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H Kettenmann

Publications and source records attributed to H Kettenmann.

At least 145 records · Page 8Linked to original sources
PubMed1989

Activation of K+ currents in cultured Schwann cells is controlled by extracellular pH.

We analyzed the pH dependence of K+ currents recorded with the patch-clamp technique from cultured Schwann cells obtained from mouse dorsal root ganglia. Currents were activated at potentials more positive than -50 mV which was close to the resting membrane potential. Current amplitudes were affected by a change in extracellular pH (pHo), being increased at alkaline, and decreased at acidic pHo. The strongest effect of a pHo change was observed on currents activated close to the resting membrane potential suggesting a functional role for the pH sensitivity of K+ currents. Analysis of the time course of current activation at different pHo values led to the conclusion that the pH-sensitivity of K+ currents in Schwann cells is due to changes in surface charges shifting the potential sensed by the gating process of the channel. The reversal potential of the currents was not affected by a change in pHo. This observation and the finding that even a strong acidification to a pHo value of 5.0 did not lead to a blockade of the fully activated channel, indicate that the pH-sensitive charges are not located in the channel pore. Under the assumption that pHo changes in a peripheral nerve are associated with nerve activity as in the optic nerve, the pH-sensitive K+ channel in Schwann cells could serve to facilitate the spatial buffering of extracellular K+.

4-Aminopyridine↗
PubMed1989

Channel expression correlates with differentiation stage during the development of oligodendrocytes from their precursor cells in culture.

Membrane currents in cultured murine oligodendrocytes and their precursors were characterized using the patch-clamp technique. Prior to recording, cells were identified by immunofluorescence using monoclonal antibodies characteristic of two types of precursor cells and two differentiation stages of oligodendrocytes. The most immature, A2B5 antigen-positive glial precursors, expressed four types of voltage-activated K+ currents and tetrodotoxin-sensitive Na+ currents. The more differentiated cells, O4 antigen-positive glial precursors, expressed similar K+ currents, but Na+ currents were recorded in only a minority of cells. In differentiated O1 and O10 antigen-positive oligodendrocytes the channels characteristic of precursor cells were no longer observed, but an inwardly rectifying K+ current was apparent. Thus, channel expression by cells of the oligodendrocyte lineage correlates with differentiation stage and is more complex in precursor cells than in oligodendrocytes.

Animals↗
PubMed1989

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit.

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.

Amino Acid Sequence↗
PubMed1989

GABA-activated Cl- channels in astrocytes of hippocampal slices.

We used kainic acid-lesioned hippocampal slices to examine glial responses to the inhibitory neurotransmitter GABA in a neuron-free environment. Slices were prepared from rats which received intracerebroventricular injections of kainic acid 1 month prior to experiments. Astrocytes (membrane potential averaged 81.4 +/- 5.5 mV; n = 46; mean +/- SD) were impaled in the CA3 region of the slice, which was completely depleted of neurons. GABA (1 mM) application by bath perfusion depolarized membrane potential from 1 to 5 mV. The GABA-induced depolarization was not affected by a tetrodotoxin (1 microM)/high-Mg2+/low-Ca2+ solution. Changing the Cl- equilibrium potential by reducing extracellular Cl- greatly increased the GABA-induced depolarization. Muscimol mimicked the GABA response, while picrotoxin (0.1 mM), an antagonist of the GABA-activated Cl- channel, resulted in a 60% blockade. The barbiturate, pentobarbital (0.1 mM), and the benzodiazepine agonist, flunitrazepam (1 mM), enhanced the depolarization by 60 and 40%, respectively. A blocker of glial GABA uptake, beta-alanine (1 mM), did not affect the GABA-induced membrane depolarization, indicating that the depolarization is not caused by electrogenic uptake of the amino acid. The pharmacological properties of the GABA response of astrocytes from the hippocampal slice is similar to that previously described for cultured astrocytes from rat cerebral hemispheres. Our data suggest that GABA receptors, which are coupled to Cl- channels, are also expressed by astrocytes in an intact tissue.

Animals↗
PubMed1988

Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits.

Human gamma-aminobutyric acid A (GABAA) receptor subunits were expressed transiently in cultured mammalian cells. This expression system allows the simultaneous characterization of ligand-gated ion channels by electrophysiology and by pharmacology. Thus, coexpression of the alpha and beta subunits of the GABAA receptor generated GABA-gated chloride channels and binding sites for GABAA receptor ligands. Channels consisting of only alpha or beta subunits could also be detected. These homomeric channels formed with reduced efficiencies compared to the heteromeric receptors. Both of these homomeric GABA-responsive channels were potentiated by barbiturate, indicating that sites for both ligand-gating and allosteric potentiation are present on receptors assembled from either subunit.

Allosteric Regulation↗
PubMed1988

Electrical coupling between astrocytes and between oligodendrocytes studied in mammalian cell cultures.

The characteristics of electrical coupling between astrocytes and between oligodendrocytes were analyzed in cell cultures derived from rodent central nervous system. Experiments were carried out by impaling one member of a glial pair with separate voltage recording and current passing electrodes (cell 1) and the other cell, a measured distance from the first, with a voltage-recording electrode (cell 2). Astrocyte pairs within 300 microns of one another were always coupled. The coupling ratio was determined for 23 astrocytic pairs various distances apart, and decreased with distance in a roughly exponential manner. The average coupling ratio of astrocytes within 100 microns of each other was 0.44 +/- 0.32. Oligodendrocytes were less strongly coupled to each other than astrocytes. Even cells immediately adjacent to one another were often uncoupled. Among coupled oligodendrocytes within 100 microns of each other, the average coupling ratio was 0.11 +/- 0.1. Current passage between pairs of astrocytes and pairs of oligodendrocytes was nonrectifying. Application of 0.5 mM BaCl2 or 44.6 mM CsCl (substituted for NaCl) depolarized and increased the input resistance of astrocytes and oligodendrocytes. These ions also increased the coupling ratio in astrocyte pairs and oligodendrocyte pairs; this effect was rapid in onset and completely reversible. Ba++ and Cs+ appear to block resting K+ conductance in glia and probably increase the coupling ratio by increasing the effective length constant of the glial membrane without any direct effect on junctional resistance. In three cases, oligodendrocyte pairs that appear uncoupled in normal solution exhibited coupling in the presence of BaCl2 or CsCl. This suggests that oligodendrocytes may be widely coupled by junctions that provide only weak electrical interaction; such junctions might be important for the exchange of small metabolically active molecules. The strong electrical coupling among astrocytes, in concert with their K+-selective membrane conductance, would provide for an electrical syncytium well designed to transport K+ away from areas of focal extracellular accumulation (i.e., the spatial buffer mechanism), and these cells, more than oligodendrocytes, may provide this function.

Animals↗
PubMed1988

Effect of benzodiazepines and pentobarbital on the GABA-induced depolarization in cultured astrocytes.

We have previously shown that cultured astrocytes from neonatal rat cerebral cortex are depolarized by GABA. The underlying ionic mechanism, activation of a Cl- conductance and responses to an agonist and antagonists were found to be similar to those of the neuronal GABAA receptor (Kettenmann et al.: Brain Research 404:1-9, 1987; Kettenmann and Schachner: Journal of Neuroscience 5:3295-3301, 1985). To characterize further the pharmacological properties of the GABA receptor we have tested the influence of pentobarbital and benzodiazepines on the GABA response. Pentobarbital potentiated and prolonged the GABA-induced depolarization and enhanced the velocity of the depolarization. Agonists of the neuronal benzodiazepine receptor, flunitrazepam, diazepam, and midazolam, increased the GABA-induced depolarization. As in neurons, an antagonist of the benzodiazepine receptor, Ro 15-1788, blocked the flunitrazepam-induced enhancement of the GABA response. In contrast to their effects on neurons, the inverse agonists Ro 22-7497 and DMCM increased the GABA-induced depolarization. The ligand of the putative peripheral benzodiazepine binding site, Ro 5-4864, did not show consistent effects on the GABA response. These studies confirm that cultured astrocytes express GABAA receptors. This receptor is similar to the neuronal GABAA receptor with regard to Cl- conductance and its pharmacological responses to muscimol, bicuculline, picrotoxin, pentobarbital, and benzodiazepine agonists and an antagonist, but it is different in its responses to inverse agonists of the benzodiazepine site. The physiological role of the glial GABAA receptor is at present unknown.

Animals↗
PubMed1988

Glutamate opens Na+/K+ channels in cultured astrocytes.

Glial cells from different brain regions and species are depolarized by the neurotransmitter glutamate. The depolarization or, if voltage-clamped at the resting membrane potential, the inward current induced by glutamate could be due either to activation of receptor-coupled ion channels or electrogenic uptake of the transmitter. In the present study we applied the patch-clamp technique in the whole-cell recording mode to analyze glutamate-induced currents in cultured astrocytes from rat cerebral hemispheres. At the resting membrane potential, glutamate induced an inward current ranging from 40 to 300 pA. This current decreased in size with depolarization and reversed at about 0 mV. The resulting current-to-voltage curve was linear and depended strongly on the transmembrane Na+ but not on the Ca++ or Cl- gradient. In the presence of glutamate, current noise increased at potentials positive or negative from the reversal potential indicating that ionic channels are activated by glutamate. Both kainate and quisqualate mimicked the effect of glutamate. We conclude that glutamate opens a Na+/K+ channel in cultured astrocytes because of activation of a receptor which shares many properties with the neuronal kainate/quisqualate receptor.

Animals↗
PubMed1988

Heterogeneity of potassium currents in cultured oligodendrocytes.

In the present study we have analyzed the membrane currents of mature oligodendrocytes in cultures from dissociated fetal mouse cerebral hemispheres and explant cultures from fetal mouse spinal cord. Both types of oligodendrocytes showed large voltage-dependent, but time-independent inward and outward currents that were partially blocked by Ba2+. In addition, time- and voltage-dependent inward and outward currents were observed in a minority of oligodendrocytes from spinal cord. All voltage-dependent currents were completely blocked by Ba2+, and inward currents were completely blocked by Cs+, suggesting that they are mediated by K+ channels. Current-voltage curves of mouse spinal cord oligodendrocytes varied from being linear to outwardly or inwardly rectifying. In contrast, oligodendrocytes cultured from mouse brain always showed an inward rectification of the current voltage relation and a lack of time-dependent currents. It thus appears that mature oligodendrocytes in explant cultures of mouse spinal cord, in contrast to oligodendrocytes from dissociated brain, consist of different cell populations that are distinguished by their expression or active state of K+ channels.

Animals↗
PubMed1988

Patch-clamp study of gamma-aminobutyric acid receptor Cl- channels in cultured astrocytes.

The membrane channels operated by gamma-aminobutyric acid (GABA) were studied in cultured astrocytes from rat cerebral hemispheres by using patch-clamp techniques. The channel properties appeared to be very similar, in many respects, to those present in neuronal cell membranes. The Cl- -selective channels were activated after the sequential binding of two GABA molecules to the receptor, as deduced from the slope of the dose-response curve. Single-channel currents displayed multiple conductance states of 12 pS, 21 pS, 29 pS, and 43 pS, with the main-state conductance being 29 pS. The gating properties could be described by a sequential reaction scheme for agonist-activated channels. GABA-induced whole-cell currents were potentiated by the benzodiazepine receptor agonist diazepam and also, to a lesser extent, by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate an inverse agonist. In neurons and chromaffin cells, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate reduces the sensitivity of the GABA receptor, indicating that neuronal and glial GABA/benzodiazepine receptor--Cl- channel complexes are different. Glial GABA receptor channels could be of functional importance in buffering extracellular Cl- in the cleft of the GABAergic synapse.

Animals↗
PubMed1988

Intracellular pH regulation in cultured mouse oligodendrocytes.

1. Intracellular pH (pHi) and the mechanism of pHi regulation have been investigated in cultured oligodendrocytes from mouse spinal cord using double-barrelled neutral-carrier H+-selective microelectrodes. The distribution of H+ was not in electrochemical equilibrium. The pHi was more alkaline than the pH of the bathing medium (pHo), namely 7.5 at pHo 7.2 at 7.6 at pHo 7.4. 2. Removal of HCO3- from the bathing medium reduced the steady-state pHi by 0.4 units. An increase in extracellular K+ caused, with a delay, an increase in pHi. A decrease in pHo to 6.2 caused an acidification of pHi by 0.5 units. 3. The pHi regulation was studied by applying and subsequently removing NH4+ which resulted in an acidification of the cell. The subsequent recovery of pHi could then be analysed. The recovery from an acidification by 1 pH unit lasted 3-10 min. In HCO3- -free solution pHi recovery was slowed. 4. In HCO3- -free solution pHi recovery was completely blocked when either Na+ was removed or when amiloride was applied indicating an exclusive activation of the Na+-H+ exchanger. 5. In the presence of HCO3-, removal of Na+ also completely blocked pHi recovery. When Na+ was readded, pHi recovered. In HCO3- -containing solution amiloride slightly slowed, but did not block pHi recovery. 6. Removal of Cl- or application of SITS, DIDS or furosemide, blockers of Cl- -coupled transport mechanisms, did not affect the pHi recovery in the presence of HCO3-. 7. In conclusion, oligodendrocytes possess two mechanisms regulating pHi, a Na+-H+ exchanger and a Na+-HCO3- co-transporter while the latter is clearly more potent. It follows that pHi regulation of oligodendrocytes is dependent on the transmembrane Na+ gradient and is strictly separated from regulation of internal Cl-.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

gamma-Aminobutyric acid opens Cl-channels in cultured astrocytes.

Cultured astrocytes from cerebral hemispheres of early postnatal rats responded to gamma-aminobutyric acid (GABA) with membrane depolarization. This depolarization was affected by changes in extracellular [Cl-] and depended on the membrane potential. The reversal potential of the GABA-induced depolarization was determined by double electrode voltage clamp or depolarization by elevated [K+]o and ranged between -38 and -53 mV. Cell input resistance decreased after addition of GABA with the same time course as the membrane depolarization. GABA responses were temperature dependent yielding a peak at about 14 degrees C. At higher temperatures a decrease in the GABA-induced depolarization was seen indicating that the depolarization may not be mediated by an enzyme-coupled carrier system. Addition of ouabain at different temperatures did not change the size of the GABA depolarization. This excludes the possibility that an electrogenic component of the temperature-sensitive Na+,K+-ATPase activity causes the decrease in GABA-dependent depolarization at higher temperatures. Intracellular [Cl-] was measured with Cl- sensitive microelectrodes and found to be higher than the value calculated for a free distribution according to the Nernst equation (-40 mV). Addition of furosemide did not alter the reversal potential, but reduced the size of the GABA-induced membrane depolarization. From these observations and previous experiments on the pharmacological properties of the membrane response we conclude that the ionic mechanism underlying the GABA-dependent membrane depolarization of astrocytes results from a transient increase in Cl- -conductance similar to that of the neuronal GABAA-receptor.

Animals↗
PubMed1987

Glial potassium uptake following depletion by intracellular ionophoresis.

The K+ uptake processes of immunologically identified oligodendrocytes from embryonic mouse spinal cord were studied in primary culture by injecting ions and recording membrane potential changes and, in some experiments, K+ ion activity with intracellular electrodes. When Na+ was injected [K+]i decreased. Immediately before and after current injection the membrane potential was close to the K+ equilibrium potential (EK) and this finding was used to study K+ uptake following its depletion by intracellular ionophoresis. The uptake of K+ following Na+ injection was blocked by ouabain and unaffected by removal of extracellular Cl- or Cl- transport blockers. This suggests that recovery comes about mostly through the activity of the Na+/K+ -ATPase stimulated by either the increase in [Na+]i or the decrease in [K+]i. Pump current could be determined by clamping at different membrane potentials and was found to increase in proportion to the depolarization of the cell resulting from [K+]i depletion. The time course of recovery of membrane potential following either Li+ or tetramethylammonium (TMA+) injection was similar to that after Na+ injection, indicating that injection of these ions to produce a comparable decrease in [K+]i leads to a similar stimulation of the Na+/K+ -ATPase. In addition, the recovery of membrane potential following injection of TMA+, but not of Na+ or Li+, was blocked when the external Na+ was removed. Internal Na+ or Li+ appears necessary for Na+/K+ -ATPase-activity, but under conditions of normal or low [Na+]i the rate of Na+/K+ -ATPase activity seems to be sensitive to [K+]i and/or membrane potential.

Animals↗
PubMed1987

K+ and Cl- uptake by cultured oligodendrocytes.

Cultured oligodendrocytes take up K+ triggered by an increase in [K+]o. Simultaneously [Cl-]i increases in the majority of the oligodendrocytes. This KCl uptake, which is not furosemide sensitive, can be explained by the following model. The first event is the entry of Cl- into the cell driven by the discrepancy between the membrane and Cl- equilibrium potential. As a consequence of the movement of negative charge across the membrane, K+ is driven into the cell. The prerequisites of this model, a passive Cl- distribution at resting membrane potential and a Cl- conductance of the membrane were found to exist in most cultured oligodendrocytes. The chloride equilibrium potential (-61 mV, SD +/- 10 mV) was slightly more positive than the membrane potential (-64 +/- 8 mV). Since cell input resistance determined with two independent electrodes increased by 11% (SD +/- 0.07) when [Cl-]o was reduced to 10 mM, part of the membrane conductance appears to be mediated by Cl-. Differences between membrane potential and Cl- equilibrium potential therefore will lead to Cl- fluxes across the membrane. In contrast with oligodendrocytes, [Cl-]i in astrocytes is significantly increased (from 20 to 40 mM) above the equilibrium distribution owing to the activity of an inward directed Cl- pump; this suggests a different mechanism of K+ uptake in these cells.

Animals↗
PubMed1985

A reversible decrease in electrical coupling of cultured mouse glial cells induced by superfusion from a micropipette.

Cultured mouse oligodendrocytes were superfused by pressure application to the rear-end of a 3-20 microns micropipette filled with normal bathing fluid. Input resistance was determined during superfusion with two separate electrodes. The input resistance increased reversibly by 90% in 26 cells tested and was unaffected in 39 cells. When pairs of oligodendrocytes were electrically coupled, coupling decreased in a reversible manner during superfusion. Therefore, the flow from a micropipette can uncouple cells, and studies involving application of substances by pressure ejection from microelectrodes must be evaluated with care.

Animals↗