Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.     

Lipid and mechano-gated 2P domain K(+) channels.

The two pore domain K(+) channels TREK and TRAAK are opened by membrane stretch. The activating mechanical force comes from the bilayer membrane and is independent of the cytoskeleton. Emerging work shows that mechano-gated TREK and TRAAK are opened by various lipids, including long chain polyunsaturated anionic fatty acids and neutral cone-shaped lysophospholipids. TREK-1 shares the properties of the Aplysia neuronal S channel, a presynaptic background K(+) channel involved in behavioral sensitization, a simple form of learning.     

TWIK-related acid-sensitive K+ channel 1 (TASK1) and TASK3 critically influence T lymphocyte effector functions

Two major K(+) channels are expressed in T cells, (i) the voltage-dependent K(V)1.3 channel and (ii) the Ca(2+)-activated K(+) channel KCa 3.1 (IKCa channel). Both critically influence T cell effector functions in vitro and animal models in vivo. Here we identify and characterize TWIK-related acid-sensitive potassium channel 1 (TASK1) and TASK3 as an important third K(+) conductance on T lymphocytes. T lymphocytes constitutively express TASK1 and -3 protein. Application of semi-selective TASK blockers resulted in a significant reduction of cytokine production and cell proliferation. Interference with TASK channels on CD3(+) T cells revealed a dose-dependent reduction ( approximately 40%) of an outward current in patch clamp recordings indicative of TASK channels, a finding confirmed by computational modeling. In vivo relevance of our findings was addressed in an experimental model of multiple sclerosis, adoptive transfer experimental autoimmune encephalomyelitis. Pretreatment of myelin basic protein-specific encephalitogenic T lymphocytes with TASK modulators was associated with significant amelioration of the disease course in Lewis rats. These data introduce K(2)P channels as novel potassium conductance on T lymphocytes critically influencing T cell effector function and identify a possible molecular target for immunomodulation in T cell-mediated autoimmune disorders.     

Neuronal background two-P-domain potassium channels: molecular and functional aspects

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.     

Regulation of recombinant human brain tandem P domain K+ channels by hypoxia: a role for O2 in the control of neuronal excitability?

The tandem P domain potassium channels, TREK1 and TASK1, are expressed throughout the brain but expression patterns do not significantly overlap. Since normal pO₂ in central nervous tissue is as low as 20 mmHg and can decrease even further in ischemic disease, it is important that the behaviour of human brain ion channels is studied under conditions of acute and chronic hypoxia. This is especially true for brain-expressed tandem P-domain channels principally because they are important contributors to neuronal resting membrane potential and excitability. Here, we discuss some recent data derived from two recombinant tandem P-domain potassium channels, hTREK1 and hTASK1. Hypoxia represents a potent inhibitory influence on both channel types and occludes the activation by arachidonic acid, intracellular acidosis and membrane deformation of TREK1. This casts doubt on the idea that TREK1 activation during brain ischemia might facilitate neuroprotection via hyperpolarising neurons in which it is expressed. Interestingly, hypoxia is unable to regulate alkalotic inhibition of TREK1 suggesting that this channel may be more intimately involved in control of excitability during physiological or pathological alkalosis.     

Enhancement of TWIK-related Acid-sensitive Potassium Channel 3 (TASK3) Two-pore Domain Potassium Channel Activity by Tumor Necrosis Factor α

TASK3 two-pore domain potassium (K2P) channels are responsible for native leak K channels in many cell types which regulate cell resting membrane potential and excitability. In addition, TASK3 channels contribute to the regulation of cellular potassium homeostasis. Because TASK3 channels are important for cell viability, having putative roles in both neuronal apoptosis and oncogenesis, we sought to determine their behavior under inflammatory conditions by investigating the effect of TNFα on TASK3 channel current. TASK3 channels were expressed in tsA-201 cells, and the current through them was measured using whole cell voltage clamp recordings. We show that THP-1 human myeloid leukemia monocytes, co-cultured with hTASK3-transfected tsA-201 cells, can be activated by the specific Toll-like receptor 7/8 activator, R848, to release TNFα that subsequently enhances hTASK3 current. Both hTASK3 and mTASK3 channel activity is increased by incubation with recombinant TNFα (10 ng/ml for 2–15 h), but other K2P channels (hTASK1, hTASK2, hTREK1, and hTRESK) are unaffected. This enhancement by TNFα is not due to alterations in levels of channel expression at the membrane but rather to an alteration in channel gating. The enhancement by TNFα can be blocked by extracellular acidification but persists for mutated TASK3 (H98A) channels that are no longer acid-sensitive even in an acidic extracellular environment. TNFα action on TASK3 channels is mediated through the intracellular C terminus of the channel. Furthermore, it occurs through the ASK1 pathway and is JNK- and p38-dependent. In combination, TNFα activation and TASK3 channel activity can promote cellular apoptosis.     

Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors

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.     

Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume

The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.     

TASK-5, a new member of the tandem-pore K(+) channel family.

TASKs are members of the recently identified K(+) channel family (KCNKx). Four TASKs (TASK1-4) identified so far form functional K(+) channels and encode background K(+) channels in various cell types. Recently, another member (TASK-5) was identified in the human genome. We cloned it and studied its tissue expression and functional properties. TASK-5 shares 51% amino acid identity with TASK-1 and TASK-3. Northern blot analysis showed that TASK-4 mRNA was expressed primarily in the adrenal gland and pancreas. Single nucleotide polymorphism (SNP) was found at amino acid position 95 that normally forms part of the K(+) channel selectivity filter. Neither form of TASK-5 showed channel activity when transfected in COS-7 cells. Exchange of C-termini of TASK-3 and TASK-5 failed to generate whole-cell currents. Thus, TASK-5 is a new member of the tandem-pore K(+) channel family but does not produce a functional plasma membrane K(+) current by itself.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the oxygen sensing type I (glomus) cells of rat carotid bodies via reduction of a background TASK-like K+ current

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice are prone to sudden neonatal death and have reduced respiratory response to hypoxia. Here we found that PACAP-38 elevated cytosolic [Ca(2+)] ([Ca(2+)](i)) in the oxygen sensing type I cells but not the glial-like type II (sustentacular) cells of the rat carotid body. This action of PACAP could not be mimicked by vasoactive intestinal peptide but was abolished by PACAP 6-38, implicating the involvement of PAC(1) receptors. H89, a protein kinase A (PKA) inhibitor attenuated the PACAP response. Simultaneous measurement of membrane potential and [Ca(2+)](i) showed that the PACAP-mediated [Ca(2+)](i) rise was accompanied by depolarization and action potential firing. Ni(2+), a blocker of voltage-gated Ca(2+) channels (VGCC) or the removal of extracellular Ca(2+) reversibly inhibited the PACAP-mediated [Ca(2+)](i) rise. In the presence of tetraethylammonium (TEA) and 4-aminopyridine (4-AP), PACAP reduced a background K(+) current. Anandamide, a blocker of TWIK-related acid-sensitive K(+) (TASK)-like K(+) channel, occluded the inhibitory action of PACAP on K(+) current. We conclude that PACAP, acting via the PAC(1) receptors coupled PKA pathway inhibits a TASK-like K(+) current and causes depolarization and VGCC activation. This stimulatory action of PACAP in carotid type I cells can partly account for the role of PACAP in respiratory disorders.     

Determinants of the anesthetic sensitivity of two-pore domain acid-sensitive potassium channels: molecular cloning of an anesthetic-activated potassium channel from Lymnaea stagnalis

Certain two-pore domain K(+) channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K(+) current I(K(An)) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662-664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427-430). Here we report the molecular cloning of a two-pore domain K(+) channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K(+) current I(K(An)). Sequence analysis and functional properties show it to be a member of the TASK family of channels with approximately 47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.     

Biophysical,pharmacological, and functional characteristics of cloned and native mammalian two-pore domain K<Superscript>+</Superscript> channels

The mammalian family of two-pore domain K⁺ (K2P) channel proteins are encoded by 15 KCNK genes and subdivided into six subfamilies on the basis of sequence similarities: TWIK, TREK, TASK, TALK, THIK, and TRESK. K2P channels are expressed in cells throughout the body and have been implicated in diverse cellular functions including maintenance of the resting potential and regulation of excitability, sensory transduction, ion transport, and cell volume regulation, as well as metabolic regulation and apoptosis. In recent years K2P channel isoforms have been identified as important targets of several widely employed drugs, including: general anesthetics, local anesthetics, neuroprotectants, and anti-depressants. An important goal of future studies will be to identify the basis of drug actions and channel isoform selectivity. This goal will be facilitated by characterization of native K2P channel isoforms, their pharmacological properties and tissue-specific expression patterns. To this end the present review examines the biophysical, pharmacological, and functional characteristics of cloned mammalian K2P channels and compares this information with the limited data available for native K2P channels in order to determine criteria which may be useful in identifying ionic currents mediated by native channel isoforms and investigating their pharmacological and functional characteristics.     

The neuronal background K2P channels: focus on TREK1

Two-pore-domain K(+) (K(2P)) channel subunits are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem and function as either homo- or heterodimeric channels. This structural motif is associated with unusual gating properties, including background channel activity and sensitivity to membrane stretch. Moreover, K(2P) channels are modulated by a variety of cellular lipids and pharmacological agents, including polyunsaturated fatty acids and volatile general anaesthetics. Recent in vivo studies have demonstrated that TREK1, the most thoroughly studied K(2P) channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.     

A role for two‐pore potassium (K2P) channels in endometrial epithelial function

The human endometrial epithelium is pivotal to menstrual cycle progression, implantation and early pregnancy. Endometrial function is directly regulated by local factors that include pH, oxygen tension and ion concentrations to generate an environment conducive to fertilization. A superfamily of potassium channels characterized by two‐pore domains (K2P) and encoded by KCNK genes is implicated in the control of the cell resting membrane potential through the generation of leak currents and modulation by various physicochemical stimuli. The aims of the study were to determine the expression and function of K2P channel subtypes in proliferative and secretory phase endometrium obtained from normo‐ovulatory women and in an endometrial cancer cell line. Using immunochemical methods, real‐time qRT‐PCR proliferation assays and electrophysiology. Our results demonstrate mRNA for several K2P channel subtypes in human endometrium with molecular expression of TREK‐1 shown to be higher in proliferative than secretory phase endometrium (P < 0.001). The K2P channel blockers methanandamide, lidocaine, zinc and curcumin had antiproliferative effects (P < 0.01) in an endometrial epithelial cancer cell line indicating a role for TASK and TREK‐1 channels in proliferation. Tetraethylammonium‐ and 4‐aminopyridine‐insensitive outwards currents were inhibited at all voltages by reducing extracellular pH from 7.4 to 6.6. Higher expression of TREK‐1 expression in proliferative phase endometrium may, in part, underlie linked to increased cell division. The effects of pH and a lack of effect of non‐specific channel blockers of voltage‐gated potassium channels imply a role for K2P channels in the regulation of human endometrial function.     

Ca2+-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca(2+) channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca(2+)-binding proteins are of particular importance as sensors of presynaptic Ca(2+), and a multiple of them are indeed utilized in the signaling of Ca(2+) channels. However, despite its conserved structure, CaM is the only known EF-hand Ca(2+)-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca(2+) channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca(2+)-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca(2+)-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca(2+), PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca(2+) channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Cyclic AMP-mediated regulation of striatal glutamate release: interactions of presynaptic ligand- and voltage-gated ion channels and G-protein-coupled receptors

The presynaptic regulation of striatal glutamate transmission was investigated using D-[3H]aspartate and mouse striatal slices. Functional changes in voltage-dependent and glutamate receptor-gated ion channels were elicited by pharmacologically modifying intracellular cyclic AMP formation via G-protein-coupled receptor stimulation. The kainate (KA)-evoked release was potentiated by the stimulatory G-protein (G(s))-coupled beta-adrenoceptor agonist isoproterenol (ISO) in a concentration-dependent manner. This effect was mimicked by the specific calmodulin (CaM) antagonists trifluoperazine and calmidazolium. Tetrodotoxin (TTX), a blocker of Na(+) channels, did not affect the basal release but inhibited to the same degree the releases evoked by kainate alone and by kainate and isoproterenol together. Vinpocetine, a blocker of voltage-dependent Na(+) channels, did not alter the basal or the evoked release. The Na(+) channel activator veratridine enhanced the basal release in a concentration-dependent manner and isoproterenol attenuated this effect. The opposite effects of isoproterenol on the kainate- and veratridine-evoked releases may reflect prevention of the cyclic AMP-protein kinase A (PKA) phosphorylation cascade in striatal glutamatergic signal transduction. In addition, the calmidazolium-induced potentiation of kainate-evoked release was thwarted by LY354740 and L-2-amino-4-phosphonobutanoate, agonists of the inhibitory G-protein (G(i))-coupled metabotropic group II and III glutamate receptors (mGluRs). Vinpocetine, which inhibits the CaM-dependent phosphodiesterase (PDE1), was likewise inhibitory. In turn, selective agonists and antagonists of the G(q)-protein-coupled group I mGluRs and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylate (AIDA), which modulate the intracellular Ca(2+) levels, did not alter the kainate-evoked release.The beta-adrenoceptor-mediated cyclic AMP accumulation seems to downregulate Na(+) channels but to enhance glutamate release by means of upregulation of kainate receptors. This regulation of presynaptic ligand- and voltage-gated ion channels is affected by the cAMP-protein kinase A-dependent phosphorylation cascade and controlled by G(i)-protein-coupled mGluRs.     

Molecular and electrophysiological characteristics of K+ conductance sensitive to acidic pH in aortic smooth muscle cells of WKY and SHR

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.     

The M1P1 loop of TASK3 K2P channels apposes the selectivity filter and influences channel function

Channels of the two-pore domain potassium (K2P) family contain two pore domains rather than one and an unusually long pre-pore extracellular linker called the M1P1 loop. The TASK (TASK1, TASK3, and TASK5) subfamily of K2P channels is regulated by a number of different pharmacological and physiological mediators. At pH 7.4 TASK3 channels are selectively blocked by zinc in a manner that is both pH(o)- and [K](o)(-)dependent. Mutation of both the Glu-70 residue in the M1P1 loop and the His-98 residue in the pore region abolished block, suggesting the two residues may contribute to a zinc binding site. Mutation of one Glu-70 residue and one His-98 residue to cysteine in TASK3 fixed concatamer channels gave currents that were enhanced by dithiothreitol and then potently blocked by cadmium, suggesting that spontaneous disulfide bridges could be formed between these two residues. Swapping the M1P1 loops of TASK1 and TASK3 channels showed that the M1P1 loop is also involved in channel regulation by pH. Therefore, the TASK3 M1P1 loop lies close to the pore, regulating TASK3 channel activity.