Biomedical subjects

W Stühmer

Publications and source records attributed to W Stühmer.

At least 37 records · Page 2Linked to original sources

PubMed1997

Calcium dependence and distribution of calcium-activated chloride channels in Xenopus oocytes.

1. The Ca(2+)-dependent Cl- current (ICl,Ca), expressed in the plasma membrane of Xenopus oocytes, was examined in excised inside-out macropatches using a rapid perfusion system. 2. Application of Ca(2+)-containing Ringer solution resulted in the activation of a current whose reversal potential shifted to the right by 51 +/- 5.2 mV when Cl- in the pipette solution was lowered from 119.3 to 10 mM. No currents were generated when Ca2+ was omitted from the solution. The current is therefore a Ca(2+)-activated Cl- one. 3. Following exposure to Ca2+, the half-time for activation of ICl,Ca was not voltage dependent, whereas deactivation was strongly so. 4. ICl,Ca was stable in the continuous presence of Ca2+ and showed no sign of inactivation or adaptation. 5. Comparison of the size of the currents (normalized to pipette resistance) from the animal and vegetal poles revealed that ICl,Ca had a highly polarized distribution. The current density was almost 10 times higher in the animal pole. 6. The results suggest that Cl- channels provide a continuous and reliable indication of submembranous Ca2+, at least in an excised patch, and the clustering of the Cl- channels renders it necessary to exert caution in interpreting results involving the kinetics of Ca2+ signalling, when ICl,Ca is used as the sole monitor of calcium.

Animals↗

PubMed1997

Mitosis-promoting factor-mediated suppression of a cloned delayed rectifier potassium channel expressed in Xenopus oocytes.

The cell cycle is the crucial process that leads to mitosis in all cell types. The dramatic redirectioning of many cellular processes during the cycle is known to involve ion channels, either changing their level of expression or their voltage dependence, as in the case of inward rectifiers. Here we describe the specific inhibition of heterologously expressed ionic channels at the onset of maturation in Xenopus oocytes. In cells expressing rat eag (R-eag) potassium channels, maturation induces a dramatic reduction in the current amplitude, which is almost complete in most cases. The key molecule in oocyte maturation, the mitosis-promoting factor (a complex of cyclin B and p34cdc2), is able to induce similar changes when injected into the oocytes.

Animals↗

PubMed1997

Molecular basis for different pore properties of potassium channels from the rat brain Kv1 gene family.

Members of the rat brain Kv1 family of cloned potassium channels are structurally highly homologous, but have diverse conductance and pharmacological characteristics. Here we present data on the effects of mutating residues K533 in the P-region and H471 in the S4-S5 linker of Kv1.4 to their equivalent residues in Kv1.1 and Kv1.6 on single-channel conductance and sensitivity to external tetraethylammonium cations (TEA+) and internal Mg2+. Exchange of residue K533 for its equivalent residue (Y) in Kv1.1 and Kv1.6 increased the single-channel conductance at both negative and positive potentials. This mutation is known to reduce the IC50 for external TEA+ from > 100 mM to 0.6 mM, almost identical to that for Kv1.1 (0.53 mM). We have now found that the additional exchange of residue H471 for the equivalent residue (K) in Kv1.6 increased the IC50 for external TEA+ from 0.6 mM (Kv1.4K533Y) to 2.39 mM; this is very close to that for wild-type Kv1.6 channels (2.84 mM). The mutation H471K alone was ineffective. We thus provide evidence that the S4-S5 linker does contribute to the channel's inner-pore region. Data on the block of Kv1 channels by internal Mg2+ indicate that while the binding site is probably situated within the deep-pore region, its exact location may be channel specific.

Animals↗

PubMed1997

Molecular characterization and pharmacological properties of the human P2X3 purinoceptor.

Using PCR and library screening techniques, a cDNA encoding an ATP ligand-gated channel has been isolated from human heart. The full-length cDNA encodes a protein 397 amino acids long which shows a high amino-acid sequence identity with the rat P2X3 purinoceptor (93%). By fluorescence in situ hybridization, the human P2X3 gene has been mapped to region q12 of chromosome 11. Tissue distribution analysis of human P2X3 receptor mRNA shows a restricted expression pattern, i.e. transcripts are limited to the spinal cord and heart. This result contrasts with the distribution of the rat P2X3 receptor which was detected exclusively in sensory neurons of trigeminal, dorsal root and nodose ganglia. Heterologous expression of human P2X3 cRNA in Xenopus oocytes generates a fast desensitizing ATP-activated channel with pharmacological properties resembling the profile of the rat homologue receptor. Thus, the order of agonist potency is 2MeSATP > ATP > alphabeta-meATP > CTP > betagamma-meATP approximately ADP. Moreover, ATP-evoked currents on human P2X3 receptor are efficiently blocked in a reversible manner by the purinoceptor antagonists, suramin and PPADS.

Adenosine Triphosphate↗

PubMed1997

The role of the IsK protein in the specific pharmacological properties of the IKs channel complex.

IKs channels are composed of IsK and KvLQT1 subunits and underly the slowly activating, voltage-dependent IKs conductance in heart. Although it appears clear that the IsK protein affects both the biophysical properties and regulation of IKs channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K+ channels are inhibited by the IKs blockers 293B, azimilide and 17-beta-oestradiol. However, IKs channels induced by the coexpression of IsK and KvLQT1 subunits have a 6-100 fold higher affinity for these blockers. Moreover, the IKs activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady-state currents through heteropolymeric IKs channels by arresting them in an open state. In summary, the IsK protein modulates the effects of both blockers and activators of IKs channels. This finding is important for the action and specificity of these drugs as IsK protein expression in heart and other tissues is regulated during development and by hormones.

Animals↗

PubMed1997

Characterization of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue.

We isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X4 receptor (87% identity). By fluorescence in situ hybridization, the human P2X4 gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X4 transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X4 receptor in Xenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embronic kidney 293 cells transfected with human P2X4 cDNA allowed us to determine the fraction of the current carried by Ca2: this was approximately 8%, demonstrating a high Ca2+ permeability. Low extracellular Zn2+ concentrations (5-10 microM) increase the apparent gating efficiency of human P2X4 by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (> 100 microM) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X4 receptor displays a very similar agonist potency profile to that of rat P2X4 (ATP > > 2-methylthio-ATP > or = CTP > alpha, beta-methylene-ATP > dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid.

Amino Acid Sequence↗

PubMed1996

Molecular cloning and functional expression of a novel rat heart P2X purinoceptor.

Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full-length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40-47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK-293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP-activated current reveals little agonist desensitization, is not activated by alpha,beta-meATP and is completely blocked by suramin and PPADS.

Amino Acid Sequence↗

PubMed1996

Cloning and tissue distribution of a novel P2X receptor from rat brain.

We have isolated the cDNA for a novel member (P2X6) of the ATP-gated ion channel family. The rat P2X6 nucleotide sequence encodes a 379 amino acid protein that conserves all the structural features of previously cloned P2X receptors, including the two putative transmembrane domains predicted by hydrophobicity plots. In situ hybridization analysis of rat brain sections showed a wide pattern of mRNA expression that is virtually identical to that already described for P2X4. Injection of P2X6 cRNA in Xenopus oocytes did not give rise to ATP-activated channels. Coexpression of P2X6 with P2X4 subunits produced currents which were not discernibly different from those of P2X4 expressed alone.

Amino Acid Sequence↗

PubMed1996

IRK(1-3) and GIRK(1-4) inwardly rectifying K+ channel mRNAs are differentially expressed in the adult rat brain.

Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K+ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1-3 and GIRK1-4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantial nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K+ channels in neurons and glial cells.

Animals↗

PubMed1996

Strategy for rapid immobilization of prey by a fish-hunting marine snail.

Some venomous animals capture prey with remarkable efficiency and speed. The purple cone, Conus purpurascens, uses two parallel physiological mechanisms requiring multiple neurotoxins to immobilize fish rapidly: neuromuscular block, and excitotoxic shock. The latter requires the newly characterized peptide kappa-conotoxin PVIIA, which inhibits the Shaker potassium channel 2-4, and beta-conotoxin PVIA5, which delays sodium-channel inactivation. Despite the extreme biochemical diversity in venoms, the number of effective strategic alternatives for prey capture are limited. How securely prey is initially tethered may strongly influence the venom strategy evolved by a predator.

Amino Acid Sequence↗

PubMed1996

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.

Adenosine Triphosphate↗

PubMed1996

Extracellular Mg2+ regulates activation of rat eag potassium channel.

The rat homologue of Drosophila ether à gogo cDNA (rat eag) encodes voltage-activated potassium (K) channels with distinct activation properties. Using the Xenopus expression system, we examined the importance of extracellular Mg2+ on the activation of rat eag. Extracellular Mg2+ at physiological concentrations dramatically slowed the activation in a dose- and voltage-dependent manner. Other divalent cations exerted similar effects on the activation kinetics that correlated with their enthalpy of hydration. Lowering the external pH also resulted in a slowing of the activation. Protons competed with Mg2+ as the effect of Mg2+ was abolished at low pH. A kinetic model for rat eag activation was derived from the data indicating that all four channel subunits undergo a Mg2+-dependent conformational transition prior to final channel activation. The strong dependence of rat eag activation on both the resting potential and the extracellular Mg2+ concentration constitutes a system for fine-tuning K channel availability in neuronal cells.

Animals↗

PubMed1995

Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene product.

Mammalian voltage-activated Shaker K+ channels associate with at least three cytoplasmic proteins: Kv beta 1, Kv beta 2 and Kv beta 3. These beta subunits contain variable N-termini, which can modulate the inactivation of Shaker alpha subunits, but are homologous throughout an aldo-keto reductase core. Human and ferret beta 3 proteins are identical with rat beta 1 throughout the core while beta 2 proteins are not; beta 2 also contains a shorter N-terminus and has no reported physiological role. We report that human beta 1 and beta 3 are derived from the same gene and that beta 2 modulates the inactivation properties of Kv1.4 alpha subunits.

Alternative Splicing↗

PubMed1995

alpha-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: molecular cloning, functional expression and alternative splicing of the alpha 7 subunit.

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovine alpha 7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat alpha 7 and 89% to chicken alpha 7 subunits. The alpha-bungarotoxin affinity of alpha 7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin alpha-bungarotoxin-sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors; the alpha 7 receptor has a pentameric structure. Upon activation with nicotinic agonists the alpha 7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription-polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine alpha 7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of alpha 7 homomer expression when coinjected with the undeleted isoform.

Alternative Splicing↗

PubMed1994

Functional expression of a rat homologue of the voltage gated either á go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart.

We have cloned a mammalian (rat) homologue of Drosophila ether á go-go (eag) cDNA, which encodes a distinct type of voltage activated potassium (K) channel. The derived Drosophila and rat eag polypeptides share > 670 amino acids, with a sequence identity of 61%, exhibiting a high degree of similarity at the N-terminus, the hydrophobic core including the pore forming P region and a potential cyclic nucleotide binding site. Rat eag mRNA is specifically expressed in the central nervous system. In the Xenopus oocyte expression system rat eag mRNA gives rise to voltage activated K channels which have distinct properties in comparison with Drosophila eag channels and other voltage activated K channels. Thus, the rat eag channel further extends the known diversity of K channels. Most notably, the kinetics of rat eag channel activation depend strongly on holding membrane potential. Hyperpolarization slows down the kinetics of activation; conversely depolarization accelerates the kinetics of activation. This novel K channel property may have important implications in neural signal transduction allowing neurons to tune their repolarizing properties in response to membrane hyperpolarization.

Amino Acid Sequence↗

PubMed1993

Ether-à-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP.

The Drosophila ether-à-go-go (eag) mutant is responsible for altered potassium currents in excitable tissue. These mutants exhibit spontaneous, repetitive firing of action potentials in the motor axons of larval neuromuscular junctions. The eag gene encodes a polypeptide that shares sequence similarities with several different ionic channel proteins, including voltage-gated potassium channels, an inward rectifier as well as cyclic-nucleotide-gated channels. These formal similarities in the derived primary sequences indicate that eag polypeptides might express a new type of ion channel. Here we report the expression by eag RNA in Xenopus oocytes of such a channel which incorporates properties of both voltage- and ligand-gated channels. The permeability of these eag channels to potassium and calcium is dependent on voltage and cyclic AMP. The ability to mediate potassium-outward and calcium-inward currents endows this channel with properties likely to be important in the modulation of synaptic efficiency in both central and peripheral nervous systems.

Animals↗

PubMed1993

Depletion of InsP3 stores activates a Ca2+ and K+ current by means of a phosphatase and a diffusible messenger.

In non-excitable cells, release of Ca2+ from the inositol 1,4,5-trisphosphate (InsP3)-sensitive store can activate Ca2+ entry. Very little is known about the signal mechanism relating store emptying to plasma membrane Ca2+ influx. It has been suggested that the signal may be either a diffusible messenger like an inositol phosphate, or the InsP3 receptor itself, which, by physically coupling to some component of Ca2+ entry in the plasma membrane, may link store release to Ca2+ entry. The nature of the Ca2+ entry pathway is also unclear. Only in mast cells has a very selective Ca2+ current been observed after store emptying. Activation of exogenous 5-hydroxytryptamine (5-HT) receptors expressed in Xenopus oocytes or direct injection of InsP3 evokes Ca2+ entry activated by InsP3 pool depletion. Here we investigate the nature of this influx pathway and find a current activated by pool depletion. This has an unusual selectivity in that it is more permeable to Ca2+ ions than to other divalent cations (Ba2+, Sr2+ or Mn2+). Moreover, a K+ permeability is also stimulated after pool depletion. The activation of this store depletion current involves both a phosphatase and an unidentified diffusible messenger. Both the Ca2+ entry pathway and the activating factors found here may be relevant to pool-depleted Ca2+ entry in a variety of non-excitable cells.

Animals↗

PubMed1993

Ca2+ oscillations and Ca2+ influx in Xenopus oocytes expressing a novel 5-hydroxytryptamine receptor.

1. We expressed a novel 5-hydroxytryptamine receptor (SRL) in Xenopus oocytes and monitored cytosolic Ca2+ through the endogenous Ca(2+)-dependent Cl- channel activity using the double electrode voltage-clamp technique. 2. 5-Hydroxytryptamine (5-HT; 200 nM) led to an initial rapid oscillatory current followed by a pronounced secondary one, which lasted long after 5-HT wash-out (20-40 min) and was not affected by the receptor antagonist yohimbine. 3. Both phases of the current were abolished by heparin demonstrating a key role for IP3-induced Ca2+ release. 4. Caffeine (10 mM) alone did not evoke a current but reduced both phases of the current evoked by 5-HT. Ryanodine had no effect. No evidence for Ca(2+)-induced Ca2+ release was found. 5. The secondary current activated by 5-HT was sensitive to changes in extracellular Ca2+, suggesting it was evoked by Ca2+ influx. Reducing external Na+ did not affect this current, demonstrating that it was rather specific for Ca2+. 6. The Ca2+ influx pathway was much more sensitive to Cd2+ than other divalent ions (Co2+, Mn2+, Sr2+, Ba2+). It was insensitive to verapamil. 7. Injection of D-myo-inositol 1,4,5-trisphosphate, 3-deoxy-3-fluoro (IP3-F; an analogue not metabolized to D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4)), evoked either an oscillatory current or a rapid current followed by a sustained secondary one. The latter was sensitive to external Ca2+ and was blocked by Cd2+. Heparin dramatically reduced the IP3-F-evoked current. 8. Perfusion in Ca(2+)-free solution, once a secondary current had been generated, significantly decreased the amount of intracellular Ca2+ mobilized by 5-HT, indicating that the Ca2+ influx pathway plays an important role in pool refilling. 9. Block of Ca2+ influx by Cd2+ in cells that were oscillating transiently increased the amplitude and then either abolished the oscillations or made them irregular. This effect was also elicited by increasing external Ca2+. 10. These results demonstrate that 5-HT, acting via IP3, both releases Ca2+ from internal stores and evokes a pronounced Ca2+ influx. This last step is activated by pool depletion and is important for both refilling of the agonist-sensitive stores and modifying the oscillatory pattern.

Animals↗