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At least 55 records · Page 3Linked to original sources
PubMed1992

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1992

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2+ channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activations curves for L-type Ca2+ currents, suggesting the presence of L-type Ca2+ "window" current. We used the on-cell, patch-clamp technique to study Ca2+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2+ channels 75% of the time. L-type channels were seen in 69% and T-type channels in 73% of these patches. With 110 mM Ba2+ as the charge carrier, the conductances of the L- and T-type Ca2+ channels were 24.2 +/- 0.8 pS (n = 9) and 9.0 +/- 0.5 pS (n = 8), respectively (mean +/- SEM). With 110 mM Ca2+ as the charge carrier, the conductance of the L-type Ca2+ channel decreased to 9.7 +/- 1.2 pS (n = 4), whereas the T-type Ca2+ channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2+ channels, although for L-type Ca2+ channel with Ba2+ as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis.

Animals↗
PubMed1992

Calcium currents in solitary hair cells isolated from frog crista ampullaris.

Some properties of Ca2+ currents in hair cells isolated from frog semicircular canals by enzymatic or mechanical treatment were studied by using the whole-cell configuration of the patch-clamp technique. After blocking the large outward K+ currents by substituting Cs+ for K+ and adding tetraethylammonium to the pipette filling solution, voltage- and time-dependent inward currents were clearly detectable in the presence of 4 mM Ca2+ in the extracellular solution. Ca2+ current was recruited at test potentials more positive than -60 mV, showed a rapid activation, and exhibited no inactivation during 150-ms depolarizing pulses. The maximal amplitude was attained at about -20 mV, with an average value of about 80 pA. When Ca2+ in the extracellular solution was replaced with Ba2+, the magnitude of inward currents increased about twofold. Ba2+ currents were blocked more effectively by Cd2+ than by Ni2+, were suppressed by 0.5 microM omega-conotoxin, and were virtually unaffected by amiloride. The dihydropyridine Bay K 8644 caused a marked voltage-dependent increase in inward currents. The present data suggest that hair cells from frog crista ampullaris are endowed with a homogeneous population of Ca2+ channels having several properties similar to those described for neuronal L channels. Since these channels are recruited in a range of potentials close to the resting level, it is suggested that they subserve the control of both resting and evoked transmitter release from the basal pole of the hair cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

The effect of ATP on Ca(2+)-dependent K+ channels of human red cells.

The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using patch-clamp techniques. Single-channel current was recorded at pH 6.5 from inside-out patches in the presence of symmetrical K+ gluconate solutions, containing both 1 microM free Ca2+ in the bath and 0.5 mM LaCl3 on the pipette side. With no ATP, the electrical activity revealed low-conductance K+ channels (25 pS), which showed inward rectification and an opening kinetics dependent on membrane potential. When ATP (1 mM) and Mg2+ (2 mM) were added together and a depolarizing potential was simultaneously applied, only a high-conductance channel (about 75 pS) was observed. This channel showed no rectifying properties and it was not found if ATP was added in the absence of Mg2+. Channel activity was enhanced by adding fluoride (10 mM) or trifluoperazine (50 microM) whilst it was reduced after incubating with dibutyryl cAMP (50 microM) or alkaline phosphatase (250 U/ml). On the other hand, when fragmented membranes from inside-out vesicles were incubated with gamma-32 P-ATP and 1 microM free Ca2+ under above conditions, only two high-molecular weight polypeptides (235 and 320 kDa) were labelled with 32P. The results suggest that ATP-mediated phosphorylation of Ca(2+)-dependent K+ channels leads to a high-conductance state.

Adenosine Triphosphate↗
PubMed1992

Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.

Adenosine Triphosphate↗
PubMed1992

Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine4 receptors.

In various species, including humans, 5-hydroxytryptamine (5-HT) has been shown to exert positive chronotropic and inotropic cardiac effects through different types of receptors. The goal of the present study was to investigate the regulation by 5-HT of voltage-gated Ca2+ channels in human atrial myocytes and to characterize the receptor involved. Cardiomyocytes isolated enzymatically and mechanically were voltage-clamped using the whole-cell configuration of the patch-clamp technique. Extracellular perfusion of 5-HT increased Ca2+ current (ICa) amplitude with a EC50 (0.1 microM) similar to that observed with isoprenaline. The effects of 5-HT were blocked by the addition of protein kinase A inhibitor in the pipette. In addition, the effects of 5-HT, isoprenaline, and intracellular cAMP on ICa were not additive. These results support the hypothesis that the inotropic effect of 5-HT in human atrial myocytes is related to an increase of ICa via an elevation of intracellular cAMP levels and stimulation of cAMP-dependent protein kinase. The effects of 5-HT were not blocked by antagonists of 5-HT1 (methiothepin), 5-HT2 (ketanserin), or 5-HT3 (ICS 205-930 at a low concentration) receptors. The benzamide derivatives renzapride and zacopride and the azabicyclobenzimidazolone derivative BIMU 8 increased ICa, but less efficiently than did 5-HT or 5-methoxytryptamine. Moreover, ICS 205-930 at high concentrations (greater than 1 microM) completely antagonized the effects of 5-HT. Thus, the pharmacology of the 5-HT receptor involved in an increase of ICa in human atrial myocytes resembles that recently described for the 5-HT4 receptor. In atrial myocytes dissociated from rat, rabbit, guinea pig, or frog, 5-HT at high concentrations had no effect on Ca2+ currents, suggesting that the distribution of 5-HT4 receptors in cardiac tissues is species dependent.

Animals↗
PubMed1992

Blocking action of terodiline on calcium channels in single smooth muscle cells of the guinea pig urinary bladder.

The blocking action of terodiline, a nonspecific organic Ca++ antagonist, on smooth muscle Ca++ channels of the guinea pig urinary bladder was investigated. Inward Ca++ currents were recorded from smooth muscle cells isolated from the urinary bladder using the whole-cell patch-clamp technique. In the absence of terodiline, a use-dependent reduction in the amplitude of inward Ca++ current was observed at a stimulus frequency of 0.2 Hz. When terodiline (1-10 microM) was applied, the use-dependent reduction was accelerated markedly, depending on the stimulus frequency. The blocking action of terodiline was also dose-dependent; the Kd value as measured at the end of 20 times repetitive stimulation at 0.2 Hz was 1.7 microM. In addition to such a use-dependent block, terodiline produced a hyperpolarizing shift in the steady-state inactivation curve. The results suggest that terodiline preferentially binds to the Ca++ channel in the open state and also in the inactivated state.

Action Potentials↗
PubMed1992

Differences in effects of forskolin and an analog on calcium currents in cardiac myocytes suggest intra- and extracellular sites of action.

The effects of forskolin (FO) and a water-soluble derivative of FO, L858051 (7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)-butyryl] forskolin), were compared on calcium currents (ICa) studied by the whole-cell patch-clamp technique in frog ventricular cardiac myocytes. Both FO and L858051 increased ICa, with half-times of 160 +/- 20 sec and 343 +/- 22 sec, respectively. The stimulation was blocked by internal perfusion with inhibitors of protein kinase A. The EC50 for stimulation of ICa was 0.3 microM for FO and 1.0 microM for L858051. The maximal stimulated current was the same for both drugs, 20.3 microA/cm2 and 23.1 microA/cm2, respectively. Internal perfusion with 30-500 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] suppressed ICa stimulation by low concentrations of FO or L858051. This suppression was due to a rightward shift in the concentration-response curve, with increases in the EC50 values to 11.4 microM for FO and 28.4 microM for L858051. Isoproterenol (ISO) was ineffective in increasing ICa after the FO-stimulated ICa had been reduced by Gpp(NH)p and FO had been washed out. In contrast, after the L858051-stimulated current had been reduced by Gpp(NH)p, ISO stimulated ICa significantly. This stimulation was blocked by inhibitors of protein kinase A and was due to a positive effect of L858051 not shared by FO. A brief application of L858051 after Gpp(NH)p had blocked the ISO response restored the ISO response for at least 30 min. This effect was mimicked by internal perfusion with low concentrations of L858051. We conclude that the ability of brief exposure of L858051, but not FO, to restore the response to ISO after Gpp(NH)p is due to the accumulation of L858051 intracellularly, due to its hydrophilicity. Because internal L858051 and FO are very ineffective in stimulating adenylyl cyclase, whereas internal L858051 can restore the ISO response blocked by Gpp(NH)p, we propose that FO compounds can affect adenylyl cyclase at two sites, one site that is accessible only from the extracellular side that stimulates catalytic activity and another that is accessible from the intracellular side that increases beta-agonist efficacy in the presence of Gpp(NH)p.

Animals↗
PubMed2026

Biophysical mechanisms underlying the generation and maintenance of rule-learning engram.

Training rodents in a particularly difficult olfactory-discrimination task results with acquisition of high-skill to perform the task superbly, termed 'rule-learning'. We show that rule-learning occurs abruptly, in a "light-bulb moment". Using whole-cell patch-clamp recordings from the piriform cortex (PC) of Fos2A-iCreER/TRAP2 mice, we target activated-neurons, expressing immediate early genes (IEG). We notice, from the onset of training, IEG-positive neurons from trained animals display enhanced intrinsic excitability. Subsequently, synaptic excitation and inhibition are enhanced in these neurons, in a coordinated, cell-wide process. Additionally, in parallel, we detect the density of IEG-expressing neurons sharply declines. Double labeling with TRAP and c-Fos reveal that nearly two-thirds of the rule-memory cell ensemble neurons are activated from the beginning of training. Silencing TRAP-expressing neurons using inhibitory DREADD leads to a complete loss of rule memory. Hence, we propose that rule learning occurs at a discrete moment and is developed through a gradual process that stabilizes the memory of the rule.

Animals↗
PubMed2026

Automated patch clamp data improve variant classification and penetrance stratification for SCN5A-Brugada syndrome.

BACKGROUND AND AIMS: Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. METHODS: All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. RESULTS: Most variants (146/252) were functionally abnormal (Z ≤ -2), with 100 having severe loss-of-function (Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. CONCLUSIONS: This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

Humans↗
PubMed1992

Stretch-activated non-selective cation channels in the antiluminal membrane of porcine cerebral capillaries.

1. Single stretch-activated (SA) channels have been studied in isolated brain capillary endothelial cells as well as in the antiluminal membrane of intact porcine cerebral capillaries using the patch-clamp recording technique. 2. The SA channels were found to be cation selective and permeable to Na+, K+, Ba2+ and Ca2+. 3. With monovalent cations in the patch pipette, the channels showed inward rectification in cell-attached patches with a single-channel conductance of 37 pS at negative and 24 pS at positive clamp potentials. 4. With either 70 mM-Ca2+ or Ba2+ in the patch pipette, the current-voltage relation was linear with slope conductances of 16 and 19 pS, respectively. 5. Mean channel open probability increased with increasing pressure and with depolarizing clamp potentials. 6. Cell swelling induced by hypotonic shock activated the SA channels in cell-attached experiments. 7. The SA channel may be involved in cell volume or blood flow regulation. The contribution of these channels to the regulation of cerebrospinal salt and water content, especially in brain oedema, is discussed.

Animals↗
PubMed1992

Structural and functional crosstalk between acetylcholine receptor and its membrane environment.

Nicotinic acetylcholine receptor (AChR) is a transmembrane protein belonging to the superfamily of rapid, ligand-operated channels. Theoretical models based on thermodynamic criteria assign portions of the polypeptide chains to the lipid bilayer region. From an experimental point of view, however, the relationship between the two moieties remains largely unexplored. Current studies from our laboratory are aimed at defining the structural, dynamic, and functional relationship between membrane lipids and AChR. We are particularly interested in establishing the characteristics of and differences between the lipids in each leaflet of the bilayer and the belt or "annular" lipids immediately surrounding AChR and the bulk bilayer lipids. We are also interested in determining the possible implications of lipid modifications on AChR channel properties. Toward these ends, fluorescence and other spectroscopic techniques, together with biochemical analyses and patch-clamp studies, are currently being undertaken. Correlations can be established between structural aspects of phospholipid packing in the immediate perimeter of AChR and other properties of these annular lipids revealed by dynamic spectroscopic and molecular modeling techniques. Lipid compositional analyses of the clonal muscle cell line BC3H-1 and chemical modification studies have been carried out by incubation of intact cells in culture and of membrane patches excised therefrom with liposomes of different lipid composition. These studies have been combined with electrophysiological measurements using the patch-clamp technique, with the aim of determining the possible effects of lipids on the channel properties of muscle-type AChR. A variety of experimental conditions, involving polar head and fatty acyl chain substitution of phospholipids and cholesterol incorporation, are being assayed in the BC3H-1 cells.

Acetylcholine↗
PubMed1992

Chromostatin receptors control calcium channel activity in adrenal chromaffin cells.

One of the functions of chromogranin A (CGA), the major soluble component of secretory granules in both adrenal medullary chromaffin cells and many other endocrine cell types appears to be that of a prohormone. CGA is the precursor of several peptides including pancreastatin, a 49-residue peptide, and a 20-residue peptide, chromostatin, which have been identified as biologically active peptides. Chromostatin produces a dose-dependent inhibition (ID50 of 5 nM) of the secretagogue-evoked catecholamine secretion from chromaffin cells. Here we report that chromostatin potently inhibits L-type calcium currents recorded with the nystatin-perforated patch technique in cultured chromaffin cells. This inhibitory effect of chromostatin on calcium currents was not observed in experiments using the classical patch-clamp whole-cell approach which induces the leakage of cytoplasmic components. Using 125I-chromostatin, we show that chromostatin exhibits a fully reversible and saturable binding to the plasma membrane of cultured chromaffin cells. Analysis of binding experiments at equilibrium indicates the existence of one class of binding sites with a Bmax of 2.7 pmol/mg of chromaffin cell proteins and an apparent Kd of 6.5 nM. This high affinity is in good correlation with the half-maximal concentration (ID50 5 nM) of chromostatin inhibiting catecholamine secretion from chromaffin cells. Specificity of the chromostatin binding was further assessed by displacement experiments with unlabeled CGA-related or -unrelated peptides. We found an excellent quantitative correlation between the affinities of the various peptides determined by binding assays and their functional potency tested on catecholamine secretion: bovine chromostatin greater than human chromostatin greater than CGA much greater than rat chromostatin, pancreastatin, CAP-14, substance P, and Leu-enkephalin. Cross-linking experiments reveal that chromostatin associates specifically with an 80-kDa plasma membrane protein. These results together with the patch-clamp experiments support the idea that chromaffin cells possess specific chromostatin receptors and that activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels through an intracellular second messenger pathway.

Adrenal Glands↗
PubMed1992

Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor.

1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.

Calcimycin↗
PubMed1992

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.

Adenosine Diphosphate Ribose↗
PubMed1992

Adenosine-dependent enhancement by methylxanthines of excitatory synaptic transmission in hippocampus of rats.

Whole-cell patch-clamp was used to investigate synaptic transmission in hippocampal slices. Excitatory post-synaptic currents (EPSCs) were facilitated by low (less than or equal to 1 microM) adenosine (Ado) concentrations, while high concentrations had well-known inhibitory effects on the EPSC. When added on the background of preapplied Ado, methylxanthines caused a large potentiation of EPSCs. At saturation, the enhanced EPSC could exceed the control almost by an order of magnitude. Pertussis toxin strongly impaired the ability of Ado to block EPSCs but did not augment the facilitatory effect. The two components of the EPSC mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors were facilitated simultaneously and in equal proportions.

Adenosine↗