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O Pongs

Publications and source records attributed to O Pongs.

At least 19 recordsLinked to original sources
PubMed1992

Cloning of a bovine voltage-gated K+ channel gene utilising partial amino acid sequence of a dendrotoxin-binding protein from brain cortex.

Several variants of fast-activating, voltage-dependent K+ channels exist in the nervous system where they control cell excitability and synaptic transmission, some of which are blocked selectively by alpha-dendrotoxin. Cloning of a K+ channel from bovine genomic DNA was achieved using a primer based on the N-terminal sequence of the larger subunit from the purified toxin acceptor, in conjunction with secondary primers, in the polymerase chain reaction. The resultant amino acid sequence is highly homologous to RCK 5 already cloned from rat brain, which yields a K+ current susceptible to alpha-dendrotoxin, when expressed in oocytes. These findings establish conclusively that the extensively characterized alpha-dendrotoxin acceptor is a K+ channel protein.

Amino Acid Sequence↗
PubMed1992

Extracellular K+ specifically modulates a rat brain K+ channel.

Extracellular potassium concentration is actively maintained within narrow limits in all higher organisms. Slight variations in extracellular potassium levels can induce major alterations of essential physiological functions in excitable tissues. Here we describe that superfusion of cultured rat hippocampal neurones with potassium-free medium leads to a decrease of a specific outward potassium current, probably carried by RCK4-type channels (RCK4 are potassium channels found in rat brain). This is confirmed by heterologous expression of these channels in Xenopus oocytes. In this system, variations of extracellular potassium in the physiological concentration range induce significant differences in current amplitude. Moreover, the current is completely suppressed in the absence of extracellular potassium. The potassium dependence of macroscopic conductance in RCK4 channels was related by site-directed mutagenesis to that lysine residue in the extracellular loop between the transmembrane segments S5 and S6 of RCK4 protein that confers resistance to extracellular blockage by tetraethylammonium. It is shown that extracellular potassium affects the number of available RCK4 channels, but not the single-channel conductance, the mean open time, or the gating charge displacement upon depolarization.

Amino Acid Sequence↗
PubMed1992

The potassium channel gene HK1 maps to human chromosome 11p14.1, close to the FSHB gene.

Transiently activating (A-type) potassium (K) channels are important regulators of action potential and action potential firing frequencies. HK1 designates the first human cDNA that is highly homologous to the rat RCK4 cDNA that codes for an A-type K-channel. The HK1 channel is expressed in heart. By somatic cell hybrid analysis, the HK1 gene has been assigned to human chromosome 11p13-p14, the WAGR deletion region (Wilms tumor, aniridia, genito-urinary abnormalities and mental retardation). Subsequent pulsed field gel (PFG) analysis and comparison with the well-established PFG map of this region localized the gene to 11p14,200-600 kb telomeric to the FSHB gene.

Animals↗
PubMed1992

Structural basis of voltage-gated K+ channel pharmacology.

Major advances have been made in understanding the domains and amino acid sidechains important for the function of voltage-gated K+ channels, by combining recombinant DNA techniques with pharmacological and electrophysiological approaches. As explained in this review by Olaf Pongs, the results of these experiments have enabled description of a detailed model of the K+ channel pore structure and provide an exciting picture of how open-channel blockers occlude the pore of K+ channels. Since the pore is a highly conserved structure among voltage-gated K+ channels, there are only limited possibilities for open K+ channel blockers to distinguish between the many distinct voltage-gated K+ channels, which have diverse kinetic and conductance properties.

Amino Acid Sequence↗
PubMed1992

Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.

Animals↗
PubMed1991

Cloned neuronal IK(A) channels reopen during recovery from inactivation.

The kinetic behaviour and functional role of potassium ion (K+) channels mediating a fast-inactivating K+ current (IK(A)) has been widely discussed. Activating in the subthreshold range of excitation, IK(A) channels are assumed to reduce the excitatory effect of depolarizing membrane currents in a time-dependent manner. Here we report that IK(A) channels not only open in response to a depolarization but open again after repolarization of the membrane. Although the current in response to the depolarization is rapidly inactivating, the current elicited by repolarization declines slowly and produces long-lasting afterhyperpolarizations under current-clamp conditions. This implies an additional physiological role for IK(A) channels, particularly those that activate positive to the threshold of excitation. The underlying biophysical mechanism was studied by fast-application of peptides corresponding to the N-terminal end of the IK(A) channel proteins. It was found to be a voltage-dependent release of the inactivation gate.

Amino Acid Sequence↗
PubMed1991

Characterization of a specific ecdysteroid receptor-DNA complex reveals common properties for invertebrate and vertebrate hormone-receptor/DNA interactions.

A gel-filtration assay has been developed with which the specific interaction between Drosophila melanogaster ecdysteroid receptor and the 20-hydroxy-ecdysone responsive element of the hsp27 gene promoter region was characterized in terms of complex formation, saturation of DNA binding and the apparent molecular mass of the complex. The hsp27 DNA-binding sequence for ecdysteroid receptor in vitro was delimited by footprinting and mutational analysis. The combined results show that ecdysteroid receptor binds as a dimer to an imperfect palindromic sequence (GGTTCAATGCACT) closely resembling the structures of the different vertebrate steroid-hormone-responsive elements reported so far.

Animals↗
PubMed1991

Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation.

Modulation of neuronal excitability by regulation of K+ channels potentially plays a part in short-term memory but has not yet been studied at the molecular level. Regulation of K+ channels by protein phosphorylation and oxygen has been described for various tissues and cell types; regulation of fast-inactivating K+ channels mediating IK(A) currents has not yet been described. Functional expression of cloned mammalian K+ channels has provided a tool for studying their regulation at the molecular level. We report here that fast-inactivating K+ currents mediated by cloned K+ channel subunits derived from mammalian brain expressed in Xenopus oocytes are regulated by the reducing agent glutathione. This type of regulation may have a role in vivo to link metabolism to excitability and to regulate excitability in specific membrane areas of mammalian neurons.

Amino Acid Sequence↗
PubMed1991

Swapping of functional domains in voltage-gated K+ channels.

Functionally significant properties of domains in the amino acid sequence of potassium (K+) channel-forming proteins have been investigated by constructing chimeric K+ channels. The N-terminal domain of ShA2 channels was responsible for the fast inactivation (IKA) and also determined a shift in the threshold of activation whereas the membrane domain determined the timecourse of slow inactivation. The binding site for dendrotoxin (DTX), but not for mast cell degranulating peptide (MCDP), is completely located on the loop between the membrane spanning segments S5 and S6 in RCK1 channels. A certain part of this region which has recently been designated as a narrow part of the pore was found to be not responsible for the differences in the single-channel current amplitude between RCK4 and RCK2 K+ channels. Interchange of the C-terminal domain did not influence activation or inactivation of the channels.

Amino Acid Sequence↗
PubMed1991

Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain.

A rat brain cDNA (Raw3) related to the Drosophila Shaw K+ channel family has been characterized. Raw3 cRNA leads to the formation of TEA-insensitive, fast inactivating (A-type) K+ channels when injected into Xenopus laevis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A-type K+ channel RCK4, Raw3 channels operate in the positive voltage range.

Amino Acid Sequence↗
PubMed1991

Gating currents of inactivating and non-inactivating potassium channels expressed in Xenopus oocytes.

The Xenopus oocyte expression system in combination with patch-clamp techniques allows the measurement of ionic currents from a single class of genetically engineered ion channels. Ionic currents in the nanoampere range from oocytes injected with cRNA, corresponding to potassium channels, can be recorded in the inside-out patch configuration. These recordings have a high time resolution at low background noise. Substitution of impermeant ions for potassium and blocking of the channel conductance with tetraethylammonium allows the recording of potassium gating currents, Ig, which is hampered in natural excitable cells by the simultaneous presence of sodium channels and a variety of different potassium channels. The "on" transients, Ig(on), are fast and can have amplitudes of up to several tens of pA. Upon repolarization to -100 mV after small depolarizations, "off" gating currents, Ig(off)g, which reverse most of the "on" charge displacement, Q(on), within 1 ms, are readily observed. However, this fast recovery of the gating charge is drastically reduced upon increasing the amplitude of the depolarizing pulse. In contrast to sodium channels, this temporary charge immobilization is complete within a few milliseconds at positive membrane potentials. Furthermore, there seems to be no direct correlation between charge immobilization and inactivation because the same phenomenon occurs for channels that do not inactivate.

Animals↗
PubMed1991

Novel potassium channels encoded by the Shaker locus in Drosophila photoreceptors.

The Shaker gene, responsible for A-type potassium channels in Drosophila muscle, encodes a large family of transcripts capable of generating a variety of kinetically distinct A channels when expressed in oocytes. We describe a distinct class of A channel encoded by the Shaker gene in a novel preparation of dissociated Drosophila photoreceptors. Whole-cell recordings reveal a rapidly inactivating A current that is absent in Shaker mutants and that can be readily isolated in cell-attached patches. Although very similar to their muscle counterparts, the photoreceptor A channels show a striking 40-50 mV negative shift in their voltage-operating range. Two mutations (ShE62 and T(1;Y)W32), which exclude only certain classes of Shaker transcripts, were used to show that photoreceptor A channels are encoded by multiple transcripts distinct from those encoding muscle A channels, while PCR techniques identified four transcripts (ShA1, ShA2, ShG1, and ShG2) in mRNA from dissected retina.

Animals↗
PubMed1991

Troponin I is encoded in the haplolethal region of the Shaker gene complex of Drosophila.

We have analyzed one of the nine complementation groups that constitute the haplolethal (HL) region of the Shaker gene complex (ShC). Five mutations, including a dominant lethal, define this complementation group: HL I. Mutant phenotypes show abnormal embryogenesis with structural defects in the nervous system and aberrant degeneration of specific adult muscles in addition to altered action potentials. HL I encodes a family of proteins with extensive homology to invertebrate troponin I (TnI). Members of this family are brought about by two alternative and two mutually exclusive exons in conjunction with two differential polyadenylation sites. Transcription analysis indicates that some isoforms are adult specific and others are synthesized throughout development, except during early metamorphosis. Certain isoforms of Drosophila TnI are expressed in specific muscles. The specificity of mutant phenotypes suggests a functional role of particular TnI isoforms in the development and the mature activity of muscle and nervous systems.

Amino Acid Sequence↗
PubMed1990

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor.

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.

Cellulose↗
PubMed1990

Heteromultimeric channels formed by rat brain potassium-channel proteins.

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.

Animals↗