in peripheral nerves and can move along axons by means of axonal transport mechanisms. Using specific antibodies directed against TREK and TRAAK peptides, we found that immunoreactivity for both potassium channels accumulates above and below a ligature in rat sciatic nerves. The process was rapid and bidirectional suggesting that the channels are associated with vesicles. This represents the first report on the axonal transport of potassium channels.

TRAAK is the sole member of the emerging class of 2P domain K+ channels to be exclusively expressed in neuronal cells. TRAAK produces baseline K+ currents which are strongly stimulated by arachidonic acid and by mechanical stretch, and which are insensitive to the classical K+ channel blockers tetraethylammonium, Ba2+, and Cs+. This report describes the immunolocalization of TRAAK in brain, spinal cord, and retina of the adult mouse. The most striking finding is the widespread distribution of the TRAAK immunoreactivity, with a prominent staining of the cerebellar cortex, neocortex, hippocampus, dentate gyrus, subiculum, the dorsal hippocampal commissure, thalamus, caudate-putamen, olfactory bulb, and several nuclei in the brainstem. Virtually all neurons express TRAAK, and the highest immunoreactivity was seen in soma, and to a lesser degree in axons and/or dendrites in most areas in brain and spinal cord. In the retina, the TRAAK protein is concentrated to the soma of ganglion cells and to the dendrites of all other neurons. Taken together, these results show a wide distribution of TRAAK, a mechanosensitive and arachidonic acid-stimulated neuron-specific baseline K+ channel, in brain, spinal cord and retina.

TREK-1 is a member of the novel structural class of K(+) channels with four transmembrane segments and two pore domains in tandem (1,2). TREK-1 is opened by membrane stretch and arachidonic acid. It is also an important target for volatile anesthetics (2,3). Here we show that internal acidification opens TREK-1. Indeed, lowering pH(i) shifts the pressure-activation relationship toward positive values and leads to channel opening at atmospheric pressure. The pH(i)-sensitive region in the carboxyl terminus of TREK-1 is the same that is critically involved in mechano-gating as well as arachidonic acid activation. A convergence, which is dependent on the carboxyl terminus, occurs between mechanical, fatty acids and acidic stimuli. Intracellular acidosis, which occurs during brain and heart ischemia, will induce TREK-1 opening with subsequent K(+) efflux and hyperpolarization.

The identification of multigene families encoding K-ATPases, K channels, and K transporters is a major step in understanding the molecular mechanisms engaged in K homeostasis. These membrane proteins, which also transport Na, H, or Cl ions, have been shown to play fundamental roles in cellular housekeeping functions (volume regulation, uptake of nutrients) and in specialized tissue functions (transepithelial transport of solutes and water, uptake of neurotransmitters, control of vascular tone). The association of mutations (especially in the K channels) with human diseases and disorders as well as the creation of animal models harboring specific gene inactivation should allow investigators to reach nonambiguous conclusions about the roles of these genes. These approaches should be complemented by techniques such as DNA array and chip hybridization and computer-based simulation in order to form an integrated view of the functional interactions between the genes underlying physiological and pathological processes.

Volatile anesthetics produce safe, reversible unconsciousness, amnesia and analgesia via hyperpolarization of mammalian neurons. In molluscan pacemaker neurons, they activate an inhibitory synaptic K+ current (IKAn), proposed to be important in general anesthesia. Here we show that TASK and TREK-1, two recently cloned mammalian two-P-domain K+ channels similar to IKAn in biophysical properties, are activated by volatile general anesthetics. Chloroform, diethyl ether, halothane and isoflurane activated TREK-1, whereas only halothane and isoflurane activated TASK. Carboxy (C)-terminal regions were critical for anesthetic activation in both channels. Thus both TREK-1 and TASK are possibly important target sites for these agents.

Mouse KCNK6 is a new subunit belonging to the TWIK channel family. This 335-amino acid polypeptide has four transmembrane segments, two pore-forming domains, and a Ca2+-binding EF-hand motif. Expression of KCNK6 transcripts is principally observed in eyes, lung, stomach and embryo. In the eyes, immunohistochemistry reveals protein expression only in some of the retina neurons. Although KCNK6 is able to dimerize as other functional two-P domain K+ channels when it is expressed in COS-7 cells, it remains in the endoplasmic reticulum and is unable to generate ionic channel activity. Deletions, mutations, and chimera constructions suggest that KCNK6 is not an intracellular channel but rather a subunit that needs to associate with a partner, which remains to be discovered, in order to reach the plasma membrane. A closely related human KCNK7-A subunit has been cloned. KCNK7 displays an intriguing GLE sequence in its filter region instead of the G(Y/F/L)G sequence, which is considered to be the K+ channel signature. This subunit is alternatively spliced and gives rise to the shorter forms KCNK7-B and -C. None of the KCNK7 structures can generate channel activity by itself. The KCNK7 gene is situated on chromosome 11, in the q13 region, where several candidate diseases have been identified.

The novel structural class of mammalian channels with four transmembrane segments and two pore regions comprise background K+ channels (TWIK-1, TREK-1, TRAAK, TASK, and TASK-2) with unique physiological functions (1-6). Unlike its counterparts, TRAAK is only expressed in neuronal tissues, including brain, spinal cord, and retina (1). This report shows that TRAAK, which was known to be activated by arachidonic acid (3), is also opened by membrane stretch. Mechanical activation of TRAAK is induced by a convex curvature of the plasma membrane and can be mimicked by the amphipathic membrane crenator trinitrophenol. Cytoskeletal elements are negative tonic regulators of TRAAK. Membrane depolarization and membrane crenation synergize with stretch-induced channel opening. Finally, TRAAK is reversibly blocked by micromolar concentrations of gadolinium, a well known blocker of stretch-activated channels. Mechanical activation of TRAAK in the central nervous system may play an important role during growth cone motility and neurite elongation.

Several K+ conductances have been identified in the kidney, with specific properties and localization in distinct cell types and membrane domains. On the other hand, several K+ channels have been characterized at the molecular level. By immunolocalization, we show that a new inward rectifying K+ channel, TWIK-1, is specifically expressed in distinct tubular segments and cell types of the rat kidney. In the proximal tubule, TWIK-1 prevails in the initial portions (convoluted part), where it is restricted to the apical (brush-border) membrane. In the collecting duct, immunofluorescence was intracellular or confined to the apical membrane and restricted to intercalated cells, i.e., in cells lacking aquaporin-2, as shown by double immunofluorescence. TWIK was also expressed in medullary and cortical parts of the thick limb of the loop of Henle, identified with an anti-Tamm-Horsfall protein antibody (double immunofluorescence). The intensity of TWIK-1 immunolabeling was unchanged in rats fed a low-Na+ or a low-K+ diet. Because TWIK-1 shares common properties with the low-conductance apical K+ channel of the collecting duct, we propose that it could play a role in K+ secretion, complementary to ROMK, another recently characterized K+ channel located in principal cells of the cortical collecting duct and in the loop of Henle.

A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18-22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+ equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+ concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mM K+. TASK-2 currents are blocked by quinine (IC50 = 22 microM) and quinidine (65% of inhibition at 100 microM) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6. 5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.

Aplysia S-type K+ channels of sensory neurons play a dominant role in presynaptic facilitation and behavioural sensitization. They are closed by serotonin via cAMP-dependent phosphorylation, whereas they are opened by arachidonic acid, volatile general anaesthetics and mechanical stimulation. We have identified a cloned mammalian two P domain K+ channel sharing the properties of the S channel. In addition, the recombinant channel is opened by lipid bilayer amphipathic crenators, while it is closed by cup-formers. The cytoplasmic C-terminus contains a charged region critical for chemical and mechanical activation, as well as a phosphorylation site required for cAMP inhibition.

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.

We have recently discovered a new class of potassium channels with two pore-forming domains and four membrane-spanning domains. When heterologously expressed, these channels produce time- and voltage-independent currents that classify them as background or leak channels. TWIK (for tandem of P domains in a weak inwardly rectifying K+ channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42-43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.

G-protein-gated inward rectifier potassium channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain, and were recently shown to be candidates for genetic mutations leading to neuronal cell death. This report describes the localization of G-protein-gated inward rectifier potassium channel-2 and G-protein-gated inward rectifier potassium channel-4 proteins in the rat brain, as assessed by immunocytochemistry. G-protein-gated inward rectifier potassium channel-2 immunoreactivity was widely distributed throughout the brain, with the strongest staining seen in the hippocampus, septum, granule cell layer of the cerebellum, amygdala and substantia nigra pars compacta. In contrast, G-protein-gated inward rectifier potassium channel-4 immunoreactivity was restricted to some neuronal populations, such as Purkinje cells and neurons of the globus pallidus and the ventral pallidum. The presence of G-protein-gated inward rectifier potassium channel-2 immunoreactivity in substantia nigra pars compacta dopaminergic neurons was confirmed by showing its co-localization with tyrosine hydroxylase by double immunocytochemistry, and also by selectively lesioning dopaminergic neurons with the neurotoxin 6-hydroxydopamine. At the cellular level both proteins were localized in neuronal cell bodies and dendrites, but clear differences were seen in the degree of dendritic staining among neuronal groups. For some neuronal groups the staining of distal dendrites (notably dendritic spines) was strong, while for others the cell body and proximal dendrites were preferentially labelled. In addition, some of the results suggest that G-protein-gated inward rectifier potassium channel-2 protein could be localized in distal axonal terminal fields. A knowledge of the distribution of G-protein-gated inward rectifier potassium channel proteins in the brain could help to elucidate their physiological roles and to evaluate their potential involvement in neurodegenerative processes in animal models and human diseases.

TASK is a new member of the recently recognized TWIK K+ channel family. This 395 amino acid polypeptide has four transmembrane segments and two P domains. In adult human, TASK transcripts are found in pancreas

The main target for degeneration associated with the weaver mutation is the cerebellum. Expression of the GIRK2 mRNA and protein was studied in cerebellum of 12- and 22-day-old normal and weaver mice. In 12-day-old mice, GIRK2 is expressed at highest levels in the external granule layer (EGL) and in lower levels in the newly forming internal granule layer (IGL). In the weaver cerebellum, a high hybridization signal and dark immunostaining was observed in the EGL due to the higher density of non-migrated cells. In 22-day-old weaver cerebella, there are only few remaining granule cells existing as scattered cells within the IGL and molecular layer. GIRK2 is expressed in these neurons but the majority of cells expressing GIRK2 in these cerebella are Purkinje cells that are also affected by the weaver mutation (position, shape) but have not died. Normal cerebellar granule neurons but not homozygous mutant neurons in primary cultures and cerebellar slices of 8-day-old mice displayed inward rectifier K+ currents. Taken together, these findings suggest that cell loss in the weaver cerebellum is not directly related to a differential content of GIRK2 in the affected neurons during development. The lethal effect of the weaver mutation in specific neurons is probably due to a combination of the abnormal function of the inward rectifier K+ channels and other factors specific to the vulnerable neurons.

It has been suggested that a mutation in a G-protein-gated inward rectifier K+ channel (GIRK2) is responsible for inducing cell death in the cerebellum of homozygous weaver (wv/wv) mutant mice. These mice also display a progressive, massive loss of mesencephalic dopaminergic neurones. Using an immunocytochemical method, we detected GIRK2-positive cell bodies and fibres in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) of control (+/+) mice. Cell counts of both GIRK2- and tyrosine hydroxylase (TH)-positive neurones demonstrated a marked loss of SNC cell bodies, especially in 12-month-old (12M) wv/wv mice. A considerable proportion of GIRK2-positive cell bodies were preserved, however. In addition, no loss of GIRK2-positive neurones was observed in the VTA of 12M wv/wv mice, despite of a significant reduction in TH-positive cell bodies. These results suggest that expression of the mutated channel is not a sufficient condition to induce cell death in the ventral mesencephalon of the wv/wv mice.

The two P domain K+ channel mTWIK-1 has been cloned from mouse brain. In Xenopus oocytes, mTWIK-1 currents are K+-selective, instantaneous, and weakly inward rectifying. These currents are blocked by Ba2+ and quinine, decreased by protein kinase C and increased by internal acidification. The apparent molecular weight of mTWIK-1 in brain is 81 kDa. A 40 kDa form is revealed after treatment with a reducing agent, strongly suggesting that native mTWIK-1 subunits dimerize via a disulfide bridge. TWIK-1 mRNA is expressed abundantly in brain and at lower levels in lung, kidney, and skeletal muscle. In situ hybridization shows that mTWIK-1 expression is restricted to a few brain regions, with the highest levels in cerebellar granule cells, brainstem, hippocampus and cerebral cortex.

Human TWIK-1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK-1, a mammalian TWIK-1-related K+ channel. Despite a low amino acid identity between TWIK-1 and TREK-1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore-forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK-1 are inwardly rectifying while K+ currents generated by TREK-1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK-1 currents are insensitive to pharmacological agents that block TWIK-1 activity such as quinine and quinidine. Extensive inhibitions of TREK-1 activity are observed after activation of protein kinases A and C. TREK-1 currents are sensitive to extracellular K+ and Na+. TREK-1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK-1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.

TWIK-1 is a new type of K+ channel with two P domains and is abundantly expressed in human heart and brain. Here we show that TWIK-1 subunits can self-associate to give dimers containing an interchain disulfide bridge. This assembly involves a 34 amino acid domain that is localized to the extracellular M1P1 linker loop. Cysteine 69 which is part of this interacting domain is implicated in the formation of the disulfide bond. Replacing this cysteine with a serine residue results in the loss of functional K+ channel expression. This is the first example of a covalent association of functional subunits in voltage-sensitive channels via a disulfide bridge.

The isk gene is expressed in many tissues. Pharmacological evidence from the inner ear suggests that isk mediates potassium secretion into the endolymph. To examine the consequences of IsK null mutation on inner ear function, and to produce a system useful for examining the role(s) IsK plays elsewhere, we have produced a mouse strain that carries a disrupted isk locus. Knockout mice exhibit classic shaker/waltzer behavior. Hair cells degenerate, but those of different inner ear organs degenerate at different times. Functionally, we show that in mice lacking isk, the strial marginal cells and the vestibular dark cells of the inner ear are unable to generate an equivalent short circuit current in vitro, indicating a lack of transepithelial potassium secretion.

In mammalian cardiac cells, a variety of transient or sustained K+ currents contribute to the repolarization of action potentials. There are two main components of the delayed-rectifier sustained K+ current, I(Kr) (rapid) and I(Ks), (slow). I(Kr) is the product of the gene HERG, which is altered in the long-QT syndrome, LQT2. A channel with properties similar to those of the I(Ks) channel is produced when the cardiac protein IsK is expressed in Xenopus oocytes. However, it is a small protein with a very unusual structure for a cation channel. The LQT1 gene is another gene associated with the LQT syndrome, a disorder that causes sudden death from ventricular arrhythmias. Here we report the cloning of the full-length mouse K(V)LQT1 complementary DNA and show that K(V)LQT1 associates with IsK to form the channel underlying the I(Ks) cardiac current, which is a target of class-III anti-arrhythmic drugs and is involved in the LQT1 syndrome.

Cloned K+ channel beta subunits are hydrophilic proteins which associate to pore-forming alpha subunits of the Shaker subfamily. The resulting alphabeta heteromultimers K+ channels have inactivation kinetics significantly more rapid than those of the corresponding alpha homomultimers. This paper reports the cloning and the brain localization of mKvbeta4 (m for mouse), a new beta subunit. This new beta subunit is highly expressed in the nervous system but is also present in other tissues such as kidney. In contrast with other beta subunits, coexpression of the mKvbeta4 subunit with alpha subunits of Shaker-type K+ channel does not modify the kinetic properties or voltage-dependence of these channels in Xenopus oocytes. Instead, mKvbeta4 associates to Kv2.2 (CDRK), a Shab K+ channel, to specifically enhance (a factor of up to 6) its expression level without changing its elementary conductance or kinetics. It is without effect on another closely related Shab K+ channel Kv2.1 (DRK1). Chimeras between Kv2.1 and Kv2. 2 indicate that the COOH-terminal end of the Kv2.2 protein is essential for its mKvbeta4 sensitivity. The functional results associated with the observation of the co-localization of mKvbeta4 and Kv2.2 transcripts in most brain areas strongly suggest that both subunits interact in vivo to form a slowly-inactivating K+ channel. A chaperone-like effect of mKvbeta4 seems to permit the integration of a larger number of Kv2.2 channels at the plasma membrane.