Tetrodotoxin-sensitive sodium current in native Xenopus oocytes

Depolarization of oocytes of Xenopus laevis usually elicits mainly passive currents, and a calcium-dependent chloride current. However, oocytes obtained from some donors show, in addition, a transient inward current on depolarization to potentials beyond ca. -40 mV. This current is abolished by tetrodotoxin at submicromolar concentrations, and is prolonged by veratrine; thus, it probably arises through sodium channels of a type similar to those found in nerve and muscle cells. However, the kinetics of the sodium currents varied between oocytes from different donors; this result suggests that genes encoding different sodium channels may be expressed in oocytes from different donors. The presence of these native channels may complicate experiments to study the expression of exogenous sodium channels encoded by foreign messenger RNAs injected into the oocyte.     

Hypotonicity activates a native chloride current in Xenopus oocytes

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G- protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium- activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.     

Induction of endogenous channels by high levels of heterologous membrane proteins in Xenopus oocytes.

Xenopus oocytes are widely employed for heterologous expression of cloned proteins, particularly electrogenic molecules such as ion channels and transporters. The high levels of expression readily obtained permit detailed investigations without interference from endogenous conductances. Injection of min K mRNA into Xenopus oocytes results in expression of voltage-dependent potassium-selective channels. Recent data show that injections of high concentrations of min K mRNA also induce a chloride current with very different biophysical, pharmacological, and regulatory properties from the min K potassium current. This led to the suggestion that the min K protein acts as an inducer of endogenous, normally silent oocyte ion channels. We now report that high levels of heterologous expression of many membrane proteins in Xenopus oocytes specifically induce this chloride current and a hyperpolarization-activated cation-selective current. The current is blocked by 4,4'-diisothiocyanostilbene-2-2'-disulphonic acid and tetraethylammonium, enhanced by clofilium, and is pH-sensitive. Criteria are presented that distinguish this endogenous current from those due to heterologous expression of electrogenic proteins in Xenopus oocytes. Together with structure-function studies, these results support the hypothesis that the min K protein comprises a potassium-selective channel.     

非洲爪蟾卵细胞膜去极化后由超极化激活的瞬间内向电流

以双微电极电压钳制技术研究了未成熟非洲爪蟾卵细胞膜的离子流,发现在较长时间的去极化（－３０ｍｖ,５秒）前脉冲后由超极化引出的一个内向电流,其潜伏期约为０．５秒,经过０．４秒左右到达高峰,随近经０．８秒左右完全回复。其幅值随超极化程度增强而增大,其翻转电位接近氯平衡电位,并随胞外ＣＬ－浓度改变而变化。降低胞外ＣＬ－浓度使其幅值增大。降低胞外Ｎａ＾＋浓度对其无明显影响,提示此内几电流可能是氯离子流。以     

中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道

本文采用双微电极电压钳方法研究了中华大蟾蜍卵母细胞内源性电压门控型离子通道的成分及其生理特性。卵母细胞去极化至 -30 mV 及更正电压时,有一持续的电压依赖性外向电流出现。钾离子通道拮抗剂四乙基氯化氨(tetraethy-lammonium chloride, TEA, 10 mmol/L)和 4- 氨基吡啶(4-aminopyridine, 4-AP, 10 mmol/L)协同作用时,该电流只能被抑制到最大电流幅度的(23.4±0.72)%。但是,上述浓度的TEA和4-AP 与氯离子通道拮抗剂5- 硝基-2, 3- 苯酚丙胺苯甲酸盐 (5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L)、无钙 Ringer 氏液或钙离子通道拮抗剂维拉帕米(40 μmol/L)协同作用时,可分别将此外向电流抑制到最大电流幅度的(2.1±0.08)%、(2.2±0.04)% 和(3.1±0.15)%。结果表明,中华大蟾蜍卵母细胞质膜上除有钾离子电流之外,还存在钙依赖性的氯离子电流。     

Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor.     

Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis

ClC chloride channels are important in diverse physiological functions such as transepithelial transport, cell volume regulation, excitability, and acidification of intracellular organelles. We have investigated the expression of CLC-7 in oocytes from Xenopus laevis with the two electrode voltage clamp technique and Western blot analysis. Using a specific antibody against CLC-7, we found an approximately 80 kDa protein in oocytes, previously injected with CLC-7-cRNA. In voltage clamp experiments on ClC-7-cRNA-injected oocytes, no current changes were detected at normal pH (7.4). However, acidification of the Ringer solution to pH values between 6 and 4 revealed strong currents which reversed at about -15 mV (30 mV positive to the normal resting potential) and showed strong outward rectification. We therefore suggest that ClC-7 in oocytes is a functional chloride current at acidic pH. Since ClC-7 is also found in neuronal tissues and was upregulated in a rat pain model, we suggest a role of CLC-7 also for nociception and pain.     

Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.     

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, IC, was activated by perfusing naive oocytes with a divalent-free recording solution. Ethanol (25-100 mM) modestly inhibited IC with 100 mM ethanol producing a 7-8% inhibition of steady state currents. The store-operated or capacitative calcium current (I(SOC)) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10-100 mM) dose-dependently inhibited the calcium-dependent chloride current (I(Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I(Cl(Ca)), I(Cl-1) and I(Cl-2). Under these conditions, ethanol (100 mM) inhibited I(Cl-1) currents to a greater extent (38%) than it did I(Cl-2) currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.     

Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes

When poly(A)+-mRNA, extracted from rat brain, was injected into Xenopus laevis oocytes, it induced the appearance of serotonin receptors in the oocyte membrane. Application of serotonin to injected oocytes elicited, after a long delay, oscillations in membrane current. The equilibrium potential of this current corresponded with the chloride equilibrium potential. It appears that rat brain mRNA encodes the translation of serotonin receptors into the oocyte membrane. The combination of serotonin with these receptors leads to the opening of membrane channels.     

中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道

我们用电压箝方法研究了中华大蟾蜍卵母细胞的膜生理特性。发现卵母细胞膜去极化至-30mV及更偏正时,有一持续的外向电流出现,该电流与去极化程度约呈正比增加,当膜电位箝在20mV时其峰值达3.7±1.4μA。该电流被钾离子通道拮抗剂TEA和4-AP抑制,TEA半抑制浓度为2.6mmol/L。氯通道拮抗剂9-AC(2.5mmol/L)无抑制作用。无钙的或钙离子浓度增加三倍的胞外灌流液均对该电流无影响、该外向电流的逆转电位随胞外钾离子浓度的改变而变化。胞外钾离子浓度增加十倍,逆转电位约增加47.3mV,而胞外钠、钙或氯离子浓度的改变对逆转电位基本上无影响,因此该电流可被认为主要是电压依赖性钾离子流。取自冬眠蟾蜍的卵母细胞经孕酮诱发成熟后,电压依赖性钾离子流减小,仅为原来的1/20-1/30,而取自全年在高温饲养的蟾蜍的卵母细胞经孕酮处理后未见成熟,其电压依赖性钾离子流仅减小至原来的三分之一。     

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.     

卵胞内微注射mRNA后胞膜上出现的新离子流

以胞内微注射技术向爪蟾卵内注射羊气管上皮细胞抽提的Poly(A)~ mRNA,以电压钳技术观察卵膜离子流的变化。注射前卵膜上主要有Ca~(2 ),Cl~-,K~ 和Na~ 流,在高钙溶液(10Ca-ND96)中,瞬间的Ca~(2 )激活Cl~-流较大,此离子流可被9-羟蒽阻断。注射mRNA后在9-羟蒽存在的情况下,向浴槽加入1mmol/L 8-Br-cAMP后新出现一个电压敏感的离子流,使电流电压曲线的零电流电位向右移;但在注射去离子水组未见此离子流。表明羊气管上皮里含有cAMP敏感的离子通道,并可被移植到爪蟾卵膜上。     

Transient potassium current in native Xenopus oocytes

Depolarization of follicle-enclosed oocytes of Xenopus laevis obtained from some donors elicits, in addition to other responses, a fast transient outward current. After holding the membrane potential at -100 mV this response begins to be activated by depolarizations to around -30 mV, and increases progressively as the voltage is raised further. A striking characteristic is that the current recovers only slowly (several seconds) from inactivation following a depolarizing pulse. Because of its outward direction and insensitivity to removal of extracellular chloride or addition of tetrodotoxin, the current probably arises largely through a flux of potassium ions. The current was abolished after treatment of oocytes with collagenase to remove enveloping cells, and although it was blocked by barium and zinc ions, tetraethylammonium was relatively ineffective. In addition, the potassium current was unaffected by 5 mM manganese, suggesting that it does not arise as a consequence of an influx of calcium into the oocyte.     

Induction of apoptosis using sphingolipids activates a chloride current in Xenopus laevis oocytes

The purpose of this studywas to investigate whether the cell shrinkage that occurs duringapoptosis could be explained by a change of the activity in iontransport pathways. We tested whether sphingolipids, which are potentpro-apoptotic compounds, can activate ionic currents in Xenopuslaevis oocytes. Apoptosis was characterized in our model by adecrease in cell volume, a loss of cell viability, and DNAcleavage. Oocytes were studied using voltage-clamp afterinjection withN,N-dimethyl-D-erythrosphingosine (DMS) or D-sphingosine (DS). DMS and DS activated afast-activating, slowly inactivating, outwardly rectifying current,similar to I_Cl-swell, a swelling-inducedchloride current. Lowering the extracellular chloride dramaticallyreduced the current, and the channel was more selective for thiocyanateand iodide (thiocyanate > iodide) than for chloride. The currentwas blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) andlanthanum but not by niflumic acid. Oocytes injected with apseudosubstrate inhibitor of protein kinase C (PKC),PKC-(19-31), exhibited the same current.DMS-activated current was abolished by preexposure with phorbolmyristate acetate. Our results suggest that induction of apoptosis inX. laevis oocytes, using sphingolipids or PKC inhibitors,activates a current similar to swelling-induced chloride currentpreviously described in oocytes.

     

Neurotensin and substance P receptors expressed in Xenopus oocytes by messenger RNA from rat brain

Xenopus oocytes were induced to acquire sensitivity to neurotensin and substance P, by injecting them with a fraction of poly(A)+ mRNA from rat brain. Non-injected oocytes, and oocytes injected with other brain mRNAs, failed to show responses, suggesting that receptors to these peptides were expressed by specific brain mRNAs. Responses to substance P and neurotensin comprised an oscillatory chloride current, and a smooth current having different ionic basis. These currents resembled those seen during activation of muscarinic and serotonergic receptors, but were not blocked by the corresponding antagonists atropine and methysergide. The responses to substance P, and to a lesser extent to neurotensin, showed a long-lasting desensitization. Similarities between the oscillatory currents evoked by the peptides acetylcholine and serotonin suggest that all these receptors may 'link in' to a common intracellular messenger pathway.     

Phosphatidic acid and lysophosphatidic acid stimulate receptor-regulated membrane currents in the Xenopus laevis oocyte.

External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus laevis oocytes. The responses were observed in oocytes from which follicular cells had been removed, indicating they were intrinsic to the oocyte itself. The lipid-induced Ca(2+)-dependent chloride currents were observed in the absence of extracellular calcium, were blocked by intracellular injection of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic acid, and could not be elicited by direct intracellular injection of the active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that the lipid activities on oocytes were potent. With repeated or prolonged administration of either active lipid, responses exhibited desensitization. These results demonstrate that the Xenopus oocyte expresses endogenous functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic acid and thus provides a powerful model for characterization of the pharmacology and transduction pathways of these responses.     

Contribution of the cytoskeleton and the phospholipase C signaling pathway to fluid stream-induced membrane currents

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I(i,st)) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I(i,st) were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I(i,st), suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP(3))-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I(i,st) in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I(i,st). The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP(3) concentration, in turn releasing calcium from InsP(3)-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.     

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A)+ mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.