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PubMed1995

Blockade of capacitive Ca2+ influx by Cl- channel blockers inhibits secretion from rat mucosal-type mast cells.

Whole-cell patch-clamp recordings of membrane currents were performed in combination with measurements of mediator secretion from mucosal-type mast cells (rat basophilic leukemia cells, subline 2H3), to determine the involvement of membrane conductances induced upon depletion of intracellular Ca2+ stores. In patch-clamp experiments, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-induced depletion of internal Ca2+ stores led to activation of two distinct membrane conductances, a Ca2+ current and a Cl- current. The Ca2+ current was blocked by 100 microM La3+, which did not affect the Cl- current. In contrast, 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid produced selective blocked of the Cl- current. Remarkably, the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), niflumic acid, and N-phenylanthranilic acid (NPAA) inhibited not only the Cl- current but also the Ca2+ current. IC50 values for the blockade of the Ca2+ inward current by NPPB, niflumic acid, and NPAA were determined to be 23, 150, and 190 microM, respectively. In secretion experiments, thapsigargin-induced depletion of internal Ca2+ stores stimulated serotonin release, which was found to be strictly dependent on extracellular Ca2+. In the presence of 100 microM La3+ secretion was almost completely inhibited. In contrast, only 50% of secretion was suppressed by 500 microM 4,4'-diisothiocyanato-2,2'-disulfonic acid, which fully blocked the Cl- current without affecting Ca2+ influx, as monitored by electrophysiological experiments. The other Cl- channel blockers produced a very different pattern for the inhibitory dose dependence of secretion, with IC50 values for NPPB, niflumic acid, and NPAA of 23, 60, and 180 microM, respectively. Taken together, these findings suggest that Ca2+ store depletion leads to concomitant activation of Cl- and Ca2+ currents. Blockade of the latter is apparently an additional mode of action for diarylaminocarboxylate-type Cl- channel blockers inhibiting mast cell secretory responses.

Animals↗

PubMed1996

Ionic channels in corneal endothelium.

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

Animals↗

PubMed1995

Capacitative Ca2+ influx and a Ca2+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells.

We have investigated the effect of genistein on the hormone-stimulated Ca2+ influx and on a 28pS nonselective cation channel in mouse pancreatic acinar cells using the Ca2+ indicator fluo3 and the patch-clamp technique. The identity of the Ca2+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca2+-dependent nonselective cation channel as a potential pathway for Ca2+ influx. Capacitative Ca2+ entry was induced by depletion of intracellular Ca2+ stores with 500nM acetylcholine or with the Ca2+ ATPase inhibitor 2,5di-tert- butylhydroquinone. In the presence of 100microM genistein, Ca2+ release was unimpaired, whereas Ca2+ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca2+-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28pS nonselective cation channel does not contribute to Ca2+ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca2+ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca2+ entry mechanism in mouse pancreatic acinar cells is unclear.

Animals↗

PubMed1988

A study of stretch-activated channels in the membrane of frog oocytes: interactions with Ca2+ ions.

1. We have carried out patch-clamp measurements on a cationic channel in the plasma membrane of the frog oocyte, which can be specifically activated by membrane stretch. The kinetics of this channel also display a distinct dependence upon membrane potential, the probability of the channel being open increasing with membrane depolarization. 2. When the patch-clamp pipette filling solution was standard Ringer solution, the single-channel current-voltage (I-V) relationship was linear, the elementary conductance being 38 pS and the reversal potential +7 mV, suggesting very poor selectivity of the channel for the various cations. 3. The I-V relationship was highly non-linear having a strong inward-going rectification when Ca2+-free solutions were used to fill the patch pipette. These solutions also resulted in a selective, inward cationic permeability through the membrane, with K+ being more permeable than Na+ greater than Li+ greater than Ba2+ greater than Ca2+. 4. Though permeant through the stretch-activated channel, Ca2+ inhibited in a concentration-dependent manner the currents carried by other cations. La3+ (0.1 mM) was also an effective channel blocker. 5. The inward current carried by individual cations at a given membrane potential increased with increasing external cation concentration up to a saturating level, this level being maximal for K+ and minimal for Ca2+. Also the half-saturating concentration was maximal for K+ and minimal for Ca2+ at all membrane potentials. 6. In the presence of a constant Ca2+ concentration (50 microM) increasing [K+] did not change the absolute level at which the current saturated; however the half-saturating K+ concentration was greatly increased, indicating competitive inhibition between Ca2+ and K+ for the same site. 7. The data are consistent with a model based on Eyring rate theory for current conduction through ionic channels, in which we assume that the ions capable of entering the channel compete for a binding site that they must first occupy before proceeding on. The possible energy profile of the stretch-activated channel was defined by optimizing the model parameters to obtain the best fit of the experimental data. Ca2+ was found to have a smaller dissociation constant and much longer occupancy time than Na+ or K+, thus accounting for its lower permeability and inhibitory effect on current conduction by other cations through the stretch-activatable channel.

Action Potentials↗

PubMed1991

Interleukin-2 lengthens extrajunctional acetylcholine receptor channel open time in mammalian muscle cells.

The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000-3,000 units/ml). Cell-attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07 +/- 0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system.

Animals↗

PubMed1998

Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1.

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (DeltaD13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and DeltaD13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys877 to Arg894.

Amino Acid Sequence↗

PubMed1993

Regulation of basolateral K channels in proximal tubule studied during continuous microperfusion.

Potassium channel activity of the basolateral membrane of the collagenase-treated rabbit proximal convoluted tubule (PCT) was studied during continuous luminal microperfusion. In cell-attached patches (high-K pipette) an inwardly rectifying potassium channel was observed with an inward slope conductance of 60.8 +/- 3.3 pS (n = 12) and outward slope conductance of 17.1 +/- 2.7 pS (n = 6). Stimulation of transcellular sodium transport with luminal glucose and alanine increased channel activity [measured as single-channel open probability (NPo)] from 0.19 +/- 0.11 to 0.44 +/- 0.09 (n = 8). This increase in channel activity was not likely to be mediated by either cell depolarization or cell swelling, because channel activity was voltage insensitive over physiological potentials and because the channel was not activated by stretch. However, channel activity was pH sensitive; reducing luminal pH from 7.4 to 6.5 reduced NPo from 0.63 +/- 0.24 to 0.26 +/- 0.16 (n = 5). Our work demonstrates the feasibility of patch clamping the basolateral membrane of microperfused nephron segments. This has allowed us to follow the activity of this potassium channel during an increase in sodium transport and show that its activity does increase during this maneuver. We conclude that: 1) it is possible to patch clamp the basolateral membrane of microperfused nephron segments, and 2) basolateral membrane of the rabbit PCT contains an inwardly rectifying, pH-sensitive potassium channel. The behavior of this channel on stimulation of transcellular sodium transport could explain the macroscopic increase in basolateral potassium conductance observed under similar conditions.

Alanine↗

PubMed2026

A novel iPSC model of Bryant-Li-Bhoj neurodevelopmental/neurodegenerative syndrome demonstrates the role of histone H3.3 in chromatin dynamics, neuronal differentiation, and maturation.

BACKGROUND: Bryant-Li-Bhoj neurodevelopmental syndrome (BLBS) is neurogenetic disorder caused by variants in H3-3A and H3-3B, the two genes that encode histone H3.3. Ninety-nine percent of individuals with BLBS show developmental delay/intellectual disability, but the mechanism by which variants in H3.3 result in these phenotypes is not yet understood, limiting the therapeutic interventions available to individuals living with BLBS. METHODS: Here, we investigate how one BLBS-causative variant, H3-3B p.Leu48Arg (L48R), affects neurodevelopment using an induced pluripotent stem cell model differentiated to 2D neural progenitor cells (NPCs), 2D forebrain neurons (FBNs), and 3D dorsal forebrain organoids (DFBOs). We employ a multi-omic approach in the 2D models to quantify the resulting changes in gene expression and chromatin accessibility. We used immunofluorescence (IF) staining to define the identities of cells in the 3D DFBOs and whole-cell patch clamp to investigate the electrophysiological properties of neurons in DFBOs. RESULTS: In the 2D systems, we found dysregulated gene expression and chromatin accessibility affecting neuronal fate, adhesion, neurotransmission, and excitatory/inhibitory balance. Immunofluorescence of DFBOs corroborated altered proportions of radial glia and mature neuronal populations. Patch clamp recordings revealed decreased electrical activity in neurons from L48R DFBOs compared to control DFBOs. CONCLUSIONS: These data provide the first mechanistic insights into the pathogenesis of BLBS from a human-derived model of neurodevelopment, which suggest that H3.3 L48R increases H3-3B expression, resulting in the hyper-deposition of H3.3 into the nucleosome, which underlies changes in gene expression and chromatin accessibility. Functionally, this causes dysregulation of cell adhesion, neurotransmission, and the balance between excitatory and inhibitory signaling. These results are a crucial step towards preclinical development and testing of targeted therapies for this and related disorders.

Histones↗

PubMed1992

Modulation of potassium channels in intact human T lymphocytes.

1. A voltage-dependent K+ channel called the 'n' type (for 'normal') is the most prevalent ion channel found in whole-cell recordings from T lymphocytes. In whole-cell patch-clamp recordings activity of the n-type channel is affected by mitogenic agents, pH, Ca2+ and temperature but not by cyclic nucleotides. Because channel properties and regulation can depend on cytoplasmic components we sought to reassess the properties of K+ channels in intact, normal human T lymphocytes using cell-attached, patch-clamp recordings. In the present study, we show that the predominant K+ channel in resting, intact cells is the n type and is affected by voltage, temperature and Ca2+ in ways similar to the disrupted cell. Moreover, K+ channels are activated by agents that raise cyclic AMP in intact cells. 2. In cell-attached recordings, we found voltage-activated K+ channels in about 60% of patches at room temperature. The channel was K+ selective as judged from the reversal potential under different Ka(+)-K+ gradients and at different resting membrane potentials. Some patches were subsequently excised and the selectivity further confirmed. The current-voltage relation was inwardly rectifying under symmetrical K+ concentrations and had a slope conductance of 9.4 pS at 50 mV depolarized and 23.8 pS at 50 mV hyperpolarized from the resting potential. From the reversal potentials under various conditions the cell resting potential was -51 +/- 1 mV in normal NaCl saline and about 0 mV when the bath contained 150 mM-KCl saline. Two other types of K+ channel were seen in resting, intact cells, but were much less common (less than 5% and 11% of patches). A large-conductance K+ channel was seen in less than 1% of inside-out patches. 3. The predominant K+ channel in intact, resting T lymphocytes was confirmed as the n type underlying the whole-cell K+ current evoked by voltage steps. In cell-attached patches there was a low, steady-state level of activity at the resting potential but activity was greatly increased by depolarizing voltage jumps. Steady-state inactivation could be removed by a hyperpolarizing pre-pulse. Ensemble currents constructed by summing channel openings during repeated voltage jumps showed sigmoid kinetics of current activation and a monoexponential decay phase. These kinetics were well fitted by a Hodgkin-Huxley-type n4j kinetic model with time constants very similar to the whole-cell current of disrupted cells. Moreover, the kinetics depended on the external K+ concentration as previous research has shown.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed2004

Oscillations of membrane potential and cytosolic Ca(2+) concentration in SUR1(-/-) beta cells.

AIMS/HYPOTHESIS: SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. METHODS: Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes. RESULTS: In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. [Ca(2+)](c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and [Ca(2+)](c) in cells from both SUR1(-/-) and SUR1(+/+) mice. CONCLUSION/INTERPRETATION: The strong dependence of Vm and [Ca(2+)](c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

Glucose induces temperature-dependent oscillations of cytoplasmic Ca2+ in single pancreatic beta-cells related to their electrical activity.

Glucose induces large amplitude oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells. The effects of temperature on these oscillations were examined by monitoring [Ca2+]i continuously in single beta-cells from ob/ob-mice using dual wavelength microfluorometry. The oscillations of [Ca2+]i disappeared when the temperature was increased above 42 degrees C and were reversibly inhibited below 30 degrees C. However, cooling did not prevent a glucose response in terms of the average rise of [Ca2+]i. Since patch clamp studies of single beta-cells have indicated a random occurrence of glucose-induced action potentials at room temperature, it was important to explore how the sugar affected the electrical activity at 37 degrees C. Using the cell-attached configuration of the patch clamp technique for such analyses, the action potentials were found to occur in bursts with durations similar to the large amplitude oscillations of [Ca2+]i.

Action Potentials↗

PubMed1999

Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels.

A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (KATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial KATP (mitoKATP) channels remains unclear. We used a pharmacological approach to distinguish mitoKATP channels from classical, molecularly defined cardiac sarcolemmal KATP (surfaceKATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit Kir6.2. SUR2A and Kir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceKATP channels are activated potently by 100 microM pinacidil but only weakly by 100 microM diazoxide; in addition, they are blocked by 10 microM glibenclamide, but are insensitive to 500 microM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoKATP channels as indexed by flavoprotein oxidation. MitoKATP channels in myocytes are activated equally by 100 microM diazoxide and 100 microM pinacidil. In contrast to its lack of effect on surfaceKATP channels, 5-hydroxydecanoate is an effective blocker of mitoKATP channels. Glibenclamide's effects on mitoKATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent Kir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoKATP channels are distinct from surface KATP channels at a molecular level.

Animals↗

PubMed2000

A mibefradil metabolite is a potent intracellular blocker of L-type Ca(2+) currents in pancreatic beta-cells.

It has been shown that mibefradil (Ro 40-5967) exerts a selective inhibitory effect on T-type Ca(2+) currents, although at higher concentrations it can antagonize high voltage-activated Ca(2+) currents. The action of mibefradil on Ca(2+) channels is use- and steady-state-dependent and the binding site of mibefradil on L-type Ca(2+) channels is different from that of dihydropyridines. By using conventional whole-cell and perforated patch-clamp techniques, we showed that mibefradil has an inhibitory effect on both T- and L-type Ca(2+) currents in insulin-secreting cells. However, the effect on L-type Ca(2+) currents was time-dependent and poorly reversible in perforated patch-clamp experiments. By using mass spectrometry, we demonstrated that mibefradil accumulates inside cells, and furthermore, a metabolite of mibefradil was detected. Intracellular application of this metabolite selectively blocked the L-type Ca(2+) current, whereas mibefradil exerted no effect. This study demonstrates that mibefradil permeates into cells and is hydrolyzed to a metabolite that blocks L-type Ca(2+) channels specifically by acting at the inner side of the channel.

Calcium Channel Blockers↗

PubMed1990

Neuronal acetylcholine receptor channels from insects: a comparative electrophysiological study.

The channel properties of nicotinic acetylcholine receptor subtypes in the nervous system of insects (Locusta migratoria) have been characterized. Single channel measurements were performed using patch-clamp techniques as well as planar lipid bilayer reconstitution approaches. In reconstitution experiments using receptor-preparations isolated from neuronal membranes by alpha-toxin affinity chromatography, a ligand-gated channel type was found, which showed a high conductance and a short mean lifetime. Patch-clamp experiments on synapse-free somata of isolated nerve cells revealed an acetylcholine-gated channel type with a lower conductance but a longer lifetime. The two different agonist-activated channel types are supposed to represent synaptic and extrasynaptic acetylcholine receptors.

Animals↗

PubMed2000

Acid and salt responses in mouse taste cells.

Acid and salt responses of taste cells induced by natural stimulation have not been investigated with exception of early studies with conventional microelectrode method, due to the toxicity of high concentration of salt or low pH of acid stimuli applied to isolated taste cells. This indicates that the application of rapid and localized stimulation to the apical membrane of taste cells is necessary for recording of natural responses to salt or acid stimuli using patch clamp technique. Recently we have developed a procedure to accomplish the quasi-natural condition including rapid, localized stimuli to the apical receptive membrane and the maintenance of taste bud polarity. In this review, we present our recent results obtained under quasi-natural condition using patch clamp techniques, comparing with the previously proposed hypothesis. One of our major finding is the fact that the acid-induced responses of taste cells in the mouse fungiform papillae are never suppressed by amiloride but an apical proton-gated conductance and a basolateral Cl(-) conductance possibly contribute to sour transduction. On the other hand, salt-induced responses are suppressed by amiloride, although the salt-induced responses recorded from a single cell involve both amiloride-sensitive and -insensitive components. Furthermore, the amiloride-insensitive component of salt responses possibly consists of multiple subcomponents including an apical sodium-gated nonselective cation conductance and a basolateral Cl(-) conductance. Recent reports also support the hypothesis that both acid and salt responses require specific receptor mechanisms of inorganic cations such as H(+) and Na(+) at the apical receptive membrane.

Acids↗

PubMed1992

Serotonin hyperpolarizes cholinergic low-threshold burst neurons in the rat laterodorsal tegmental nucleus in vitro.

Serotonergic suppression of cholinergic neuronal activity implicated in the regulation of rapid eye movement sleep and its associated phenomenon, pontogeniculooccipital waves, has long been postulated, but no direct proof has been available. In this study, intracellular and whole-cell patch-clamp recording techniques were combined with enzyme histochemistry to examine the intrinsic electrophysiological properties and response to serotonin (5-HT) of identified cholinergic rat laterodorsal tegmental nucleus neurons in vitro. Sixty-five percent of the recorded neurons demonstrated a prominent low-threshold burst, and of these, 83% were cholinergic. In current-clamp recordings 64% of the bursting cholinergic neurons tested responded to the application of 5-HT with a membrane hyperpolarization and decrease in input resistance. This effect was mimicked by application of the selective 5-HT type 1 receptor agonist carboxamidotryptamine maleate. Whole-cell patch-clamp recordings revealed that the hyperpolarizing response was mediated by an inwardly rectifying K+ current. Application of 5-HT decreased excitability and markedly modulated the discharge pattern of cholinergic bursting neurons: during a 5-HT-induced hyperpolarization these neurons exhibited no rebound burst after hyperpolarizing current input and a burst in response to depolarizing current input. In the absence of 5-HT, the relatively depolarized cholinergic bursting neurons responded to an identical hyperpolarizing current input with a burst and did not produce a burst after depolarizing current input. These data provide a cellular and molecular basis for the hypothesis that 5-HT modulates rapid eye movement sleep phenomenology by altering the firing pattern of bursting cholinergic neurons.

Action Potentials↗

PubMed1998

Activation of a Cl--conductance by protein kinase-dependent phosphorylation in cultured rat retinal pigment epithelial cells.

While chloride conductances are involved in signals of the electroretinogram generated by the retinal pigment epithelium (RPE), patch-clamp experiments of freshly isolated or cultured RPE cells have shown that potassium conductances predominate. The purpose of this study was to investigate mechanisms which activate Cl--conductances in RPE cells. Membrane currents of cultured rat RPE cells were measured using the whole-cell configuration of the patch-clamp technique under extra- and intracellular K+-free conditions. The bath solution was hyperosmolal to the pipette solution to prevent hypoosmotic swelling. Exchange of the physiological intracellular fluid by a pipette solution with physiological levels of ATP (2 mm) induced a continuous increase of membrane conductance. Conductance was blocked by DIDS (1 mm), and showed a reversal potential close to the Nernst potential for Cl-. When the experiments were carried out under conditions in which all cations, and not only potassium, were replaced by NMDG, the same responses could be observed. Current activation was independent of extracellular calcium. Chloride currents were also induced when ATPgammaS or AMP-PNP were used instead of ATP. In the presence of AMP-PNP currents were 10 times smaller than in the presence of ATP or ATPgammaS. In cells preincubated with staurosporine or chelerythrine no currents were induced. Establishing the whole-cell configuration with ATP and with myristoylated PKC substrate in addition, no voltage-dependent currents were activated. We conclude that ATP hydrolysis leads to activation of chloride currents via PKC in the whole-cell configuration. The perforated patch configuration, with the intracellular compartment intact, no currents were induced under otherwise identical experimental conditions. Inhibition of phosphatase by calyculin (10 nm) in the perforated-patch configuration did not change membrane conductance. In the intact cell, chloride conductance is possibly inhibited by a cytosolic factor which is washed out when the whole-cell configuration is established.

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

PubMed1988

Na channels and two types of Ca channels in rat pancreatic B cells identified with the reverse hemolytic plaque assay.

The reverse hemolytic plaque assay (RHPA) was used to study the secretory properties of single rat pancreatic B cells, and to identify insulin-secreting cells for patch-clamp experiments. In secretion studies using the RHPA, we find that the percentage of secreting B cells and the amount of insulin secreted per B cell increase as the glucose concentration is raised from 0 to 20 mM. Using the whole-cell variation of the patch-clamp technique, we find that identified B cells have three types of channels capable of carrying inward current: (a) tetrodotoxin-sensitive, voltage-dependent Na channels, which are nearly completely inactivated at -40 mV, (b) fast deactivating (FD) Ca channels, and (c) slowly deactivating (SD) Ca channels. We have shown that Na channels are functionally significant to the B cell, because tetrodotoxin partially inhibits glucose-induced insulin secretion. The properties of FD and SD Ca channels differ in several respects. FD channels deactivate at -80 mV, with a time constant of 129 microseconds, they are half-maximally activated near +15 mV, they do not inactivate during 100 ms, they conduct Ba2+ better than Ca2+, and they are very sensitive to washout during intracellular dialysis. SD channels, on the other hand, deactivate with a time constant of 2.8 ms, they are half-maximally activated near -5 mV, they inactivate rapidly, they conduct Ba2+ and Ca2+ equally well, and they are insensitive to washout.

Animals↗