Identification and functional characterization of zebrafish K2P10.1 (TREK2) two-pore-domain K+ channels

Two-pore-domain potassium (K_2P) channels mediate K⁺ background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K_2P currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K_2P channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K_2P10.1 channel. K_2P10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK_2P10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K_2P10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K_2P10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K⁺ selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK_2P10.1, induced robust activation of zK_2P10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK_2P10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K_2P10.1 two-pore-domain K⁺ channels that exhibit structural and functional properties largely similar to human K_2P10.1. We conclude that the zebrafish represents a valid model to study K_2P10.1 function in vivo.     

Recombinant hTASK1 is an O(2)-sensitive K(+) channel

Hypoxic inhibition of background K(+) channels is crucial to O(2) sensing by chemoreceptor tissues, but direct demonstration of O(2) sensitivity by any member of this K(+) channel family is lacking. HEK293 cells were transfected with a pcDNA3.1-hTASK1 construct; expression of hTASK1 was verified using RT-PCR and immunocytochemistry. Whole-cell K(+) currents of cells stably expressing hTASK-1 were, as anticipated, extremely sensitive to extracellular pH, within the physiological range (IC(50) approximately 7.0). All cells expressing this signature pH sensitivity were acutely modulated by pO(2); reduction of pO(2) from 150 to <40 mmHg (at pH 7.4) caused rapid and reversible suppression of pH-sensitive K(+) currents. Furthermore, these two regulatory signals clearly acted at the same channel, since the magnitude of the O(2)-sensitive current was dependent on the extracellular pH. These data represent the first direct verification that hTASK1 is O(2)-sensitive and reinforce the idea that this K(+) channel is key to O(2) sensing in chemoreceptors.     

Enhancement of HERG K(+) currents by Cd(2+) destabilization of the inactivated state

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 &mgr;M) of extracellular Cd(2+) increased outward currents through HERG channels; 200 &mgr;M Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 &mgr;M did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >/=500 &mgr;M shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.     

TWIK-2, an inactivating 2P domain K+ channel

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.     

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.     

A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

By analysis of whole cell membrane currents in Na(+)-absorbing H441 human airway epithelial cells, we have identified a K(+) conductance (G(K)) resistant to Ba(2+) but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K(+) current (I(Bl)) whereas Ba(2+) has only a weak inhibitory effect. I(Bl) was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in I(Bl), acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K(+) channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (I(SC)), and basolateral bupivacaine, lidocaine, quinidine, and Ba(2+) inhibited I(SC) at concentrations similar to those needed to inhibit I(Bl), suggesting that the K(+) channels underlying I(Bl) are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K(+) (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie G(K) in unstimulated cells and so maintain the driving force for Na(+) absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.     

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.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

急性低温/再复温对大鼠心室肌膜电位和钾电流的影响

本文旨在研究急性低温/再复温对大鼠心室肌膜电位和钾电流的影响.膜电位和膜电流分别在全细胞膜片钳的电压钳和电流钳模式下记录.当细胞外灌流液从25℃降低到4℃后,一过性外向电流(transient outward current, Ito)完全消失,膜电位为 60mV时的稳态外向K 电流(sustained outward K current, Iss)和膜电位为-120mV时的内向整流K 电流(inward rectifier K current, IK1)分别降低(48.5±14.1)%和(35.7±18.2)%,同时,膜电位绝对值降低.当细胞外灌流液从4℃再升高到36℃后,膜电位出现一过性超级化,然后恢复到静息电位水平;在58个细胞中,有36个细胞伴随复温出现ATP-敏感性K (ATP-sensitive K , KATP)通道的激活.再复温引起的上述变化可以被Na /K -ATP酶抑制剂哇巴因(100μmol/L)所抑制.再复温引起的KATP通道激活也能被蛋白激酶A抑制剂H-89(100μmol/L)所抑制.在细胞膜电位被钳制在0mV时,当细胞外灌流液温度从25℃降低到4℃后,细胞的体积没有发生明显改变,但当再复温引起KATP通道激活后,细胞很快发生皱缩,同时细胞内部出现许多折光较强的斑点.上述结果表明急性低温/再复温对大鼠心室肌膜电位和K 电流有明显影响,并提示KATP通道激活可能与心肌低温/再复温损伤有关.     

Temperature-dependent expression of sarcolemmal K(+) currents in rainbow trout atrial and ventricular myocytes

Temperature has a strong influence on the excitability and the contractility of the ectothermic heart that can be alleviated in some species by temperature acclimation. The molecular mechanisms involved in the temperature-induced improvement of cardiac contractility and excitability are, however, still poorly known. The present study examines the role of sarcolemmal K(+) currents from rainbow trout (Oncorhynchus mykiss) cardiac myocytes after thermal acclimation. The two major K(+) conductances of the rainbow trout cardiac myocytes were identified as the Ba(2+)-sensitive background inward rectifier current (I(K1)) and the E-4031-sensitive delayed rectifier current (I(Kr)). In atrial cells, the density of I(K1) is very low and the density of I(Kr) is remarkably high. The opposite is true for ventricular cells. Acclimation to cold (4 degrees C) modified the two K(+) currents in opposite ways. Acclimation to cold increases the density of I(Kr) and depresses the density of I(K1). These changes in repolarizing K(+) currents alter the shape of the action potential, which is much shorter in cold-acclimated than warm-acclimated (17 degrees C) trout. These results provide the first concrete evidence that K(+) channels of trout cardiac myocytes are adaptable units that provide means to regulate cardiac excitability and contractility as a function of temperature.     

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.     

ATP-dependent activation of K(Ca) and ROMK-type K(ATP) channels in human submandibular gland ductal cells.

[Ca(2+)](i) and membrane current were measured in human submandibular gland ductal (HSG) cells to determine the regulation of salivary cell function by ATP. 1-10 microM ATP activated internal Ca(2+) release, outward Ca(2+)-dependent K(+) channel (K(Ca)), and inward store-operated Ca(2+) current (I(SOC)). The subsequent addition of 100 microM ATP activated an inwardly rectifying K(+) current, without increasing [Ca(2+)](i). The K(+) current was also stimulated by ATP in cells treated with thapsigargin in a Ca(2+)-free medium and was blocked by glibenclamide and tolbutamide, but not by charybdotoxin. This suggests the involvement of a Ca(2+)-independent, sulfonylurea-sensitive K(+) channel (K(ATP)). UTP mimicked the low [ATP] effects, while benzoyl-ATP activated internal Ca(2+) release, a Ca(2+) influx pathway, and K(Ca). Thus, ATP acts via P(2U) (P2Y(2)) and P(2Z) (P2X(7)) receptors to increase [Ca(2+)](i) and activate K(Ca), but not K(ATP). Importantly, (i) ROMK1 and the cystic fibrosis transmembrane regulator protein (but not SUR1, SUR2A, or SUR2B) and (ii) cAMP-stimulated Cl(-) and K(+) currents were detected in HSG cells. These data demonstrate for the first time that a ROMK-type K(ATP) channel is present in salivary gland duct cells that is regulated by extracellular ATP and possibly by the cystic fibrosis transmembrane regulator. This reveals a potentially novel mechanism for K(+) secretion in these cells.     

Cellular localization of THIK-1 (K(2P)13.1) and THIK-2 (K(2P)12.1) K channels in the mammalian kidney.

K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney.     

Ca(2+)-activated K+ channel inhibition by reactive oxygen species

We studied the effect of H(2)O(2) on the gating behavior of large-conductance Ca(2+)-sensitive voltage-dependent K(+) (K(V,Ca)) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) alpha-subunit. Exposure of the intracellular side of K(V,Ca) channels to H(2)O(2) (4-23 mM) leads to a time-dependent decrease of the open probability (P(o)) without affecting the unitary conductance. H(2)O(2) did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H(2)O(2) action. Desferrioxamine (60 microM) and cysteine (1 mM) completely inhibited the effect of H(2)O(2), indicating that the decrease in P(o) was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H(2)O(2). However, DTT did completely reverse the decrease in P(o) induced by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of K(V,Ca) channel activity promoted by DTT suggests that H(2)O(2) treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing hSlo channels showed that H(2)O(2) induced a decrease in current mediated by a decrease both in the number of active channels and P(o).     

Activating of ATP-dependent K+ channels comprised of K(ir) 6.2 and SUR 2B by PGE2 through EP2 receptor in cultured interstitial cells of Cajal from murine small intestine.

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE(2) might affect the electrical properties of cultured ICC by activating ATP-dependent K(+) channels and, the EP receptor subtypes and the subunits of ATP-dependent K(+) channels involved in these activities were identified. In addition, the regulation of intracellular Ca(2+) ([Ca(2+)](i)) mobilization may be involved the action of PGE(2) on ICC. Treatments of ICC with PGE(2) inhibited electrical pacemaker activities in the same manner as pinacidil, an ATP-dependent K(+) channel opener and PGE(2) had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K(+) channels exist in ICC and that these are composed of K(ir) 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP(2) receptor agonist) showed the actions on pacemaker currents in the same manner as PGE(2). However sulprostone (a mixed EP(1) and EP(3) agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP(2) receptor in ICC. To investigate cAMP involvement in the effects of PGE(2) on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE(2) on pacemaker currents, and PGE(2) did not stimulate cAMP production. Also, we found PGE(2) inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. These observations indicate that PGE(2) alters pacemaker currents by activating the ATP-dependent K(+) channels comprised of K(ir) 6.2-SUR 2B in ICC and this action of PGE(2) are through EP(2) receptor subtype and also the activation of ATP-dependent K(+) channels involves intracellular Ca(2+) mobilization.     

CO(2) inhibits specific inward rectifier K(+) channels by decreases in intra- and extracellular pH

Hypercapnia has been shown to affect cellular excitability by modulating K(+) channels. To understand the mechanisms for this modulation, four cloned K(+) channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO(2) for 4-6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pH(i), pH(o)) dropped during CO(2) exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pH(i) produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pH(i) level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO(2) without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K(+) channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO(2), and (3) these effects are independent of cytosol-soluble factors.     

Effects of lomefloxacin and norfloxacin on pancreatic beta-cell ATP-sensitive K(+) channels

In patients administered lomefloxacin alterations in blood glucose concentrations have been observed in some cases and lomefloxacin has previously been shown to augment insulin release from rat pancreatic islets at micromolar concentrations. The aim of the present study was to compare the effects of two structurally related fluoroquinolones, lomefloxacin and norfloxacin, on ATP-sensitive K(+) (K(ATP)) currents from the clonal insulinoma cell line RINm5F using the whole-cell configuration of the patch-clamp technique. The application of lomefloxacin concentration-dependently blocked K(ATP) currents from RINm5F cells with a half-maximally inhibitory concentration of 81 microM, whereas the application of norfloxacin (at concentrations up to 300 microM) had only minor effects on K(ATP) currents. Block of pancreatic beta-cell K(ATP) currents could be mediated by interaction of lomefloxacin either with the regulatory subunit (SUR1) or with the pore-forming subunit (Kir6.2). We favour the latter hypothesis, since some fluoroquinolones have recently been shown to block the pore-forming subunit of the cardiac rapid delayed rectifier K(+) current I(Kr) (which is encoded by HERG (human ether-a-go-go-related gene)). Thus, as demonstrated for cardiac HERG channels in previous studies and for pancreatic beta-cell K(ATP) channels in the present study, fluoroquinolones differ markedly in their potencies to inhibit K(+) channel activity.