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Xenopus Laevis Oocytes (xenopus + laevi_oocyte)
Selected AbstractsLocalization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevisEVOLUTION AND DEVELOPMENT, Issue 6 2003Yvonne M. Beckham SummaryEleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3, untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui. [source] Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Paola Imbrici Abstract Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker -like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kv,1.x subunits. Here we show that the human Kv,1.1 and Kv,1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kv,1.1 and Kv,1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements. [source] Functional characterization of compound heterozygosity for GlyR,1 mutations in the startle disease hyperekplexiaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2002Ruth Rea Abstract The human disease hyperekplexia is characterized by excessive startle reactions to auditory and cutaneous stimuli. In its familial form, hyperekplexia has been associated with both dominant and recessive mutations of the GLRA1 gene encoding the glycine receptor ,1 subunit (GlyR,1), which mediates inhibitory transmission in the spinal cord and brainstem. Here we have examined the functional consequences of two amino acid substitutions found in a compound heterozygous family, R252H and R392H, to investigate the mechanisms determining this inheritance pattern. When expressed in Xenopus laevis oocytes, both mutations were non-functional. Neither mutant affected the electrophysiological properties of wild type GlyR,1 when co-expressed. We introduced a green fluorescent protein tag to mutant subunits and found that both mutant proteins were detectable. Evidence that subcellular localization differed from wild type was significant for one of the mutants. Thus, an effective loss of functional GlyR,1-mediated current underlies hyperekplexia in this family, whereas a partial loss is asymptomatic. [source] The product of the gene GEF1 of Saccharomyces cerevisiae transports Cl, across the plasma membraneFEMS YEAST RESEARCH, Issue 8 2007Angélica López-Rodríguez Abstract Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl, channel that remained permanently open and was blocked by nitro-2-(3-phenylpropylamino) benzoic acid and niflumic acid. NPPB induced petite -like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O2 consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl, through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined. [source] Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletionHEPATOLOGY, Issue 3 2003Olga Coll The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 ± 0.3 mmol/L and maximum velocity of 1.9 ± 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A2C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A2C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion. [source] Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in RasJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Susan E. SadlerArticle first published online: 10 JUL 200 Abstract Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction. © 2001 Wiley-Liss, Inc. [source] Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathwayJOURNAL OF PINEAL RESEARCH, Issue 3 2001Cirstin Blumenau Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1,100 ,mol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P<0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway. [source] Acetylcholine and Alcohol Sensitivity of Neuronal Nicotinic Acetylcholine Receptors: Mutations in Transmembrane DomainsALCOHOLISM, Issue 12 2002Cecilia M. Borghese Background The effect of n-alcohols on glycine and ,-aminobutyric acid type A receptors depends on two specific amino acids (AAs) located in the transmembrane domains (TM) 2 and 3. Our aim was to assess whether the corresponding AAs in the neuronal nicotinic acetylcholine receptor (nAChR) also formed a binding pocket for alcohols. Methods We made single AA substitutions in the homologous sites in rat neuronal nAChR ,2 and ,4 (,L261 and ,L283) and expressed them in Xenopus laevis oocytes in combination with ,4 wild type. The effect of different n-alcohols was studied in ,4(L261A),4 and ,4(L283A),4 nAChRs. The effect of ethanol, propanol, and octanol on acetylcholine (ACh) responses was studied in ,2(L261X),4 and ,2(L283X),4 nAChRs. Results Most of the mutations in the ,2 subunit, in either the 261 or the 283 position, induced changes in ACh sensitivity and increased alcohol action, but none was able to reduce ethanol potentiation. In ,4(L283A),4, enhancement of potentiation by short-chain alcohols was observed, as well as a change from inhibition to potentiation for long-chain alcohols. The exposure of the AAs was assessed through the action of a charged thiol-specific reagent on ,2(L261C),4 and ,2(L283C),4, and these experiments suggest that the AA in TM2 is located in a water-accessible position, whereas the AA in TM3 is inaccessible. However, a noncharged thiol-specific reagent did not affect either ACh responses or ethanol effect on ,2(L261C),4. Conclusions The AAs located at positions 261 and 283 of the ,2 and ,4 nAChR subunits do not seem to form a binding pocket for alcohols. Additional studies are required to determine whether alcohols act on a site near these AAs or on sites unrelated to the TM2-TM3 site found in glycine and ,-aminobutyric acid type A receptors. [source] Voltage-induced morphological modifications in oocyte membranes containing exogenous K+ channels studied by electrochemical scanning force microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2008Andrea Alessandrini Abstract We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation of morphological modifications occurring in plasma membranes containing voltage-gated ion channels, on membrane potential variation. Membrane patches of Xenopus laevis oocytes microinjected with exogenous KAT1 cRNA, deposited by a stripping method at the surface of a derivatized gold film in inside-out configuration, have been imaged by SFM in an electrochemical cell. A potentiostat was used to maintain a desired potential drop across the membrane. Performing imaging at potential values corresponding to open (,120 mV) and closed (+20 mV) states for KAT1, morphological differences in localized sample zones were observed. Particularly, cross-shaped features involving a significant membrane portion appear around putative channel locations. The reported approach constitutes the first demonstration of an SPM-based experimental technique suitable to investigate the rearrangements occurring to the plasma membrane containing voltage-gated channels on transmembrane potential variation. Microsc. Res. Tech., 2008. © 2007 Wiley-Liss, Inc. [source] The molecular interactions of pyrethroid insecticides with insect and mammalian sodium channels,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 10 2001Horia Vais Abstract Recent progress in the cloning of , (para) and , (TipE) Na channel sub-units from Drosophila melanogaster (fruit fly) and Musca domestica (housefly) have facilitated functional expression studies of insect Na channels in Xenopus laevis oocytes, assayed by voltage clamp techniques. The effects of Type I and Type II pyrethroids on the biophysical properties of these channels are critically reviewed. Pyrethroid resistance mutations (termed kdr and super-kdr) that reduce the sensitivity of the insect Na channel to pyrethroids have been identified in a range of insect species. Some of these mutations (eg L1014F, M918T and T929I) have been incorporated into the para Na channel of Drosophila, either individually or in combination, to investigate their effects on the sensitivity of this channel to pyrethroids. The kdr mutation (L1014F) shifts the voltage dependence of both activation and steady-state inactivation by ,5,mV towards more positive potentials and facilitates Na channel inactivation. Incorporation of the super-kdr mutation (M918T) into the Drosophila Na channel also increases channel inactivation and causes a >100-fold reduction in deltamethrin sensitivity. These effects are shared by T929I, an alternative mutation that confers super-kdr -like resistance. Parallel studies have been undertaken using the rat IIA Na channel to investigate the molecular basis for the low sensitivity of mammalian brain Na channels to pyrethroids. Rat IIA channels containing the mutation L1014F exhibit a shift in their mid-point potential for Na activation, but their overall sensitivity to permethrin remains similar to that of the wild-type rat channel (ie both are 1000-fold less sensitive than the wild-type insect channel). Mammalian neuronal Na channels have an isoleucine rather than a methionine at the position (874) corresponding to the super-kdr (M918) residue of the insect channel. Replacement of the isoleucine of the wild-type rat IIA Na channel with a methionine (I874M) increases deltamethrin sensitivity 100-fold. In this way, studies of wild-type and mutant Na channels of insects and mammals are providing a molecular understanding of kdr and super-kdr resistance in insects, and of the low pyrethroid sensitivity of most mammalian Na channels. They are also giving valuable insights into the binding sites for pyrethroids on these channels. © 2001 Society of Chemical Industry [source] Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+ exchangeTHE JOURNAL OF PHYSIOLOGY, Issue 18 2010Kenneth B. Gagnon Na+,K+,2Cl, cotransport (NKCC) mediates the movement of two Cl, ions for one Na+ and one K+ ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl, uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K+ uptake with only a minimal increase in bumetanide-sensitive Cl, uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K+/K+ and Cl,/Cl, exchange. Although significant stimulation of K+/K+ exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K+/K+ exchange. [source] Taurine uptake across the human intestinal brush-border membrane is via two transporters: H+ -coupled PAT1 (SLC36A1) and Na+ - and Cl, -dependent TauT (SLC6A6)THE JOURNAL OF PHYSIOLOGY, Issue 4 2009Catriona M. H. Anderson Taurine is an essential amino acid in some mammals and is conditionally essential in humans. Taurine is an abundant component of meat and fish-based foods and has been used as an oral supplement in the treatment of disorders such as cystic fibrosis and hypertension. The purpose of this investigation was to identity the relative contributions of the solute transporters involved in taurine uptake across the luminal membrane of human enterocytes. Distinct transport characteristics were revealed following expression of the candidate solute transporters in Xenopus laevis oocytes: PAT1 (SLC36A1) is a H+ -coupled, pH-dependent, Na+ - and Cl, -independent, low-affinity, high-capacity transporter for taurine and ,-alanine; TauT (SLC6A6) is a Na+ - and Cl, -dependent, high-affinity, low-capacity transporter of taurine and ,-alanine; ATB0,+ (SLC6A14) is a Na+ - and Cl, -dependent, high-affinity, low-capacity transporter which accepts ,-alanine but not taurine. Taurine uptake across the brush-border membrane of human intestinal Caco-2 cell monolayers showed characteristics of both PAT1- and TauT-mediated transport. Under physiological conditions, Cl, -dependent TauT-mediated uptake predominates at low taurine concentrations, whereas at higher concentrations typical of diet, Cl, -independent PAT1-mediated uptake is the major absorptive mechanism. Real-time PCR analysis of human duodenal and ileal biopsy samples demonstrates that PAT1, TauT and ATB0,+ mRNA are expressed in each tissue but to varying degrees. In conclusion, this study is the first to demonstrate both taurine uptake via PAT1 and functional coexpression of PAT1 and TauT at the apical membrane of the human intestinal epithelium. PAT1 may be responsible for bulk taurine uptake during a meal whereas TauT may be important for taurine supply to the intestinal epithelium and for taurine capture between meals. [source] Human solute carrier SLC6A14 is the ,-alanine carrierTHE JOURNAL OF PHYSIOLOGY, Issue 17 2008Catriona M. H. Anderson The ,-alanine carrier was characterized functionally in the 1960s to 1980s at the luminal surface of the ileal mucosal wall and is a Na+ - and Cl, -dependent transporter of a number of essential and non-essential cationic and dipolar amino acids including lysine, arginine and leucine. ,-Alanine carrier-like function has not been demonstrated by any solute carrier transport system identified at the molecular level. A series of experiments were designed to determine whether solute carrier SLC6A14 is the molecular correlate of the intestinal ,-alanine carrier, perhaps the last of the classical intestinal amino acid transport systems to be identified at the molecular level. Following expression of the human SLC6A14 transporter in Xenopus laevis oocytes, the key functional characteristics of the ,-alanine carrier, identified previously in situ in ileum, were demonstrated for the first time. The transport system is both Na+ and Cl, dependent, can transport non-,-amino acids such as ,-alanine with low affinity, and has a higher affinity for dipolar and cationic amino acids such as leucine and lysine. N -methylation of its substrates reduces the affinity for transport. These observations confirm the hypothesis that the SLC6A14 gene encodes the transport protein known as the ,-alanine carrier which, due to its broad substrate specificity, is likely to play an important role in absorption of essential nutrients and drugs in the distal regions of the human gastrointestinal tract. [source] Mg2+ and memantine block of rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2008David C. Wrighton N -methyl- d -aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg2+ and memantine that are dependent on the identity of the NR2 subunit present in the receptor,channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg2+ and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg2+ block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a ,10-fold reduction in Mg2+ potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a ,2-fold increase in Mg2+ potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg2+ also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg2+ is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding. [source] CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005G. Nagel The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR,ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. [source] Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2001Cyril Goudet Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 ,M), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels. British Journal of Pharmacology (2001) 134, 1195,1206; doi:10.1038/sj.bjp.0704361 [source] Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant ,7 and ,4,2 neuronal nicotinic acetylcholine receptors in Xenopus oocytesBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2001Kristen M Coates Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the ,7 subunit or ,4 and ,2 subunits expressed in Xenopus laevis oocytes. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human ,7 and ,4,2 nAChRs, with IC50 values of 20 and 50 ,M respectively. Inhibition of the ,7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the ,4,2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both ,7 (IC50 value of 122 nM) and ,4,2 (IC50 value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar. Ketamine is a noncompetitive inhibitor at both the ,7 and ,4,2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose-response curve at the ,7 nAChR suggesting competitive inhibition. Ketamine causes both voltage-dependent and use-dependent inhibition, only in the ,4,2 nAChR. Since ,7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic. British Journal of Pharmacology (2001) 134, 871,879; doi:10.1038/sj.bjp.0704315 [source] | |||||||||||||