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O Pongs

Publications and source records attributed to O Pongs.

At least 127 records · Page 7Linked to original sources
PubMed1990

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.

Animals↗
PubMed1990

Alternative Shaker transcripts express either rapidly inactivating or noninactivating K+ channels.

Two members of the Shaker K+ channel family designated ShA2 and ShD2 were characterized in the Xenopus oocyte expression system. The predicted amino acid sequences of ShA2 and ShD2 differ only in the amino terminus, which is located intracellularly according to the present topological model of K+ channels. The differing amino termini have profound effects on the electrophysiological and pharmacological properties of the K+ channel. Most markedly, the nature of the amino terminus determines whether the K+ channel mediates rapidly inactivating or noninactivating K+ currents. It also affects the 4-aminopyridine, tetraethylammonium, and charybdotoxin sensitivities of the K+ channels. These results suggest that the amino terminus of Shaker proteins affects K+ channel structures on both sides of the membrane.

4-Aminopyridine↗
PubMed1990

Genetic analysis of the Shaker gene complex of Drosophila melanogaster.

The Shaker complex (ShC) spans over 350 kb in the 16F region of the X chromosome. It can be dissected by means of aneuploids into three main sections: the maternal effect (ME), the viable (V) and the haplolethal (HL) regions. The mutational analysis of ShC shows a high density of antimorphic mutations among 12 lethal complementation groups in addition to 14 viable alleles. The complex is the structural locus of a family of potassium channels as well as a number of functions relevant to the biology of the nervous system. The constituents of ShC seem to be linked by functional relationships in view of the similarity of the phenotypes, antimorphic nature of their mutations and the behavior in transheterozygotes. We discuss the relationship between the genetic organization of ShC and the functional coupling of potassium currents with the other functions encoded in the complex.

Action Potentials↗
PubMed1989

A member of the steroid hormone receptor gene family is expressed in the 20-OH-ecdysone inducible puff 75B in Drosophila melanogaster.

Drosophila melanogaster DNA has been cloned which encompasses a major part of the 20-OH-ecdysone inducible puff 75B. One 20-OH-ecdysone responsive transcription unit was detected which is expressed into two transcripts which accumulate upon the incubation of salivary glands of 3rd instar larvae with 20-OH-ecdysone. This accumulation is correlated with the 20-OH-ecdysone induced activity of puff 75B. 75B cDNA analysis indicates that the activity of puff 75B leads to the synthesis of a protein which belongs to the steroid and thyroid hormone receptor superfamily. The highest similarity of the derived 75B protein sequence was found to the DNA and ligand binding domains of human retinoic acid receptor. A study of the tissue distribution in larvae revealed that 75B mRNA is present in most, if not all 20-OH-ecdysone target tissues. It is proposed that 75B protein is a DNA-binding protein playing a key role in mediating the regulation of the larval molt by 20-OH-ecdysone.

Amino Acid Sequence↗
PubMed1989

Characterization of a putative transcription factor gene expressed in the 20-OH-ecdysone inducible puff 74EF in Drosophila melanogaster.

Drosophila melanogaster DNA has been cloned which encompasses the major part of the 20-OH-ecdysone inducible puff 74EF. One 20-OH-ecdysone responsive transcription unit was detected which gives rise to two alternative transcripts. The expression of one transcript in salivary glands of 3rd instar larvae is correlated with the 20-OH-ecdysone induced activity of puff 74EF. Corresponding cDNA analysis indicates that the two transcripts are translated into two different proteins which have alternative amino terminal ends. The carboxy terminal domain of the 74E proteins is similar to the carboxy terminal sequences of ets-oncoproteins suggesting that the 74E proteins represent alternative transcription factors. It is proposed that the activity of the 20-OH-ecdysone inducible puff 74EF leads to a switch in the synthesis of a transcription factor.

Amino Acid Sequence↗
PubMed1989

Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+-channel agonist and antagonist.

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an insideout orientation. Digitonin-solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside-out orientation. Ca2+ selectively stimulates binding of the Ca2+-channel antagonist [3H]PN200-110 to dihydropyridine receptor in the outside-out but not the inside-out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+-specific binding site of dihydropyridine receptor is 2-3 microM. The data demonstrate that binding Ca2+ to the extracellular high-affinity Ca2+-binding site is required for binding dihydropyridines to dihydropyridine receptor. This binding is inhibited, however, by 1-10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+-channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside-out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+-specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+-channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200-110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.

Animals↗
PubMed1989

The interference of truncated with normal potassium channel subunits leads to abnormal behaviour in transgenic Drosophila melanogaster.

The Shaker locus of Drosophila melanogaster encodes a family of A-type potassium channel subunits. Shaker mutants behave as antimorphs in gene dosage tests. This behaviour is due to the production of truncated A-channel subunits. We propose that they interfere with the function of their normal counterpart by forming multimeric A-channel structures. This hypothesis was tested by constructing transgenic flies carrying a heat-inducible gene encoding a truncated A-type potassium channel subunit together with a normal wild type doses of A-type potassium channel subunits. The altered subunit leads at larval, pupal or adult stages to the transformation of wild type into Shaker flies. The transformed flies exhibited a heat-inducible abnormal leg shaking behaviour and a heat-inducible facilitated neurotransmitter release at larval neuromuscular junctions. By the overexpression of an aberrant A-channel subunit the normal behaviour of transgenic D. melanogaster can be altered in a predictable way.

Amino Acid Sequence↗
PubMed1989

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.

4-Aminopyridine↗
PubMed1989

Antibodies against Drosophila potassium channels identify membrane proteins across species.

Shaker is a complex locus (ShC) in Drosophila that encodes components of the K+ channel responsible for the IA current. We have raised antibodies against synthetic peptides of selected sequences from the Sh products. One of the antisera identifies a 71 kDa protein band in immunoblots from Drosophila neural membrane proteins. We demonstrate that this protein is encoded within the viable (V) region of the ShC since deletions and breakpoints in this part of the complex eliminate this band from the immunoblots. Certain Sh mutations abolish the production of this product while other do not seem to interfere with it. The same antiserum identifies bands of different apparent molecular weight (Mr) in membrane extracts of nervous systems of a variety of organisms including vertebrates.

Animals↗
PubMed1989

Characterization of an estradiol-stimulated mRNA in the brain of adult male rats.

Differential hybridization of a cDNA library from rat C6 glioma cells with cDNA probes from naive C6 glioma cells and from cells exposed to 17 beta-estradiol identified cDNAs of an mRNA stimulated by 17 beta-estradiol. This mRNA designated ESP1 mRNA, reached maximal levels after 8 h of treatment with 17 beta-estradiol. The stimulation was not suppressed by cycloheximide. Dexamethasone treatment of C6 glioma cells did not induce ESP1 mRNA. It codes for a 164 amino acids long peptide. The sequence is similar in part to that of CRIP protein, a probably member of the ferredoxin superfamily. The conservation of primary structure suggests a role of ESP1 peptide in oxygen consumption. ESP1 mRNA expression is sexually dimorphic in body tissue, whereas it is expressed to comparative levels in the brain of adult males and females. This suggests that 17 beta-estradiol stimulates the expression of the ESP1 gene in the brain of both gender.

Animals↗
PubMed1988

Potassium channels expressed from rat brain cDNA have delayed rectifier properties.

Injection into Xenopus oocytes of RNA synthesized in vitro using the rat brain cDNA RCK1 as a template or nuclear injection of the cDNA results in the expression of functional potassium channels. These channels exhibit properties similar to those of the non-inactivating delayed rectifier channel found in mammalian neurons and other excitable cells.

4-Aminopyridine↗
PubMed1988

Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila.

The Shaker locus of Drosophila contains a very large transcription unit. It is expressed predominantly in the nervous system by multiple, differential as well as alternative, splicing mechanisms into different, but functionally related proteins. The structure of the Shaker transcription unit and the properties of the encoded Shaker protein family provide a molecular basis for A channel diversity in excitable cells.

Amino Acid Sequence↗
PubMed1988

Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems.

Voltage-sensitive potassium channels are found in vertebrate and invertebrate central nervous systems. We have isolated a rat brain cDNA by cross-hybridization with a probe of the Drosophila Shaker gene complex. Structural conservation of domains of the deduced protein indicate that the rat brain cDNA encodes a voltage-sensitive potassium channel. Of the deduced amino acid sequence, 82% is homologous to the Drosophila Shaker protein indicating that voltage-sensitive potassium channels have been highly conserved during evolution. Selective pressure was highest on sequences facing the intracellular side and on proposed transmembrane segments S4-S6, suggesting that these domains are crucial for voltage-dependent potassium channel function. The corresponding rat mRNA apparently belongs to a family of mRNA molecules which are preferentially expressed in the central nervous system.

Amino Acid Sequence↗
PubMed1988

Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes.

Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca2+ channel) has been purified as a 340-kDa protein complex to approximately 80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two-step chromatographic purification procedure. The equilibrium dissociation constant of [3H](+)-PN200-110 binding (Kd; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approximately 300 kDa. The 195-kDa subunit was affinity-labeled with [3H]azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).

Affinity Labels↗
PubMed1987

Molecular organization of the maternal effect region of the Shaker complex of Drosophila: characterization of an I(A) channel transcript with homology to vertebrate Na channel.

We have cloned 215-kb DNA containing the maternal effect region (ME) of the Shaker gene complex (shC) at 16F of the Drosophila X chromosome. Five translocation and deletion breakpoints have been mapped on the cloned DNA allowing a correlation of the genetic map to transcription units. The ME region spans 100 kb. The genetic behavior of this region correlates with the occurrence of maternal RNAs in this part of the ShC. Two transcripts have been identified in the vicinity of chromosomal rearrangements which cause a Sh phenotype. These are a 4.5-kb transcript interrupted by T(x;2)B27 and a 2-kb transcript interrupted by T(X;3)Sh and T(X;Y)W32. The latter transcript is derived from a primary transcript which spans >65 kb genomic DNA. The cDNA-sequencing data show that this Shaker (I(A)channel) gene can encode a protein of 35 kd with three alpha-helical membrane-spanning sequences near its carboxyl terminus. These have a striking homology with membrane-spanning sequences of the vertabrate Na channel.

Journal Article↗
PubMed1986

Upstream sequences modulate in vitro transcription from Drosophila yolk protein genes I and II.

A Drosophila transcription system was employed to study transcription in vitro from templates carrying yolk polypeptide I and II (YPI, YPII) promoter regions. The transcription start sites of YPI and YPII genes are linked by 1225 base pairs of intergenic DNA and the genes are transcribed in divergent directions. Transcription was studied in vitro from templates carrying YPI or YPII promoter regions separately. We have successively deleted intergenic DNA upstream of the YPI or the YPII transcription start site and have assayed the dependence of transcription efficiency an template concentration. The results indicate that in vitro YPI and YPII transcript synthesis is controlled by separate cis-acting DNA sequence elements, which are located between 86 and 159 base pairs upstream of the YPI transcription start site and between 161 and 341 base pairs upstream of the YPII transcription start site. These elements stimulate in vitro transcription up to fivefold. Transcription was also studied with templates which contained both YP promoters. The transcription data indicate that the intergenic DNA contains additional DNA motif(s), which apparently modulate in vitro transcription coordinately from both YP promoters in an orientation-dependent manner.

Animals↗