ATP crossing the cell plasma membrane generates an ionic current in Xenopus oocytes

The presence of ATP within cells is well established. However, ATP also operates as an intercellular signal via specific purinoceptors. Furthermore, nonsecretory cells can release ATP under certain experimental conditions. To measure ATP release and membrane currents from a single cell simultaneously, we used Xenopus oocytes. We simultaneously recorded membrane currents and luminescence. Here, we show that ATP release can be triggered in Xenopus oocytes by hyperpolarizing pulses. ATP release (3.2 +/- 0.3 pmol/oocyte) generated a slow inward current (2.3 +/- 0.1 microA). During hyperpolarizing pulses, the permeability for ATP(4-) was more than 4000 times higher than that for Cl(-). The sensitivity to GdCl(3) (0. 2 mm) of hyperpolarization-induced ionic current, ATP release and E-ATPase activity suggests their dependence on stretch-activated ion channels. The pharmacological profile of the current inhibition coincides with the inhibition of ecto-ATPase activity. This enzyme is highly conserved among species, and in humans, it has been cloned and characterized as CD39. The translation, in Xenopus oocytes, of human CD39 mRNA encoding enhances the ATP-supported current, indicating that CD39 is directly or indirectly responsible for the electrodiffusion of ATP

Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS

The Cytotoxicity of Epsilon toxin from Clostridium perfringens on lymphocytes is mediated by MAL protein expression

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the blood-brain barrier, binds to myelin, and, hence, has been suggested to be a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, myelin and lymphocyte (MAL) protein has been identified to be a key protein in the cytotoxic effect of Etx; however, the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL protein through a direct Etx-MAL protein interaction. Experiments on lymphocytic cell lines revealed that MAL protein-expressing T cells, but not B cells, are sensitive to Etx and reveal that the toxin may be used as a molecular tool to distinguish subpopulations of lymphocytes. The overall results open the door to investigation of the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis

Differential voltage-dependent K+ channel responses during proliferation and activation in macrophages

Voltage-dependent K+ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K+ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-alpha mimicked LPS effects, and studies with TNF-alpha receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-alpha-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses

Botulinum neurotoxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release,was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods.To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata

Neurone membrane and aging: electrophysiological aspects

Introducción: las membranas celulares están formadas por lípidos y proteínas. Por su naturaleza hidrofóbica, los fosfolípidos aíslan el medio intracelular y el extracelular. Desarrollo: el medio extracelular es una solución enriquecida en iones de sodio, mientras que el medio intracelular es rico en iones de potasio. De acuerdo con las leyes de la química, la distribución asimétrica de iones produce una pequeña diferencia de potencial entre el interior y el exterior celular. Todas las células del cuerpo humano presentan esta pequeña diferencia de potencial que se sitúa alrededor de ­60 mV. Otro componente de las membranas celulares son las proteínas. Algunas de ellas -las bombas iónicas o ATPasas- mantienen la distribución asimétrica de los iones. Otras proteínas de membrana -los canales iónicos- permiten el paso selectivo de iones entre el medio intracelular y extracelular. Las técnicas de electrofisiología celular (patch clamp) permiten medir el paso de iones a través de una sola molécula. Cada canal iónico permite el paso de una sola especie iónica. Las células contienen canales iónicos que son transitoriamente permeables a los iones sodio, potasio, calcio y cloruro. La excitabilidad neuronal depende de los canales iónicos. Las neuronas presentan un distinto comportamiento eléctrico en las distintas fases de su vida. Los cambios en la actividad eléctrica de las neuronas vienen determinados por la actividad de los canales iónicos y su interacción con su entorno lipídico. Conclusión: se realizan esfuerzos investigadores para relacionar los cambios de comportamiento de los canales iónicos en el proceso de envejecimiento de las membranas neuronale

Botulinum neurotoxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release,was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods.To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata

Neurone membrane and aging: electrophysiological aspects

Introducción: las membranas celulares están formadas por lípidos y proteínas. Por su naturaleza hidrofóbica, los fosfolípidos aíslan el medio intracelular y el extracelular. Desarrollo: el medio extracelular es una solución enriquecida en iones de sodio, mientras que el medio intracelular es rico en iones de potasio. De acuerdo con las leyes de la química, la distribución asimétrica de iones produce una pequeña diferencia de potencial entre el interior y el exterior celular. Todas las células del cuerpo humano presentan esta pequeña diferencia de potencial que se sitúa alrededor de ­60 mV. Otro componente de las membranas celulares son las proteínas. Algunas de ellas -las bombas iónicas o ATPasas- mantienen la distribución asimétrica de los iones. Otras proteínas de membrana -los canales iónicos- permiten el paso selectivo de iones entre el medio intracelular y extracelular. Las técnicas de electrofisiología celular (patch clamp) permiten medir el paso de iones a través de una sola molécula. Cada canal iónico permite el paso de una sola especie iónica. Las células contienen canales iónicos que son transitoriamente permeables a los iones sodio, potasio, calcio y cloruro. La excitabilidad neuronal depende de los canales iónicos. Las neuronas presentan un distinto comportamiento eléctrico en las distintas fases de su vida. Los cambios en la actividad eléctrica de las neuronas vienen determinados por la actividad de los canales iónicos y su interacción con su entorno lipídico. Conclusión: se realizan esfuerzos investigadores para relacionar los cambios de comportamiento de los canales iónicos en el proceso de envejecimiento de las membranas neuronale

Regulation of exocytotic fusion by cell inflation

We have inflated patch-clamped mast cells by 3.8 +/- 1.6 times their volume by applying a hydrostatic pressure of 5-15 cm H2O to the interior of the patch pipette. Inflation did not cause changes in the cell membrane conductance and caused only a small reversible change in the cell membrane capacitance (36 +/- 5 fF/cm H2O). The specific cell membrane capacitance of inflated cells was found to be 0.5 microF/cm2. High-resolution capacitance recordings showed that inflation reduced the frequency of exocytotic fusion events by approximately 70-fold, with the remaining fusion events showing an unusual time course. Shortly after the pressure was returned to 0 cm H2O, mast cells regained their normal size and appearance and degranulated completely, even after remaining inflated for up to 60 min. We interpret these observations as an indication that inflated mast cells reversibly disassemble the structures that regulate exocytotic fusion. Upon returning to its normal size, the cell cytosol reassembles the fusion pore scaffolds and allows exocytosis to proceed, suggesting that exocytotic fusion does not require soluble proteins. Reassembly of the fusion pore can be prevented by inflating the cells with solutions containing the protease pronase, which completely blocked exocytosis. We also interpret these results as evidence that the activity of the fusion pore is sensitive to the tension of the plasma membrane