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Biomedical subjects

O Pongs

Publications and source records attributed to O Pongs.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cloning and functional expression of rat eag2, a new member of the ether-à-go-go family of potassium channels and comparison of its distribution with that of eag1.

A second mammalian gene for the ether-à-go-go (eag) potassium channel has been cloned from the rat, and its predicted protein sequence is 70% identical to that of rat ether-à-go-go1 with a further 10% conservatively replaced residues. The rat eag2 mRNA was predominantly expressed in neural tissue and was not detected in adult skeletal, cardiac, or smooth muscle. Within the brain, its distribution overlaps that of rat ether-à-go-go1 in specific regions within the cortex and olfactory bulb, but was differentially distributed in other locations, being scanty within the cerebellum, and most notably present in the thalamus, inferior colliculus, and certain brainstem nuclei. Heterologous expression of rat eag2 in HEK-293 cells gave rise to a voltage-gated, noninactivating potassium current, active at the cells' resting potential and blocked by low nanomolar concentrations of cytosolic calcium. Thus, in neurones, this current is likely to impart a modulation in membrane conductance, which is sensitively responsive to resting internal calcium, and levels of electrical activity.

Animals↗
PubMed2000

Effects of antiarrhythmic drugs on cloned cardiac voltage-gated potassium channels expressed in Xenopus oocytes.

The effects of 17 commonly used antiarrhythmic drugs on the rapidly activating cardiac voltage-gated potassium channels (Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1 and Kv4.2) were studied in the expression system of the Xenopus oocyte. A systematic overview on basic properties was obtained using a simple and restricted experimental protocol (command potentials 10 mV and 50 mV positive to the threshold potential; concentration of 100 micromol/l each). The study revealed that 8 of 17 drugs yielded significant effects (changes >10% of control) on at least one type of potassium channel in the oocyte expression system. These drugs were ajmaline, diltiazem, flecainide, phenytoin, propafenone, propranolol, quinidine and verapamil, whereas the effects of adenosine, amiodarone, bretylium, disopyramide, lidocaine, mexiletine, procainamide, sotalol and tocainide were negligible. The drug effects were characterized by reductions of the potassium currents (except for quinidine and ajmaline). A voltage-dependence of drug effect was found for quinidine, verapamil and diltiazem. The different effect of the drugs was not related to the fast or slow current inactivation of the potassium channels (except for verapamil). Profiles of the individual drug effects at the different potassium channel types were identical for propafenone and flecainide and differed for all other substances. The study demonstrates marked differences in sensitivity to antiarrhythmic drugs within the group of voltage-operated cardiac potassium channel types. Taking the restrictions of the oocyte system into consideration, the findings suggest that several antiarrhythmic drugs exert significant effects at rapidly activating cardiac potassium channels.

Animals↗
PubMed2000

Structural elements determining activation kinetics in Kv2.1.

Voltage-dependent K+ channels open when depolarizing the membrane voltage. Among the different alpha-subunits, the time course of current activation spreads over a wide range. The structural basis underlying this diversity is not known. We constructed multiple chimeras between two voltage-dependent K+ channels, the rapidly activating Kv1.2 and the slowly activating Kv2.1, and we focused on the C-terminal half of the core region. The general strategy was to substitute parts of Kv2.1 by corresponding parts of Kv1.2 and to test for an acceleration of activation. We identified three regions which contribute to the determination of the activation kinetics: the S5-pore linker, the deep pore, and the S4-segment.

Amino Acid Sequence↗
PubMed2000

Solution structure of hpTX2, a toxin from Heteropoda venatoria spider that blocks Kv4.2 potassium channel.

HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel. We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge. A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected. Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin. The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue. Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (L24). The high density in aromatic side chains of the putative functional surface as well as the lack of an asparagine is proposed to be the structural basis of the specificity of HpTX2 toward Kv4.2 channel.

Amino Acid Sequence↗
PubMed1999

NMR structure and functional characteristics of the hydrophilic N terminus of the potassium channel beta-subunit Kvbeta1.1.

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming alpha-subunit or certain auxiliary beta-subunits. Upon coexpression, Kvbeta1.1 was found to endow non-inactivating members of the Kv1alpha family with fast inactivation via its unique N terminus. Here we investigated structure and functional properties of the Kvbeta1.1 N terminus (amino acids 1-62, betaN-(1-62)) using NMR spectroscopy and patch clamp recordings. betaN-(1-62) showed all hallmarks of N-type inactivation: it inactivated non-inactivating Kv1.1 channels when applied to the cytoplasmic side as a synthetic peptide, and its interaction with the alpha-subunit was competed with tetraethylammonium and displayed an affinity in the lower micromolar range. In aequous and physiological salt solution, betaN-(1-62) showed no well defined three-dimensional structure, it rather existed in a fast equilibrium of multiple weakly structured states. These structural and functional properties of betaN-(1-62) closely resemble those of the "unstructured" ID from Shaker B, but differ markedly from those of the compactly folded ID of the Kv3.4 alpha-subunit.

Amino Acid Sequence↗
PubMed1999

Voltage-gated potassium channels: from hyperexcitability to excitement.

The superfamily of voltage-activated potassium channels may express structurally and functionally diverse voltage-activated potassium channels in the nervous system. The roles of some voltage-activated potassium channel types, e.g. rapidly inactivating (transiently active type) channels and muscarine sensitive muscarine sensitive channels, are beginning to be understood. They may significantly influence dendritic action-potential back-propagation, signal to noise ratios in presynaptic excitability or the responsiveness of a neuron to synaptic input. Inherited disorders related to changes in excitability (episodic ataxia, epilepsy, heart arrhythmia) or to defects in sensory perception (hearing loss) have been associated with mutations in a few voltage-activated potassium channel genes. Most likely, more voltage-activated potassium channel genes will be linked to related disorders in the near future.

Animals↗
PubMed1999

Functional and molecular aspects of voltage-gated K+ channel beta subunits.

Voltage-gated potassium channels (Kv) of the Shaker-related superfamily are assembled from membrane-integrated alpha subunits and auxiliary beta subunits. The beta subunits may increase Kv channel surface expression and/or confer A-type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kv alpha and Kv beta subunits depends on the presence or absence of several domains including the amino-terminal N-type inactivating and NIP domains and the Kv alpha and Kv beta binding domains. Loss of function of Kv beta 1.1 subunits leads to a reduction of A-type Kv channel activity in hippocampal and striatal neurons of knock-out mice. This reduction may be correlated with altered cognition and motor control in the knock-out mice.

Alternative Splicing↗
PubMed1999

Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by muristeroneA.

Ecdysteroids play an important role during insect development. We report here the isolation and characterisation of an Ecdysone receptor (EcR) homologue from Heliothis virescens (HvEcR) and present evidence supporting the HvEcR active role as an active component of the native insect receptor. Alignment of the deduced amino acid sequence of HvEcR with those of EcRs from other species confirmed its membership of this family and showed that it is closely related to the B1 isoform of Drosophila melanogaster. Northern blot analysis showed that two transcripts (6.0 and 6.5 kb) were recognised by a probe spanning the DNA and ligand binding domains of the HvEcR. Genomic Southern blots showed that the HvEcR is encoded by a single copy gene. Two lines of evidence towards the functional activity of the HvEcR are presented. In vitro transcribed and translated HvEcR showed specific binding to hsp27 and pall response elements in the presence of CfUSP. Stable expression of HvEcR in 293 cells induced reporter gene activity in the presence of muristeroneA in a dose dependant manner while dexamethasone failed to activate.

ATP-Binding Cassette Transporters↗
PubMed1999

Potassium current suppression in patients with peripheral nerve hyperexcitability.

Acquired neuromyotonia (Isaac's syndrome) is considered to be an autoimmune disease, and the pathomechanism of nerve hyperexcitability in this syndrome is correlated with anti-voltage-gated K(+) channel (VGKC) antibodies. The patch-clamp technique was used to investigate the effects of immunoglobulins from acquired neuromyotonia patients on VGKCs and voltage-gated Na(+) channels in a human neuroblastoma cell line (NB-1). K(+) currents were suppressed in cells that had been co-cultured with acquired neuromyotonia patients' immunoglobulin for 3 days but not for 1 day. The activation and inactivation kinetics of the outward K(+) currents were not altered by these immunoglobulins, nor did the immunoglobulins significantly affect the Na(+) currents. Myokymia or myokymic discharges, with peripheral nerve hyperexcitability, also occur in various neurological disorders such as Guillain-Barré syndrome and idiopathic generalized myokymia without pseudomyotonia. Immuno-globulins from patients with these diseases suppressed K(+) but not Na(+) currents. In addition, in hKv 1.1- and 1.6-transfected CHO (Chinese hamster ovary)-K1 cells, the expressed VGKCs were suppressed by sera from acquired neuromyotonia patients without a change in gating kinetics. Our findings indicate that nerve hyperexcitability is mainly associated with the suppression of voltage-gated K(+) currents with no change in gating kinetics, and that this suppression occurs not only in acquired neuromyotonia but also in Guillain-Barré syndrome and idiopathic generalized myokymia without pseudomyotonia.

Adolescent↗
PubMed1999

Structural and functional characterization of Kv6.2 a new gamma-subunit of voltage-gated potassium channel.

We have cloned and functionally expressed Kv6.2, a new member of the Kv6 subfamily of voltage-gated potassium channel subunits. The human Kv6.2 (KCNF2) gene was mapped at 18q22-18q23. Kv6.2 mRNA is preferentially expressed in rat and human myocard. Rat and human Kv6.2 subunits appear to be unable to form functional Kv channels in a heterologous expression system, but, when coexpressed with Kv2.1 alpha subunits, heteromultimeric Kv channels were formed mediating voltage-activated delayed-rectifier type outward currents. Their kinetics and conductance-voltage relationship were different from those mediated by homomultimeric Kv2.1 channels. Yeast two-hybrid reporter assays indicated that Kv6.2 amino-termini are able to interact specifically with the Kv2.1 amino-terminus. It is proposed that this protein protein interaction underlies Kv2.1/Kv6.2 subunit assembly and the expression of functional heteromultimeric Kv2.1/Kv6.2 channels. The most resiliant feature of the Kv2.1/Kv6.2 channels was their submicromolar sensitivity to the antiarrhythmic drug propafenone. The data suggest that delayed-rectifier type channels containing Kv6.2 subunits may contribute to cardiac action potential repolarization.

Amino Acid Sequence↗
PubMed1999

Characterization of human Kv4.2 mediating a rapidly-inactivating transient voltage-sensitive K+ current.

A human cDNA for the voltage-sensitive potassium channel subunit Kv4.2 has been cloned and functionally characterized. The human Kv4.2 (KCND2) gene was mapped at 7q31-32. Kv4.2 mRNA is prominently expressed in human brain. Relatively high concentrations of Kv4.2 mRNA occurred in mRNA preparations of amygdala, caudate nucleus, cerebellum, hippocampus, substantia nigra, and thalamus. Kv4.2 mRNA was not detected in human heart, kidney, liver, lung, pancreas, and skeletal muscle. The derived Kv4.2 open reading frame consists of 630 amino acids. In comparison to rat Kv4.2, the human Kv4.2 sequence is highly conserved showing amino acid sequence differences at five positions only. The Kv4.2 subunits were expressed heterologously in human embryonic kidney (293) cells and mediated a rapidly inactivating, A-type outward K+ current. The gating kinetics of the Kv4.2-mediated currents were very similar to those of rat Kv4.2-mediated currents. Both the Kv4.2 and Kv4.3 subunits have been implicated in mediating the transient outward K+ current Ito in rodent cardiac myocytes. In contrast we did not detect Kv4.2. but solely Kv4.3 mRNA in human heart RNA preparations. This may suggest that Kv4.2 subunits do not contribute to the rapid transient outward K+ current of atrial and ventricular myocytes in humans.

Amino Acid Sequence↗
PubMed1999

Bacillus stearothermophilus lctB gene gives rise to functional K+ channels in Escherichia coli and in Xenopus oocytes.

We have cloned a small K+ channel subunit (LctB) of the gram-positive bacterium Bacillus stearothermophilus (B. stearo.). The B. stearo. LctB protein is only 134 amino acids long. The sequence contains a typical K+ channel P-domain with a K+ channel GYGD signature sequence and two hydrophobic, possibly membrane-spanning segments M1 and M2. Unexpectedly, LctB K+ channels exhibited properties which differed markedly from the ones reported for KcsA channels of the gram-positive bacterium Streptomyces lividans. LctB channels, when expressed in E. coli, were targeted to the outer membrane and not like KcsA channels to the inner membrane. After reconstitution in black lipid membrane, LctB channels mediated K+ currents at neutral pH. They were apparently not gated by pH like KcsA channels. Also, LctB cRNA produced functional LctB channels in the Xenopus oocyte expression system in marked contrast to KcsA. The results demonstrated that heterologous expression produced functional LctB channels both in E. coli and in Xenopus oocytes. It is proposed that bacterial LctB subunits can be properly handled by the Xenopus oocyte leading to the occurrence of functional LctB K+ channels in the oocyte plasma membrane.

Amino Acid Sequence↗
PubMed1998

Coexpression of the KCNA3B gene product with Kv1.5 leads to a novel A-type potassium channel.

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral alpha-subunits associated with auxiliary cytoplasmic beta-subunits. In this study we have cloned the human Kvbeta3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B, KCNA2B, and KCNA3B gene structures showed that the three Kvbeta genes have very disparate lengths varying from >/=350 kb (KCNA1B) to approximately 7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvbeta subunits, are very similar. The Kvbeta1 and Kvbeta2 splice variants are generated by alternative use of 5'-exons. Mouse Kvbeta4, a potential splice variant of Kvbeta3, is a read-through product where the open reading frame starts within the sequence intervening between Kvbeta3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvbeta4-like open reading frame. Human Kvbeta3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvbeta3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvbeta3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

Alternative Splicing↗
PubMed1998

Cloning and functional expression of rat ether-à-go-go-like K+ channel genes.

1. Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophila ether-à-go-go-like K+ channel (elk). 2. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas. 3. Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage-activated K+ channels with novel properties. 4. RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at -90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG-mediated currents. 5. RELK2 channels produced currents with a fast inactivation component and HERG-like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.

Amino Acid Sequence↗
PubMed1998

Migrating transformed MDCK cells are able to structurally polarize a voltage-activated K+ channel.

Cell migration of transformed renal epithelial cells (MDCK-F) depends-in addition to cytoskeletal mechanisms-on the polarized activity of a Ca2+-sensitive K+ channel in the rear part of the cells. However, because of the lack of specific markers for this channel we are not able to determine whether a polarized distribution of the channel protein underlies its functional polarization. To determine whether the migrating MDCK-F cells have retained the ability to target K+ channels to distinct membrane areas we stably transfected the cells with the voltage-dependent K+ channel Kv1.4. Stable expression and insertion into the plasma membrane could be shown by reverse transcription-PCR, genomic PCR, Western blot, and patch-clamp techniques, respectively. The distribution of Kv1.4 was assessed with indirect immunofluorescence by using conventional and confocal microscopy. These experiments revealed that Kv1.4 is expressed only in transfected cells where it elicits the typical voltage-dependent, rapidly inactivating K+ current. The Kv1.4 protein is clustered at the leading edge of protruding lamellipodia of migrating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion channels into the plasma membrane in an asymmetric way, which reflects the polarization of migrating cells in the plane of movement. These findings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane areas.

Animals↗
PubMed1998

NIP domain prevents N-type inactivation in voltage-gated potassium channels.

Shaker-related voltage-gated K+ (Kv) channels are assembled from ion-conducting K(v)alpha subunits, which are integral membrane proteins, and auxiliary K(v)beta subunits. This leads to the formation of highly diverse heteromultimeric Kv channels that mediate outward currents with a wide range of time courses for inactivation. Two principal inactivation mechanisms have been recognized: C-type inactivation correlated with carboxy-terminal K(v)alpha-subunit structures, and N-type inactivation conferred by 'ball' domains in the amino termini of certain K(v)alpha and K(v)beta subunits. Assembly of heteromultimers with one or more K(v)alpha- and/or K(v)beta ball domains appears to be an essential principle of the generation of A-type Kv channel diversity. Here we show that, unexpectedly, the presence of K(v)alpha- or K(v)beta-ball domains does not dominate the gating phenotype in heteromultimers containing Kv1.6alpha subunits. These heteromultimers mediate non-inactivating currents because of the dominant-negative activity of a new type of N-type inactivation-prevention (NIP) domain present in the Kv1.6 amino terminus. Mutations in the NIP domain lead to loss of function, and its transfer to another K(v)alpha subunit leads to gain of function. Our discovery of the NIP domain, which neutralizes the activity of K(v)alpha- and K(v)beta-inactivation gates, establishes a new determinant for the gating behaviour of heteromultimeric Kv channels.

Amino Acid Sequence↗
PubMed1998

RERG is a molecular correlate of the inward-rectifying K current in clonal rat pituitary cells.

The rat homologue of the human ether-ä-go-go-related gene (r-erg) was cloned from rat brain using homology screening. RERG has a 96% amino acid identify to HERG. Membrane currents recorded in CHO cells after previous injection of r-erg showed that the voltage- and time-dependent properties are indistinguishable from h-erg-induced currents expressed in the same system. RT-PCR revealed the presence of r-erg mRNA in clonal rat pituitary cells (GH3/B6 cells). These cells exhibit a voltage-dependent inward-rectifying K current (IK, IR) which is highly sensitive to the class III antiarrhythmic E-4031. IK, IR recorded in GH3/B6 cells and ERG currents in CHO cells were compared using similar experimental conditions (same pulse protocols and isotonic KCl as extracellular solution). The voltage- and time-dependent properties of both currents were found to be almost identical. These results strongly suggest that RERG channels mediate IK, IR in GH3/B6 cells.

Amino Acid Sequence↗