Molecular mapping of the conductance activity linked to tAE1 expressed in Xenopus oocyte

It was previously shown that expressed in Xenopus oocyte the trout (tAE1) and the mouse (mAE1) anion exchangers behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with an anion conductance. In order to identify the structural domains involved in the induction of tAE1 channel activity, chimeras have been prepared between mouse and trout AE1. As some constructs were not expressed at the plasma membrane, skate exchanger (skAE1) was used instead of mouse exchanger to complete the structure-function analysis. The present paper shows that skAE1, highly similar to mAE1, does not induce a chloride conductance when expressed in Xenopus oocyte. Construct expression analysis showed that only tAE1 transmembrane domain is linked to the anion conductance. More precisely, we identified two regions composed of helices 6, 7 and 8 and putative helices 12 and 13 which are required for this function.     

Bestrophin genes are expressed in Xenopus development

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.     

Short-term exposure to waterborne free silver has acute effects on membrane current of Xenopus oocytes

Waterborne free silver can cause osmo- and ionoregulatory disturbances in freshwater organisms. The effects of a short-term exposure to extracellular Ag⁺ ions on membrane currents were investigated in voltage-clamped defolliculated Xenopus oocytes. At a holding potential of − 60 mV, ionic silver (1 μM Ag⁺) increased inward currents (=I_Ag) from − 8 ± 2 nA to − 665 ± 41 nA (n = 74; N = 27). I_Ag activated within 2 min of silver exposure and then rose impetuously. This current was largely reversible by washout and repeatable. I_Ag reversed around − 30 mV and rectified slightly at more positive potentials. Na⁺-free bath conditions reduced the silver-induced current to a smaller but sustained current. The response to silver was abolished by the Cl⁻ channel blockers DIDS and SITS, whereas niflumic acid strongly potentiated I_Ag. Intraoocyte injection of AgNO₃ to about 1 mM [Ag]_i strongly potentiated I_Ag. Extracellular application of either dithiothreitol (DTT), a compound known to reduce disulfide bridges, or l-cysteine abolished Ag⁺-activated increase of membrane current. In contrast, n-ethylmaleimide (NEM) which oxidizes SH-groups potentiated I_Ag. Hypoosmotic bath solution significantly increased I_Ag whereas hyperosmolar conditions attenuated I_Ag. The activation of I_Ag was largely preserved after chelation of cytosolic Ca²⁺ ions with BAPTA/AM. Taken together, these data suggest that Xenopus oocytes are sensitive to short-term exposure to waterborne Ag⁺ ions and that the elicited membrane currents result from extra- and intracellular action of Ag⁺ ions on peptide moieties at the oocyte membrane but may also affect conductances after internalization.     

Effects of ketamine on glucose uptake by glucose transporter type 3 expressed in Xenopus oocytes: The role of protein kinase C

We investigated the effects of ketamine on the type 3 facilitative glucose transporter (GLUT3), which plays a major role in glucose transport across the plasma membrane of neurons. Human-cloned GLUT3 was expressed in Xenopus oocytes by injection of GLUT3 mRNA. GLUT3-mediated glucose uptake was examined by measuring oocyte radioactivity following incubation with 2-deoxy-d-[1,2-³H]glucose. While ketamine and S(+)-ketamine significantly increased GLUT3-mediated glucose uptake, this effect was biphasic such that higher concentrations of ketamine inhibited glucose uptake. Ketamine (10 μM) significantly increased V_max but not K_m of GLUT3 for 2-deoxy-d-glucose. Although staurosporine (a protein kinase C inhibitor) increased glucose uptake, no additive or synergistic interactions were observed between staurosporine and racemic ketamine or S(+)-ketamine. Treatment with ketamine or S(+)-ketamine partially prevented GLUT3 inhibition by the protein kinase C activator phorbol-12-myrisate-13-acetate. Our results indicate that ketamine increases GLUT3 activity at clinically relevant doses through a mechanism involving PKC inhibition.     

Pro-apoptotic activity and mono-/diubiquitylation of Xenopus Bid in egg extracts

Apoptosis in Xenopus egg extracts is carried out by maternally stockpiled materials, but the contributions of endogenous apoptosis regulators are still poorly characterized. Here we examined the physiological role of Xenopus Bid (xBid), a pro-apoptotic BH3-only member of Bcl-2 family proteins. We found that endogenous xBid was a physiological accelerator of apoptosis in egg extracts. Interestingly, xBid was mono-/diubiquitylated but not degraded by proteasome in egg extracts, and we identified three ubiquitylated Lys residues in the N-terminal propeptide region. Comparison with human Bid suggested that mono-/diubiquitylation is a specific feature of xBid.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Substrate specificity of a chimera made from Xenopus SGLT1-like protein and rabbit SGLT1

To characterize the sugar translocation pathway of Na⁺/glucose cotransporter type 1 (SGLT1), a chimera was made by substituting the extracellular loop between transmembrane domain (TM) 12 and TM13 of Xenopus SGLT1-like protein (xSGLT1L) with the homologous region of rabbit SGLT1. The chimera was expressed in Xenopus oocytes and its transport activity was measured by the two-microelectrode voltage-clamp method. The substrate specificity of the chimera was different from those of xSGLT1L and SGLT1. In addition the chimera's apparent Michaelis-Menten constant (K_m) for myo-inositol, 0.06 mM, was about one fourth of that of xSGLT1L, 0.25 mM, while the chimera's apparent K_m for d-glucose, 0.8 mM, was about one eighth of that of xSGLT1L, 6.3 mM. Our results suggest that the extracellular loop between TM12 and TM13 participates in the sugar transport of SGLT1.     

Calpain2 protease: A new member of the Wnt/Ca pathway modulating convergent extension movements in Xenopus

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development. We show that Calpain2 is expressed maternally with elevated expression in neural tissues and that Calpain2 activity is spatially and temporally regulated. Using a Calpain inhibitor, a dominant negative and a morpholino oligonoucleotide we demonstrate that impaired Calpain2 activity results in defective convergent extension both in mesodermal and neural tissues. Specifically, Calpain2 downregulation results in loss of tissue polarity and blockage of mediolateral intercalation in Keller explants without affecting adherens junction turnover. We further show that Calpain2 is activated in response to Wnt5a and that the inhibitory effect of Wnt5a expression on animal cap elongation can be rescued by blocking Calpain2 function. This suggests that Calpain2 activity needs to be tightly regulated during convergent extension. Finally we show that expression of Xdd1 blocks the membrane translocation of Calpain2 suggesting that Calpain2 activation is downstream of Dishevelled. Overall our data show that Calpain2 activation through the Wnt/Ca²⁺ pathway and Dishevelled can modulate convergent extension movements.     

Translational repression by the oocyte-specific protein P100 in Xenopus

The translational regulation of maternal mRNAs is one of the most important steps in the control of temporal-spatial gene expression during oocyte maturation and early embryogenesis in various species. Recently, it has become clear that protein components of mRNPs play essential roles in the translational regulation of maternal mRNAs. In the present study, we investigated the function of P100 in Xenopus oocytes. P100 exhibits sequence conservation with budding yeast Pat1 and is likely the orthologue of human Pat1a (also called PatL2). P100 is maternally expressed in immature oocytes, but disappears during oocyte maturation. In oocytes, P100 is an RNA binding component of ribosome-free mRNPs, associating with other mRNP components such as Xp54, xRAP55 and CPEB. Translational repression by overexpression of P100 occurred when reporter mRNAs were injected into oocytes. Intriguingly, we found that when P100 was overexpressed in the oocytes, the kinetics of oocyte maturation was considerably retarded. In addition, overexpression of P100 in oocytes significantly affected the accumulation of c-Mos and cyclin B1 during oocyte maturation. These results suggest that P100 plays a role in regulating the translation of specific maternal mRNAs required for the progression of Xenopus oocyte maturation.     

The involvement of lethal giant larvae and Wnt signaling in bottle cell formation in Xenopus embryos

Lethal giant larvae (Lgl) plays a critical role in establishment of cell polarity in epithelial cells. While Frizzled/Dsh signaling has been implicated in the regulation of the localization and activity of Lgl, it remains unclear whether specific Wnt ligands are involved. Here we show that Wnt5a triggers the release of Lgl from the cell cortex into the cytoplasm with the concomitant decrease in Lgl stability. The observed changes in Lgl localization were independent of atypical PKC (aPKC), which is known to influence Lgl distribution. In ectodermal cells, both Wnt5a and Lgl triggered morphological and molecular changes characteristic of apical constriction, whereas depletion of their functions prevented endogenous and ectopic bottle cell formation. Furthermore, Lgl RNA partially rescued bottle cell formation in embryos injected with a dominant negative Wnt5a construct. These results suggest a molecular link between Wnt5a and Lgl that is essential for apical constriction during vertebrate gastrulation.     

Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels

Fast Na⁺ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in L_III-IV of the rat brain type IIA Na⁺ channel. We have analyzed macroscopic and single channel currents from Na⁺ channels with mutations within and adjacent to hydrophobic clusters in L_III-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in L_III-IV also affected the latency to first opening, indicating coupling between L_III-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na⁺ channels.     

Trim36/Haprin plays a critical role in the arrangement of somites during Xenopus embryogenesis

The TRIM family members contain a tripartite motif (TRIM), which includes RING, B-box, and coiled-coil domains, or collectively RBCC. They have been implicated in a variety of biological processes, such as the regulation of differentiation and development, and oncogenesis. In this study, we discovered a novel function of the TRIM family in early development. We report the expression of Trim36/Haprin during Xenopus laevis early embryogenesis and its involvement in somite formation. Temporal expression analysis indicated that Trim36/Haprin was present throughout embryogenesis. Spatial expression analysis showed that its expression was mainly confined to the nervous system and a portion of the posterior somite. Morpholino-mediated knockdown of Trim36/Haprin markedly and specifically inhibited the somite formation. We conclude that Trim36/Haprin plays an important role in the arrangement of somites during their formation.     

Minor-class splicing occurs in the nucleus of the Xenopus oocyte

A small fraction of premessenger RNA introns in certain eukaryotes is excised by the minor spliceosome, which contains low-abundance small nuclear ribonucleoproteins (snRNPs). Recently, it was suggested that minor-class snRNPs are localized to and function in the cytoplasm of vertebrate cells. To test whether U12-type splicing occurs in the cytoplasm of Xenopus oocytes, we performed microinjections of the well-characterized P120 minor-class splicing substrate into the nucleus or into the cytoplasm. Our results demonstrate that accurate splicing of this U12-dependent intron occurs exclusively in the nuclear compartment of the oocyte, where U12 and U6atac snRNPs are primarily localized. We further demonstrate that splicing of both a major-class and a minor-class intron is inhibited after nuclear envelope breakdown during meiosis.     

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na⁺-independent chloride-bicarbonate exchanger genes and multiple Na⁺-bicarbonate cotransporter and Na⁺-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na⁺-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.     

Molecular Analysis of Potential Hinge Residues in the Inactivation Gate of Brain Type IIA Na+ Channels

During inactivation of Na⁺ channels, the intracellular loop connecting domains III and IV is thought to fold into the channel protein and occlude the pore through interaction of the hydrophobic motif isoleucine-phenylalanine-methionine (IFM) with a receptor site. We have searched for amino acid residues flanking the IFM motif which may contribute to formation of molecular hinges that allow this motion of the inactivation gate. Site-directed mutagenesis of proline and glycine residues, which often are components of molecular hinges in proteins, revealed that G1484, G1485, P1512, P1514, and P1516 are required for normal fast inactivation. Mutations of these residues slow the time course of macroscopic inactivation. Single channel analysis of mutations G1484A, G1485A, and P1512A showed that the slowing of macroscopic inactivation is produced by increases in open duration and latency to first opening. These mutant channels also show a higher probability of entering a slow gating mode in which their inactivation is further impaired. The effects on gating transitions in the pathway to open Na⁺ channels indicate conformational coupling of activation to transitions in the inactivation gate. The results are consistent with the hypothesis that these glycine and proline residues contribute to hinge regions which allow movement of the inactivation gate during the inactivation process of Na⁺ channels.     

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2×RBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.