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W Stühmer

Publications and source records attributed to W Stühmer.

At least 19 recordsLinked to original sources
PubMed2005

Role of voltage-gated potassium channels in cancer.

Ion channels are being associated with a growing number of diseases including cancer. This overview summarizes data on voltage-gated potassium channels (VGKCs) that exhibit oncogenic properties: ether-à-go-go type 1 (Eag1). Normally, Eag1 is expressed almost exclusively in tissue of neural origin, but its ectopic expression leads to uncontrolled proliferation, while inhibition of Eag1 expression produces a concomitant reduction in proliferation. Specific monoclonal antibodies against Eag1 recognize an epitope in over 80% of human tumors of diverse origins, endowing it with diagnostic and therapeutic potential. Eag1 also possesses unique electrophysiological properties that simplify its identification. This is particularly important, as specific blockers of Eag1 currents are not available. Molecular imaging of Eag1 in live tumor models has been accomplished with dye-tagged antibodies using 3-D imaging techniques in the near-infrared spectral range.

Animals↗
PubMed2000

Tracking chromaffin granules on their way through the actin cortex.

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a approximately 300-nm(1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D(3) was about 10,000 times lower than expected for free diffusion. Granules located approximately 60 nm beneath the plasma membrane moved on random tracks (D(3) approximately 10(-10) cm(2)s(-1)) with several reversals in direction before they approached their docking site at angles larger than 45 degrees. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.

Actins↗
PubMed2000

Cytoskeletal interactions determine the electrophysiological properties of human EAG potassium channels.

The electrophysiological properties of ether a go-go (EAG) potassium channels are modified during the cell cycle when they are expressed in heterologous systems. In Chinese hamster ovary (CHO) mammalian somatic cells we found that the cell-cycle-dependent modulation of human EAG (hEAG) channels occurs during the M phase. This modulation has three components: reduction in current density, increased sensitivity to block by intracellular sodium, and increased selectivity for potassium ions. In this work, these three properties have been used to define the mitotic phenotype of EAG currents. The signaling pathway leading to such changes of channel properties is unknown. We tested the hypothesis that cytoskeletal interactions might affect the electrophysiological changes observed during the cell cycle. The disruption of actin filaments induces a significant increase in current density, without inducing the cell-cycle-related phenotype. In contrast, disturbance of the microtubules, achieved by pharmacological means or by mechanical excision of the membrane patch, does induce the cell-cycle-related phenotype. Our results demonstrate that hEAG channels establish complex interactions with cytoskeletal elements, and that these interactions strongly influence the properties of the channels. We also conclude that the electrophysiological changes observed during the cell cycle are most likely due to reorganization of the cytoskeleton during the G2/M transition.

Actin Cytoskeleton↗
PubMed2000

Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence.

The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and--close to the coverslip--can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles ('vesicles') of approximately 300 nm diameter in the "footprint" region of living neuroendocrine cells grown on the interface.

Cytoplasmic Granules↗
PubMed1999

Oncogenic potential of EAG K(+) channels.

We have investigated the possible implication of the cell cycle-regulated K(+) channel ether à go-go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune-depressed mice. These data provide evidence for the oncogenic potential of EAG.

Animals↗
PubMed1999

Evanescent-wave microscopy: a new tool to gain insight into the control of transmitter release.

Evanescent-wave excitation was used to visualize individual fluorescently labelled vesicles in an optical slice near the plasma membrane of bovine adrenal chromaffin cells. A standard upright microscope was modified to accommodate the optics used for directing a laser beam under a supracritical angle on to the glass-water interface on top of which the cells are grown. Whereas epi-illumination images appeared blurred and structureless, evanescent-wave excitation highlighted acridine orange-labelled vesicles as individual pinpoints. Three-dimensional (3D) trajectories of individual vesicles were obtained from time-resolved image stacks and used to characterize vesicles in terms of their average fluorescence F and mobility, expressed here as the 3D diffusion coefficient D(3). Based on the single-vesicle analysis, two groups of vesicles were identified. Transitions between these states were studied before and after stimulation of exocytosis by repetitive or maintained membrane depolarizations by elevated extracellular [K+]. Findings were interpreted as sequential transitions between the previously characterized pools of vesicles preceding the fusion step. The observed approach of vesicles to their docking sites was not explained in terms of free diffusion: most vesicles moved unidirectionally as if directed to their binding sites at the plasma membrane. Vesicle mobility at the membrane was low, such that the sites of docking and fusion were in close vicinity. Both the rim region and confined areas in the centre of the footprint region were the site of intense vesicle trafficking.

Acridine Orange↗
PubMed1999

Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles.

In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied "footprint" region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site.

Animals↗
PubMed1999

Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors.

The suramin analogue 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X1-P2X4 as well as heteromultimeric P2X2/P2X3 receptors has been characterized. P2X1 receptors were most sensitive to inhibition by NF023 with IC50 values of 0.24 and 0.21 microM for the rat and human homologue, respectively. P2X3 receptors have an intermediate sensitivity with IC50 values of 8.5 and 28.9 microM for rat and human subtypes, respectively and P2X2 was the least sensitive subtype (IC50 > 50 microM). P2X4 receptors were insensitive to NF023 at concentrations up to 100 microM. Coexpression of rat P2X3 with rat P2X2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X3 receptors (alphabeta meATP as agonist; IC50 = 1.4 and 1.6 microM, respectively). NF023 inhibited P2X1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 microM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP (K(B) = 1.1 +/- 0.2 microM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X1 receptors heterologously expressed in Xenopus oocytes.

Adenosine Triphosphate↗
PubMed1998

Cell cycle-related changes in the conducting properties of r-eag K+ channels.

Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K+ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na+ ions. Manipulation of the intracellular Na+ concentration indicates that the site of Na+ block is located approximately 45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na+ and variable permeability to Cs+. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K+ channels are modulated during cell cycle-related processes.

Animals↗
PubMed1998

ATP binding site of P2X channel proteins: structural similarities with class II aminoacyl-tRNA synthetases.

The extracellular loop of P2X channel proteins contains a sequence stretch (positions 170-330) that exhibits similarities with the catalytic domains of class II aminoacyl-tRNA synthetases as shown by secondary structure predictions and sequence alignments. The arrangement of several conserved cysteines (positions 110-170) shows similarities with metal binding regions of metallothioneins and zinc finger motifs. Thus, for the extracellular part of P2X channel proteins a metal binding domain and an antiparallel six-stranded beta-pleated sheet containing the ATP binding site are very probable. The putative channel forming H5 part (positions 320-340) shows similarities with the enzyme motif 1 responsible for aggregation of subunits to the holoenzyme.

Adenosine Triphosphate↗
PubMed1998

kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel.

kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. kappa-Conotoxin PVIIA has the following structure. This is the first Conus peptide known to target K+ channels. [structure: see text] Although the Shaker K+ channel is sensitive to kappa-PVIIA, the rat brain Kv1.1 subtype is resistant. Chimeras between Shaker and the Kv1.1 K+ channels were constructed and expressed in Xenopus oocytes. Only channels containing the putative pore-forming region between the fifth and sixth transmembrane domains of Shaker retained toxin sensitivity, indicating that the toxin target site is in this region of the channel. Evidence is presented that kappa-PVIIA interacts with the external tetraethyl-ammonium binding site on the Shaker channel. Although both kappa-PVIIA and charybdotoxin inhibit the Shaker channel, they must interact differently. The F425G Shaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes kappa-PVIIA sensitivity. The precursor sequence of kappa-PVIIA was deduced from a cDNA clone, revealing a prepropeptide comprising 72 amino acids. The N-terminal region of the kappa-PVIIA prepropeptide exhibits striking homology to the omega-, muO-, and delta-conotoxins. Thus, at least four pharmacologically distinct superfamilies of Conus peptides belong to the same "O" superfamily, with the omega- and kappa-conotoxins forming one branch, and the delta- and muO-conotoxins forming a second major branch.

Amino Acid Sequence↗
PubMed1998

Structure and function of voltage-gated ion channels.

Voltage-gated ion channels are key molecules for the generation of electrical signals in cells. They are integral membrane proteins which are activated by a depolarized membrane potential resulting in a conformational change, allowing ions to permeate. Voltage-gated ion channels can either be inactivated from this open state by an additional conformational change which leads to a nonconducting state of the channel, or they may be deactivated by a repolarized membrane potential. Following the first successful cloning of voltage-gated ion channels in 1984 the combination of molecular biological and electrophysiological techniques has been very fruitful in the investigation of the structure and function of these membrane proteins. From these studies a molecular picture of the structural elements important for the activity of voltage-gated ion channels has been established. This has assisted in clarifying the molecular basis of the electrical excitability of cells.

Amino Acid Sequence↗
PubMed1998

The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM).

We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the 'visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles.

Animals↗
PubMed1998

Modulation of the Ca2+-activated K+ current sIAHP by a phosphatase-kinase balance under basal conditions in rat CA1 pyramidal neurons.

The slow Ca2+-activated K+ current, sIAHP, underlying spike frequency adaptation, was recorded with the whole cell patch-clamp technique in CA1 pyramidal neurons in rat hippocampal slices. Inhibitors of serine/threonine protein phosphatases (microcystin, calyculin A, cantharidic acid) caused a gradual decrease of sIAHP amplitude, suggesting the presence of a basal phosphorylation-dephosphorylation turnover regulating sIAHP. Because selective calcineurin (PP-2B) inhibitors did not affect the amplitude of sIAHP, protein phosphatase 1 (PP-1) or 2A (PP-2A) are most likely involved in the basal regulation of this current. The ATP analogue, ATP-gamma-S, caused a gradual decrease in the sIAHP amplitude, supporting a role of protein phosphorylation in the basal modulation of sIAHP. When the protein kinase A (PKA) inhibitor adenosine-3', 5'-monophosphorothioate, Rp-isomer (Rp-cAMPS) was coapplied with the phosphatase inhibitor microcystin, it prevented the decrease in the sIAHP amplitude that was observed when microcystin alone was applied. Furthermore, inhibition of PKA by Rp-cAMPS led to an increase in the sIAHP amplitude. Finally, an adenylyl cyclase inhibitor (SQ22, 536) and adenosine 3',5'-cyclic monophosphate-specific type IV phosphodiesterase inhibitors (Ro 20-1724 and rolipram) led to an increase or a decrease in the sIAHP amplitude, respectively. These findings suggest that a balance between basally active PKA and a phosphatase (PP-1 or PP-2A) is responsible for the tonic modulation of sIAHP, resulting in a continuous modulation of excitability and firing properties of hippocampal pyramidal neurons.

Adenylyl Cyclases↗
PubMed1997

Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment.

1. Rat eag potassium channels (r-eag) were expressed in Xenopus oocytes. They gave rise to delayed rectifying K+ currents with a strong Cole-Moore effect. 2. Deletions in the N-terminal structure of r-eag either shifted the activation threshold to more negative potentials and slowed the activation kinetics (delta 2-190, delta 2-12 and delta 7-12) or resulted in a shift to more positive potentials and faster activation kinetics (delta 150-162). 3. The impact of the deletion delta 7-12 was investigated in more detail: it almost abolished the Cole-Moore effect and markedly slowed down channel deactivation. 4. Unlike wild-type channels, the deletion mutants delta 7-12 exhibited a rapid inactivation which, in combination with the slow deactivation, resulted in current characteristics which were similar to those of the related potassium channel HERG. 5. Both the slowing of deactivation and the inactivation induced by the deletion delta 7-12 were compensated by a single histidine-to-arginine change in the S4 segment, while this mutation (H343R) only had minor effects on the gating kinetics of the full-length r-eag channel. 6. These results demonstrate a functional role of the N-terminus in the voltage-dependent gating of potassium channels which is presumably mediated by an interaction of the N-terminal protein structure with the S4 motif during the gating process.

Amino Acid Sequence↗