Channels In Oocytes Research Articles

Plasmin improves blood-gas barrier function in oedematous lungs by cleaving epithelial sodium channels.

Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. Here, we have tested whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid. Human lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, and protein docking were combined for identifying plasmin cleavage sites. Plasmin improved lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin activated αβγENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (αΔ432-444, γΔ131-138, γΔ178-193, and γΔ410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human γENaC proteins. In addition, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both α and γ subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Plasmin can cleave ENaC to improve blood-gas exchange by resolving oedema fluid and could be a potent therapy for oedematous lungs.

Isolation and characterization of a novel member of the ACC ligand-gated chloride channel family, Hco-LCG-46, from the parasitic nematode Haemonchus contortus

The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.

Ca2+ Signaling and Homeostasis in Mammalian Oocytes and Eggs.

Changes in the intracellular concentration of calcium ([Ca2+]i) represent a vital signaling mechanism enabling communication between and among cells as well as with the environment. Cells have developed a sophisticated set of molecules, "the Ca2+ toolkit," to adapt [Ca2+]i changes to specific cellular functions. Mammalian oocytes and eggs, the subject of this review, are not an exception, and in fact the initiation of embryo devolvement in all species is entirely dependent on distinct [Ca2+]i responses. Here, we review the components of the Ca2+ toolkit present in mammalian oocytes and eggs, the regulatory mechanisms that allow these cells to accumulate Ca2+ in the endoplasmic reticulum, release it, and maintain basal and stable cytoplasmic concentrations. We also discuss electrophysiological and genetic studies that have uncovered Ca2+ influx channels in oocytes and eggs, and we analyze evidence supporting the role of a sperm-specific phospholipase C isoform as the trigger of Ca2+ oscillations during mammalian fertilization including its implication in fertility.

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, β, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models.Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize δ802-ENaC channel activity. Immunoblotting was utilized to analyse δ802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR.Results: The results indicate that δ802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide α-13 distinguishes δ802- from α-type ENaC channels. Modified proteolysis of γ-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and α-13 peptide. Expression of δ802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. δ802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells.Conclusion: The humanized mouse line conditionally expressing human δ802-ENaC is a novel model for studying the expression and function of this protein in vivo .

Voltage-gated sodium channels as targets for pyrethroid insecticides

The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

TRPV3 channels mediate Ca2+ influx induced by 2-APB in mouse eggs

Fertilization in mammals is initiated when a sperm fuses with a mature MII oocyte, also known as egg, and triggers a plethora of finely controlled processes identified as egg activation. The completion of all events of egg activation is driven by and depends on a series of repetitive calcium (Ca2+) increases (Ca2+ oscillations), which rely on Ca2+ influx from the extracellular media. Ca2+ channels on the egg plasma membrane (PM) are thought to mediate this influx. The TRP Ca2+ channel TRPV3 is differentially expressed during oocyte maturation, being most active at the MII stage. Specific stimulation of TRPV3 channels promotes Ca2+ influx sufficient to induce egg activation and parthenogenesis. Here, we explore the function and distribution dynamics of the TRPV3 channel protein during maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the expression levels of the channel and showed that the TRPV3 protein is synthesized and translocated to the PM during maturation. We demonstrated that 2-APB at the concentrations used here to promote Ca2+ influx in eggs, specifically and reversibly targets TRPV3 channels without blocking IP3R1. Finally, we found that the activity of TRPV3 channels is dependent upon an intact actin cytoskeleton, suggesting an actin-based regulation of its expression and/or function on the PM. Collectively, our results show TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The need of an intact actin cytoskeleton for the function of TRPV3 channels in oocytes is a novel finding and suggests the rearrangements of actin that occur during maturation could regulate both the presence on the PM and/or the function of TRPV3 and of other Ca2+ channels involved in oocyte maturation and fertilization.

Ivermectin modulation of pH-sensitive chloride channels in the silkworm larvae of Bombyx mori

The pH-sensitive chloride channels (pHCls) are expressed widely in the insect nervous system, but their physiological roles and pesticide sensitivity in Lepidoptera are poorly understood. Here, cDNAs of pHCl variants “A” and “B” were isolated from the head of silkworm (Bombyx mori) larvae and expressed in Xenopus laevis oocytes to characterize their functions and examine their pesticide sensitivity. Variant “A” possesses four entire transmembrane domains (TMs), while variant “B” lacks a part of the TM and the TM3–TM4 linker. Only the A variant formed a chloride channel in oocytes which was activated in response to an increase of pH in the extracellular solution. Neither fipronil nor γ-benzenehexachloride had a significant blocking effect on the A variant when tested at 10μM. By contrast, macrolide ivermectin activated it at acidic pH but blocked it at pH7.6 at concentrations higher than 300nM, indicating a likely contribution to in vivo toxicity.

An Internally Modulated, Thermostable, pH-sensitive Cys Loop Receptor from the Hydrothermal Vent Worm Alvinella pompejana

Cys loop receptors (CLRs) are commonly known as ligand-gated channels that transiently open upon binding of neurotransmitters to modify the membrane potential. However, a class of cation-selective bacterial homologues of CLRs have been found to open upon a sudden pH drop, suggesting further ligands and more functions of the homologues in prokaryotes. Here we report an anion-selective CLR from the hydrothermal vent annelid worm Alvinella pompejana that opens at low pH. A. pompejana expressed sequence tag databases were explored by us, and two full-length CLR sequences were identified, synthesized, cloned, expressed in Xenopus oocytes, and studied by two-electrode voltage clamp. One channel, named Alv-a1-pHCl, yielded functional receptors and opened upon a sudden pH drop but not by other known agonists. Sequence comparison showed that both CLR proteins share conserved characteristics with eukaryotic CLRs, such as an N-terminal helix, a cysteine loop motif, and an intracellular loop intermediate in length between the long loops of other eukaryotic CLRs and those of prokaryotic CLRs. Both full-length Alv-a1-pHCl and a truncated form, termed tAlv-a1-pHCl, lacking 37 amino-terminal residues that precede the N-terminal helix, formed functional channels in oocytes. After pH activation, tAlv-a1-pHCl showed desensitization and was not modulated by ivermectin. In contrast, pH-activated, full-length Alv-a1-pHCl showed a marked rebound current and was modulated significantly by ivermectin. A thermostability assay indicated that purified tAlv-a1-pHCl expressed in Sf9 cells denatured at a higher temperature than the nicotinic acetylcholine receptor from Torpedo californica.

The &lt;em&gt;Xenopus&lt;/em&gt; Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

A hyperpolarization-activated ion current of amphibian oocytes

A comparative analysis of a hyperpolarization-activated ion current present in amphibian oocytes was performed using the two-electrode voltage-clamp technique in Xenopus laevis, Xenopus tropicalis, and Ambystoma mexicanum. This current appears to be driven mainly by Cl(-) ions, is independent of Ca(2+), and is made evident by applying extremely negative voltage pulses; it shows a slow activating phase and little or no desensitization. The pharmacological profile of the current is complex. The different channel blocker used for Cl(-), K(+), Na(+) and Ca(2+) conductances, exhibited various degrees of inhibition depending of the species. The profiles illustrate the intricacy of the components that give rise to this current. During X. laevis oogenesis, the hyperpolarization-activated current is present at all stages of oocytes tested (II-VI), and the amplitude of the current increases from about 50nA in stage I to more than 1μA in stage VI; nevertheless, there was no apparent modification of the kinetics. Our results suggest that the hyperpolarization-activated current is present both in order Anura and Urodela oocytes. However, the electrophysiological and pharmacological characteristics are quite perplexing and seem to suggest a mixture of ionic conductances that includes the activation of both anionic and cationic channels, most probably transiently opened due to the extreme hyperpolarizion of the plasma membrane. As a possible mechanism for the generation of the current, a kinetic model which fits the data suggests the opening of pores in the plasma membrane whose ion selectivity is dependent on the extracellular Cl(-) concentration. The extreme voltage conditions could induce the opening of otherwise latent pores in plasma membrane proteins (i.e., carriers), resembling the ´slippage´ events already described for some carriers. These observations should be valuable for other groups trying to express cloned, voltage-dependent ion channels in oocytes of amphibian in which hyperpolarizing voltage pulses are applied to activate the channels.

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na(+) reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 μmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na(+) current by 52% and increased the membrane density of Na(+) channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 μmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na(+) reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

Blocking effects of acehytisine on pacemaker currents (If) in sinoatrial node cells and human HCN4 channels expressed in Xenopus laevis oocytes

Ethnopharmacological relevanceThe root of Aconitum coreanum (Levl.) Raipaics has been extensively used to treat various kinds of disorders including cardiovascular disease in China for a long time. According to recent studies, its antiarrhythmic actions are attributable to the active component, acehytisine. However, the underlying mechanism remains poorly understood. Aim of the studyThe effects of acehytisine on the spontaneous activity in sinoatrial nodes and the electropharmacological action of this drug on If in pacemaker cells and hHCN4 channels in oocytes were to be investigated. Materials and methodsSinoatrial nodes were cut from rabbit heart, and transmembrane potentials were recorded by standard microelectrode technique. A whole-cell patch clamp technique was employed to record If isolated enzymatically from rabbit sinoatrial node pacemaker cells. Human HCN4 channels were heterologously expressed in Xenopus oocytes and studied using the two-electrode voltage clamp technique. ResultsAcehytisine decreased the pacemaker rate of firing and slope of diastolic depolarization, modified the action potential configurations and blocked If in rabbit sinoatrial node cells and hHCN4 channels expressed in Xenopus oocytes in a concentration-dependent, voltage-independent and non-use-dependent manner. Its electropharmacological properties were consistent with those of a close-state blocker. ConclusionOur findings are likely to shed light on the clinical application of acehytisine in the treatment of cardiovascular disorders.

A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

Plasmin activates human epithelial sodium channels in oocytes

Salt absorption via epithelial sodium channels (ENaC) is a rate limiting process that is driven by basolaterally located Na/K-ATPase in epithelial cells. In injured lungs, plasminogen activator inhibitor (PAI-1) level is extremely high, possibly eliminating plasmin content in lung edema fluid. Plasmin cleaved heterologous mouse γ ENaC (Passero CJ, et.al JBC 283:36586–36591, 2008). To examine the regulation of human ENaC by plasmin expressed in oocytes, we incubated oocytes with plasmin and evaluated ENaC activity using dual-electrode voltage clamp technique. We found that plasmin (10μg/ml for 1 hour) stimulates ENaC currents up to 13 fold (from −1656.58 ± 127 nA to – 21711.189 ± 1797 nA, N=8, P<0.05). Plasmin activates αβγ ENaC activity in a time-dependent manner with maximal activation in one hour. To identify the potential proteolytic domains in α and γ subunits, we applied plasmin to six deletion mutants, termed, αΔ173–178, αΔ201–204, αΔ432–444, γΔ131–138, γΔ178–193 and γΔ410–422. Interestingly, four deletion mutations (αΔ432–444, γΔ131–138, γΔ178–193 and γΔ410–422) lost their responses to plasmin. Furthermore, several critical amino acid residues are able to reduce the stimulatory effects of plasmin on ENaC, including γR135M, γR137M, γK179M and γk181M. Our results suggest that plasmin may up-regulate heterologous human αβγ ENaC channels via multiple proteolytic domains in both α and γ subunits. Supported by NIH grants HL87017 and HL95435.

IP3 Receptor Channels are Clustered Before IP3 Exposure with No Discernible Effect on Single-Channel Gating Properties

A recent patch clamp study reported that recombinant rat type 3 IP3R Ca2+ release channels expressed in IP3R deficient DT40-KO cells are randomly distributed in the ER/outer nuclear membrane until exposure to low [IP3] induces their rapid clustering [Rahman et al (2009) Nature458: 655-659]. In sub-optimal ligand conditions, clustered channels gated identically and independently, but with a lower open probability (Po) than lone channels regardless of cluster size. In contrast, in optimal ligand conditions, Po for clustered and lone channels were the same, but positive cooperative gating was consistently detected in patches with two active channels. Seeking to verify these surprising observations, we acquired current records from the same channels expressed in the same cell system with identical protocols and ligand conditions. The records were analyzed with the same algorithm to characterize gating behaviors of lone and clustered channels separately. For comparison, all nuclear patch-clamp current records previously acquired under comparable ligand conditions for recombinant rat type 3 IP3R channels expressed in Xenopus oocytes, endogenous Xenopus type 1 IP3R channels in oocytes and endogenous IP3R channels in insect Sf9 cells were similarly analyzed. We found that rat type 3 IP3R channels in DT40-KO cells are clustered without exposure to IP3, like all IP3R channels investigated before. For all IP3R channels examined, we detected no significant differences between channel Po of lone and clustered channels, in sub-optimal or optimal ligand conditions. Furthermore, in two-channel current records, the same pattern of channel gating was detected, in both sub-optimal and optimal ligand conditions, with only a small fraction (< 15%) revealing positive cooperative gating behavior. Thus, single channel behavior in all ligand conditions is independent of whether the IP3R channel is in single or multi-channel patches.

ENaC Channels in Oocytes from Xenopus laevis and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na⁺ currents (INa_(amil)). We observed a marked reduction in INa_(amil) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa_(amil) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa_(amil) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

Cocaine produces D 2R-mediated conformational changes in the adenosine A 2AR-dopamine D 2R heteromer

Adenosine A 2A receptors (A 2ARs) and dopamine D 2 receptors (D 2Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A 2ARs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A 2AR and D 2LR formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D 2R homodimers and in A 2AR–D 2R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET max between A 2AR–D 2LR heterodimers and D 2LR homodimers, but not A 2AR homodimers, indicating its effect on D 2R. Cocaine was evaluated with regard to D 2R binding using a human D 2LR stable expressing CHO cell line and was found to produce an increase in the affinity of hD 2LR for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPγS. However, cocaine failed to modulate D 2R agonist-induced inhibition of cAMP in stable hD 2LR CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D 2LR, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D 2R homodimers and A 2AR–D 2R heterodimers.

10.1007/s11174-008-1006-4

A review of the data on the presence, localization, and supposed role of aquaporin water channels in oocytes of Xenopus laevis, oogenesis and maturation of teleosts Sparus auratus and Oncorhynchus mykiss, oogenesis and oocyte maturation of rats and mice, and spermatogenesis of several mammalians.