Molecular cloning and expression of a cyclic AMP-activated chloride conductance regulator: a novel ATP-binding cassette transporter.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated, cAMP-activated chloride channel located in the apical membrane of many epithelial secretory cells. Here we report cloning of a cAMP-activated epithelial basolateral chloride conductance regulator (EBCR) that appears to be a basolateral CFTR counterpart. This novel chloride channel or regulator shows 49% identity with multidrug resistance-associated protein (MRP) and 29% identity with CFTR. On expression in Xenopus oocytes, EBCR confers a cAMP-activated chloride conductance that is inhibited by the chloride channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamine)benzoic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Northern blot analysis reveals high expression in small intestine, kidney, and liver. In kidney, immunohistochemistry shows a conspicuous basolateral localization mainly in the thick ascending limb of Henle's loop, distal convoluted tubules and to a lesser extent connecting tubules. These data suggest that in the kidney EBCR is involved in hormone-regulated chloride reabsorption.

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis.

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Novel retinoic acid receptor ligands in Xenopus embryos.

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.

P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues.

Retinoid X receptor-selective ligands produce malformations in Xenopus embryos.

Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus, both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis, whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus, RAR- and RXR-selective ligands regulated the expression of the Xlim-1, gsc, and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless, RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo, consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species.

Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum.

Release of Ca2+ stored in endoplasmic reticulum is a ubiquitous mechanism involved in cellular signal transduction, proliferation, and apoptosis. Recently, sphingolipid metabolites have been recognized as mediators of intracellular Ca2+ release, through their action at a previously undescribed intracellular Ca2+ channel. Here we describe the molecular cloning and characterization of a protein that causes the expression of sphingosyl-phosphocholine-mediated Ca2+ release when its complementary RNA is injected into Xenopus oocytes. SCaMPER (for sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) is an 181 amino acid protein with two putative membrane-spanning domains. SCaMPER is incorporated into microsomes upon expression in SO cells or after translation in vitro. It mediates Ca2+ release at 4 degrees C as well as 22 degrees C, consistent with having ion channel function. The EC50 for Ca2+ release from Xenopus oocytes is 40 microM, similar to sphingosyl-phosphocholine-mediated Ca2+ release from permeabilized mammalian cells. Because Ca2+ release is not blocked by ryanodine or La3+, the activity described here is distinct from the Ca2+ release activity of the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. The properties of SCaMPER are identical to those of the sphingolipid-gated Ca2+ channel that we have previously described. These findings suggest that SCaMPER is a sphingolipid-gated Ca2+-permeable channel and support its role as a mediator of this pathway for intracellular Ca2+ signal transduction.

A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis.

We have analyzed cyclin E1, a protein that is essential for the G1/S transition, during early development in Xenopus embryos. Cyclin E1 was found to be abundant in eggs, and after fertilization, until the midblastula transition (MBT) when levels of cyclin E1 protein, and associated kinase activity, were found to decline precipitously. Our results suggest that the reduced level of the cyclin E1 protein detected after the MBT does not occur indirectly as a result of degradation of the maternally encoded cyclin E1 mRNA. Instead, the stability of cyclin E1 protein appears to play a major role in reduction of cyclin E1 levels at this time. Cyclin E1 protein was found to be stable during the cleavage divisions but degraded with a much shorter half-life after the MBT. Activation of cyclin E1 protein turnover occurs independent of cell cycle progression, does not require ongoing protein synthesis, and is not triggered as a result of the ratio of nuclei to cytoplasm in embryonic cells that initiates the MBT. We therefore propose that a developmental timing mechanism measures an approximately 5-hr time period, from the time of fertilization, and then allows activation of a protein degradative pathway that regulates cyclin E1. Characterization of the timer suggests that it might be held inactive in eggs by a mitogen-activated protein kinase signal transduction pathway.

Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.

Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo.

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.

Heteromeric connexons in lens gap junction channels.

Gap junction channels are formed by paired oligomeric membrane hemichannels called connexons, which are composed of proteins of the connexin family. Experiments with transfected cell lines and paired Xenopus oocytes have demonstrated that heterotypic intercellular channels which are formed by two connexons, each composed of a different connexin, can selectively occur. Studies by Stauffer [Stauffer, K. A. (1995) J. Biol. Chem. 270, 6768-6772] have shown that recombinant Cx26 and Cx32 coinfected into insect cells may form heteromeric connexons. By solubilizing and subfractionating individual connexons from ovine lenses, we show by immunoprecipitation that connexons can contain two different connexins forming heteromeric assemblies in vivo.

Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway.

An inhibitor of p34cdc2/cyclin B that regulates the G2/M transition in Xenopus extracts.

The activity of maturation-promoting factor (MPF), a protein kinase complex composed of p34cdc2 and cyclin B, is undetectable during interphase but rises abruptly at the G2/M transition to induce mitosis. After the synthesis of cyclin B, the suppression of MPF activity before mitosis has been attributed to the phosphorylation of p34cdc2 on sites (threonine-14 and tyrosine-15) that inhibit its catalytic activity. We previously showed that the activity of the mitotic p34cdc2/cyclin B complex is rapidly suppressed when added to interphase Xenopus extracts that lack endogenous cyclin B. Here we show that a mutant of p34cdc2 that cannot be inhibited by phosphorylation (threonine-14-->alanine, tyrosine-15-->phenylalanine) is also susceptible to inactivation, demonstrating that inhibitory mechanisms independent of threonine-14 and tyrosine-15 phosphorylation must exist. We have partially characterized this inhibitory pathway as one involving a reversible binding inhibitor of p34cdc2/cyclin B that is tightly associated with cell membranes. Kinetic analysis suggests that this inhibitor, in conjunction with the kinases that mediate the inhibitory phosphorylations on p34cdc2, maintains the interphase state in Xenopus; it may play an important role in the exact timing of the G2/M transition.

Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.