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Results for “patch clamp”

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PubMed1990

Single-channel data and missed events: analysis of a two-state Markov model.

Patch-clamp recording permits investigation of the gating kinetics of single ion channels. Careful statistical analysis of kinetic data can yield clues as to the molecular events underlying channel gating. However, it is important that such analysis should take full account of the limitations that arise from the finite time resolution of patch-clamp recording techniques. Single-ion-channel data are generally interpreted in terms of Markov process models of channel gating mechanisms. Experimental channel records suffer from time interval omission, i.e. failure to detect brief channel openings and closings. This leads to an identifiability problem when analysing single-channel data, i.e. different gating mechanisms provide equally convincing descriptions of the same experimental data. We consider a two-state Markov model of receptor-channel gating in which the channel opening rate is proportional to the agonist concentration, C in equilibrium with OA. By using computer-simulated data, the approximate likelihood of the data is maximized to yield parameter estimates for the model. At a single agonist concentration there is an identifiability problem in that two pairs of parameter estimates are obtained. The 'true' parameter estimates cannot be distinguished from the 'false' ones. By considering data corresponding to a range of agonist concentrations one may identify the 'true' parameter estimates as those that do not change as the agonist concentration is increased. Alternatively, one may identify the 'true' parameter estimates directly by maximizing a global likelihood, the latter being obtained by simultaneous consideration of data obtained at several different agonist concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Effects of lysophosphatidylcholine on electrophysiological properties and excitation-contraction coupling in isolated guinea pig ventricular myocytes.

Lysophosphoglyceride accumulation in ischemic myocardium has been implicated as a cause of arrhythmias. We examined the effects of lysophosphatidylcholine (LPC) in isolated guinea pig ventricular myocytes. In paced myocytes loaded with the Ca2+ indicator Indo-1-AM and studied at room temperature, 20 microM LPC caused an initial positive inotropic effect followed by spontaneous automaticity, a decline in active cell shortening, and progressive diastolic shortening (contracture) leading to cell death. These changes were accompanied by a progressive increase in cytosolic [Ca2+]i. In patch-clamped myocytes dialyzed internally with high EGTA concentrations, LPC caused membrane depolarization, shortening of the action potential duration, and abnormal automaticity as seen in multicellular preparations. Voltage clamp experiments revealed the appearance of a nonselective leak conductance without significant changes in the delayed rectifier K+ current, inward rectifier K+ current, L-type Ca2+ current, and T-type Ca2+ current. Pretreatment with 20 mM caffeine and [Ca2+]o-free solution did not prevent the leak current. In patch clamped myocytes loaded with 0.1 mM Fura-2 salt, the [Ca2+]i transient induced by either voltage clamps or brief caffeine exposure remained normal until the nonselective leak current developed. The Na(+)-Ca2+ exchange current elicited during caffeine-induced [Ca2+]i transients also did not appear to be altered by LPC. Qualitatively similar results were obtained in myocytes studied at 35 degrees C. The membrane detergent saponin (0.005% wt/wt) mimicked all of the effects of LPC. We conclude that under these experimental conditions the effects of LPC are most compatible with a detergent action causing membrane leakiness with resultant depolarization, [Ca2+]i overload, and contracture.

Animals↗

PubMed1993

Calcium-dependent regulation of cholecystokinin secretion and potassium currents in STC-1 cells.

Secretory and electrophysiological properties of STC-1 cells, a cholecystokinin-secreting cell line, were examined with a radioimmunoassay and patch-clamp recording techniques. Stimulation of cholecystokinin secretion was seen after exposure to agents anticipated to increase the level of intracellular calcium, including thapsigargin (8 microM), bombesin (50 nM), potassium-induced depolarization (50 mM), or after blockade of potassium channels with barium chloride (2 mM). The secretory effects of these agents were blocked by pretreatment with the calcium channel blocker diltiazem (1 microM). Whole cell patch-clamp recordings showed a hyperpolarizing shift in reversal potential after exposure to either thapsigargin (8 microM) or bombesin (50 nM) from a control value of -27 +/- 3 to -57 +/- 7 or -48 +/- 6 mV, respectively. This shift was in the direction of the reversal potential for potassium and was blocked by barium chloride (5 mM). Single-channel recordings from cell-attached membrane patches showed an inwardly rectifying potassium channel with channel open probability modulated by bombesin. These results indicate that in STC-1 cells a potassium current is increased by agents that stimulate CCK secretion, presumably by increasing the level of cytosolic calcium. STC-1 cells may serve as a model system to study the electrophysiological and secretory mechanisms involved in the release of cholecystokinin.

Animals↗

PubMedDate not supplied

[Effect of xymedone on high-voltage-activated Ca2+ currents in pyramidal neurons of the entorhinal cortex].

The effect of xymedon on Ca2+ currents in entorhinal cortex LIII pyramidal neurons was studied using brain slices from 10-17-day old rats, which were analyzed by means of the infrared video assisted whole cell patch clamp recording. The sample slices were superfused with artificial cerebrospinal fluid containing tetrodotoxin, 4-aminopyridine, and tetraethylammonium for the blocking of Na+ and K+ channels, respectively. Xymedone was added to artificial cerebrospinal fluid and to all extracellular solutions. The slices were exposed to different concentrations of xymedone for 3 hours followed by patch-clamp recordings. Control recordings were run with the vehicle. Xymedone in a concentration of 0.01 mM decreased the maximum voltage-dependent Ca2+ current amplitude by 39.8 %, while 1 mM of xymedone inhibited the Ca2+ currents almost completely. The obtained data showed for the first time that xymedone exhibits a calcium channel blocker activity in neurons. Possible neuroprotective mechanisms of xymedone are discussed.

Animals↗

PubMed1998

Noise analysis of the quinine-induced current in frog taste receptor cells.

It has been shown that quinine, a bitter tasting substance, induces a cationic membrane current in frog taste receptor cells. We analyzed the quinine-induced current fluctuation to further characterize the cationic channel conducting the current. Taste cells were enzymatically isolated from fungiform papillae of the bullfrog tongue. Membrane currents were recorded under voltage-clamp by using the whole-cell patch clamp technique. A Cs(+)-containing pipette solution was used to block K+ currents. When quinine was bath applied, it induced a steady inward current in cells held at -60 mV. The current data were digitized at a sampling frequency of 2 kHz after having been low-pass filtered (cut-off frequency, 1 kHz). The spectral density distribution was calculated by 1024 points fast Fourier transform (FFT), and was fitted by a sum of two Lorentzian functions, with corner frequencies at 10-20 Hz and at 90-120 Hz. The single-channel conductance calculated from the variance versus mean current plot was 5 pS in a normal saline solution containing 1.8 mM Ca2+. In a nominally Ca(2+)-free solution, the single-channel current was doubled. Our previous study has shown that the quinine-induced current is enhanced in low extracellular [Ca2+]. This enhancement could be partly attributable to the Ca2+ suppression of the single-channel conductance.

Analgesics, Non-Narcotic↗

PubMed2005

Activation of muscarinic K+ channels by arecaidine propargyl ester in isolated guinea-pig atrial myocytes.

Arecaidine propargyl ester (APE) was developed as a potential candidate compound for the treatment of Alzheimer's disease. APE has been shown to have cardiovascular effects. APE produces negative chronotropic and inotropic effects in isolated atria. However, the ionic mechanisms underlying the cardiovascular effects of APE in guinea-pig atria are unclear. The aims of this study were: (1) to examine the shortening effect of APE on action potential duration (APD) and to compare the difference in potency between APE and muscarine in isolated single guinea-pig atrial myocytes by using the current clamp method, (2) to examine by using patch clamp techniques the ionic mechanisms underlying the cardiac effects of APE, and (3) to determine whether the cardiac effects caused by APE affect the usefulness of APE as a potential candidate for the treatment of Alzheimer's disease. The APE significantly reduced the APD in guinea-pig atria and produced no direct effect on ventricular myocytes. APE is approximately 20 times as potent as muscarine in shortening the APD. Attenuation of the APD was consistently accompanied by a hyperpolarization of the resting membrane potential in a concentration-dependent manner. The APE activated muscarinic K+ channels and increased potassium conductance in guinea-pig atrial myocytes. In the cell-attached configuration, the APE contained in the pipette increased the channel-opening probability and decreased the closed-state time interval. The proposal that APE can be used as a potential remedy for the treatment of Alzheimer's disease should be taken into consideration the undesirable cardiovascular side effects that APE causes at lower concentrations.

Animals↗

PubMed2005

Fluctuations in Xenopus oocytes protein phosphorylation levels during two-electrode voltage clamp measurements.

The biophysical and pharmacological properties of ion channels and transporters are often studied in exogenous expression systems using either the two-electrode voltage clamp (TEVC) in Xenopus oocytes or the patch clamp techniques. Cells machinery is trusted to produce active proteins that are correctly phosphorylated and glycosylated. However, native physiological cellular processes that might be altered during the course of the experiment are often ignored. Here, we detected and quantified the effects of various electrophysiological recording conditions on the phosphorylation levels of Xenopus oocytes proteins, including membrane proteins, as phosphorylation/dephosphorylation events modulate ion channels gating and cell surface expression. Two strategies were chosen to determine relative protein phosphorylation levels: a direct detection with a phospho-Ser/Thr PKA substrate antibody, and a functional method employing two different leak potassium channels as indicators, chosen based on their opposite responses to protein kinase phosphorylation. We report that holding potential, and bath solution properties such as pH, osmolarity, temperature and ion composition, dramatically affect protein phosphorylation levels in Xenopus oocytes. Our results might explain some of the fluctuations in the biophysical properties of expressed channels, often observed during electrophysiological measurements. Minimizing possible misinterpretations could be achieved using either mutated, kinase insensitive, channels or kinases/phosphatases modulators.

Animals↗

PubMed1991

Prostaglandin E2 activates Ca2+ channels in bovine adrenal chromaffin cells.

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.

Adrenal Medulla↗

PubMed2006

Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC-1 channel and by increasing intracellular calcium.

BACKGROUND AND PURPOSE: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage-gated chloride channel CLC-1, in controlling muscle excitability, the availability of ligands modulating CLC-1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non-steroidal anti-inflammatory drugs (NSAID). EXPERIMENTAL APPROACH: Rat muscle Cl(-) conductance (gCl) and heterologously expressed CLC-1 currents were evaluated by means of current-clamp (using two-microelectrodes) and patch-clamp techniques, respectively. Fura-2 fluorescence was used to determine intracellular calcium concentration, [Ca(2+)](i), in native muscle fibres. KEY RESULTS: NFA inhibited native gCl with an IC(50) of 42 muM and blocked CLC-1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca(2+)](i) in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC-1. A structure-activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca-dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation. CONCLUSIONS AND IMPLICATIONS: These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC-1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium-dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side-effects.

Animals↗

PubMed2000

Corticosterone impairs cultured hippocampal neurons and facilitates Ca2+ influx through voltage-dependent Ca2+ channel.

AIM: To investigate the effect of corticosterone (Cor) on the viability of cultured hippocampal neurons as well as voltage-dependent Ca2+ channel (VDCC) on the membrane of the hippocampal neurons. METHODS: The primary cultured hippocampal neurons were prepared and the viability of hippocampal neurons was determined by MTT assays. Inward Ca2+ currents of VDCC on the membrane of the hippocampal neurons were measured with the whole-cell patch-clamp technique. RESULTS: Treatment with Cor concentration-dependently reduced the survival of hippocampal neurons. The IC50 of Cor was 3.2 mumol.L-1. Neurons from cerebral cortex were affected only by high concentrations of Cor (10 mumol.L-1 and 0.1 mmol.L-1) with the IC50, 85 mumol.L-1, 20 times larger than the former. Whole-cell patch-clamp experiment showed that Cor (1 mumol.L(-1)-0.1 mmol.L-1) sprayed to the surface of the hippocampal neurons instantly facilitated Ca2+ influx through VDCC with the maximal elevation of 53%, 191%, and 84% above the baseline respectively and this effect was shown to be concentration-independent. In addition, changing the membrane potentials from -40 mV to -10 mV did not affect the facilitating effect of Cor on the Ca2+ influx, indicating that Cor-induced Ca2+ influx was membrane potential-independent. CONCLUSION: Cor markedly facilitated Ca2+ influx into the hippocampal neurons, which may be one of the important mechanisms underlying the neurotoxicity of Cor to hippocampus.

Animals↗

PubMed1992

[Activation of ATP-sensitive K+-channels of cardiomyocytes by endogenous cardiopeptides].

The "patch-clamp" method was applied to rat ventricular cardiomyocytes to study the effect of endogenous cardiopeptides (a component of a cardiologic medicine "cardialin") on single K(+)-channel currents. It was noted that ATP-sensitive K(+)-channel activity increased only in the presence of 10(-8)-10(-6) mg.ml-1 peptide solution in the patch-clamp micropipette. To check up the suggestion that G-protein takes part in a direct reaction of K(+)-channel by external ligand we studied the effect of GTP-gamma-S (0.1 mM) in the presence of Mg2+ (1 mM) on the K(+)-channel in "run-down" state in the "inside-out" configuration. It was demonstrated experimentally that reactivation of the ATP-sensitive K(+)-channel develops in the presence of GTP-analogue.

Adenosine Triphosphate↗

PubMed2006

[Electrophysiological effects of hyperpolarization activated cyclic nucleotide gated channel 4 overexpression in neonatal cardiomyocytes mediated by recombinant adenovirus].

OBJECTIVE: To explore the electrophysiological effects of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4) overexpression in rat neonatal cardiomyocytes mediated by recombinant adenovirus. METHODS: Ventricular cardiomyocytes were obtained from 20 neonatal rats. Recombinant adenovirus carrying HCN4 gene, AdHCN4, a dominant isoform of hyperpolarization activated cyclic nucleotide gated cation channel gene in cardiac transduction system, was constructed and used to transfect the neonatal rat ventricular cardiomyocytes. Untransfected cardiomyocytes were used as controls. RT-PCR and immunofluorescence cytochemistry were used to detect the HCN4 mRNA and protein expression. The electrophysiological characteristics of infected cardiomyocytes were studied by patch clamp. RESULTS: The mRNA and protein expression levels of HCN4 in AdHCN4 infected cardiomyocytes were both markedly higher than those of the control cardiomyocytes. AdHCN4 caused a significant increase in The spontaneous rate in the transfected cardiomyocytes was 92.5 + 7.4 bpm, significantly higher than that of the control cells (68.9 + 6.2 bpm, P < 0.05). Patch clamp experiments showed that the pacemaker current density in the AdHCN4 infected cardiomyocytes was 115.7 + 7.8 pA/pF, significantly higher than that of the untransfected cells (7.2 + 0.6 pA/pF, P < 0.05). CONCLUSION: Overexpression of HCN4 can enhance the autorhythmicity of neonatal cardiomyocytes and significantly increase the spontaneous beat rate. HCN4 channel gene may represent a candidate gene in gene therapy for bradycardia diseases.

Action Potentials↗

PubMed1994

Stereoselective and non-stereoselective actions of isoflurane on the GABAA receptor.

1. Acutely dissociated cerebellar Purkinje neurones from 8-14 day old rats were studied under voltage clamp in the whole-cell patch-clamp configuration. Cl- currents induced by bath application of gamma-aminobutyric acid (GABA) were measured (using symmetrical Cl- solutions) at both low (2 microM) non-desensitizing and high (300 microM) desensitizing concentrations of GABA. 2. At 2 microM GABA, the bicuculline-sensitive Cl- currents were potentiated by racemic isoflurane and both of its optical isomers. Isoflurane had no effect on membrane current in the absence of GABA. The dose-response data for potentiation by racemic isoflurane could be fitted with a Hill equation with an EC50 = 320 +/- 20 microM isoflurane and a Hill coefficient of h = 2.7 +/- 0.4 (means +/- s.e.mean). 3. The potentiations produced by the optical isomers of isoflurane at 2 microM GABA were stereoselective at moderate and high anaesthetic concentrations. The maximum stereoselectivity, about two fold, occurred at the EC50 concentration for general anaesthesia (310 microM isoflurane), with S(+)-isoflurane being more effective than R(-)-isoflurane. At sub-anaesthetic concentrations, the stereoselectivity was less marked and vanished at the lowest concentration used (77 microM isoflurane). 4. The sustained residual current remaining after exposure of neurons to a desensitizing concentration of GABA (300 microM) was inhibited non-stereoselectively, but only at high concentrations of isoflurane. The ratio of inhibitions by S(+)- and R(-)-isoflurane (mean +/- s.e.mean) was 1.14 +/- 0.21 at 770 microM isoflurane. At the EC50 concentration for general anaesthesia, however, the inhibition was barely significant. 5. The above results are discussed in relation to the possible role of the GABAA receptor channel in general anaesthesia.

Anesthesia, General↗

PubMed1996

Actions of general anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. NIE-115 mouse neuroblastoma cells were studied under voltage clamp in the whole-cell patch-clamp configuration. Peak currents induced by bath application of 5-hydroxytryptamine (5-HT) were inwardly rectifying, reversed at 0.4 +/- 0.2 mV (mean +/- s.e.mean), and were approximately half-inhibited (at 1 microM 5-HT) by 2 nM of the 5-HT3 selective antagonist MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). 2. Peak inward currents activated by a low concentration of 5-HT at a holding potential of -50 mV were potentiated by volatile general anaesthetics. At their human minimum alveolar concentrations (MACs), the degree of potentiation increased in the order isoflurane < halothane < enflurane < methoxyflurane. Potentiation by methoxyflurane was independent of membrane potential in the range -70 mV to +40 mV. The reversal potential was the same in the presence and absence of methoxyflurane. 3. Methoxyflurane shifted the 5-HT dose-response curve to lower 5-HT concentrations, without significantly changing the Hill coefficient or maximum response. The EC50 concentration for 5-HT decreased from 1.86 +/- 0.02 microM to 1.07 +/- 0.11 microM (means +/- s.e.mean) due to the presence of 1 MAC (270 microM) methoxyflurane. 4. In contrast to the volatile anaesthetics, the barbiturate anaesthetic, thiopentone, inhibited the 5-HT3 receptor. Hill analysis of thiopentone dose-response data gave an average IC50 = 117 +/- 8 microM thiopentone and Hill coefficient = 1.6 +/- 0.2 (means +/- s.e.mean). These parameters were not significantly different for data obtained at 5-HT concentrations above and below the control EC50 concentration for 5-HT, consistent with non-competitive inhibition. 5. The n-alcohols occupied an intermediate position between the volatile and barbiturate anaesthetics. The lower alcohols (butanol and hexanol) potentiated 5-HT responses at low alcohol concentrations but inhibited them at high concentrations. In contrast, the higher alcohols (octanol, decanol, dodecanol, tridecanol, tetradecanol and pentadecanol) produced no potentiation, but only inhibition, at all alcohol concentrations. 6. Inhibition of the 5-HT3 receptor by the n-alcohols exhibited a cutoff in potency similar to those previously found for tadpoles, luciferase enzymes and a neuronal nicotinic acetylcholine receptor channel.

Alcohols↗

PubMed1992

Single-channel and whole-cell recordings from non-dissociated sympathetic neurones in rabbit coeliac ganglia.

A procedure is described for performing patch-clamp recordings on mammalian sympathetic neurones within intact ganglia. The plasma membrane of superficial neurones was cleaned by blowing (1.5-3 h) a gentle stream of Ringer saline onto ganglia, the connective sheath of which was previously softened by a short protease treatment. This procedure preserved the intraganglionic connectivity so that the neurones could be activated either synaptically or antidromically by stimulating the appropriate nerves. Depending on the duration of the mechanical cleaning step, recordings were performed on either the neurones or the satellite glial cells covering the neuronal cell bodies. The applicability of the various configurations of the patch-clamp technique to studying sympathetic neurones is illustrated by recordings of whole-cell voltage, whole-cell currents and single-channel currents in cell-attached and excised patches. With these techniques, the resolution of the membrane current recordings is higher than with conventional microelectrodes. The results obtained show that mammalian sympathetic neurones have a very high input resistance (0.5 G omega), are electronically compact and may display pacemaker activity. These techniques provide a useful tool for studying the synaptic transmission and neuromodulation mechanisms operating within the sympathetic ganglia.

Animals↗

PubMed1995

A rapidly activating type of outward rectifier K+ current and A-current in rat suprachiasmatic nucleus neurones.

1. The properties of calcium-independent (i.e. persisting in the absence of external calcium) depolarization-activated potassium currents in suprachiasmatic nucleus (SCN) neurones (n = 75) were studied under voltage-clamp conditions with whole-cell patch-clamp recordings in rat hypothalamic slices (150-175 microns). 2. Two distinct types of potassium currents were found. One was a rapidly activating and slowly inactivating type of outward rectifier (named IK(FR) for fast rectifier potassium current), similar to a potassium current described in cardiac muscle, and the other was a transient A-current (IA). 3. The rates of activation and deactivation of IK(FR) were voltage dependent. Time constants of activation fitted to n4 kinetics and declined from 3.5 ms (at -20 mV) to 1.1 ms (at 60 mV). Inactivation had a biexponential time course with voltage-independent time constants of 0.3 s (minor component) and 3.0 s (major component) between 10 and 50 mV. IK(FR) was activated above -40 mV with a V1/2 (membrane potential at half-maximal activation) of 14 +/- 2 mV and slope factor of -17 +/- 1 mV reaching a conductance (not maximal) of 10.8 +/- 1.7 nS at 60 mV. Steady-state inactivation had a slope factor of 11 +/- 1 mV. 4. IK(FR) was highly selective for K+ (PNa/PK = 0.002). Tetraethylammonium (TEA) reduced IK(FR) reversibly (IC50 = 20 mM), while 4-aminopyridine (4-AP) at 10 mM had little effect. The remaining current in 30 mM TEA was similar to that in control conditions, indicating that TEA reduced IK(FR) rather than revealed an additional TEA-resistant current. 5. The rate of IA activation was voltage dependent with time to peak declining from 8.5 ms (at -40 mV) to 3.6 ms (at 60 mV). Inactivation had a biexponential time course with voltage-dependent and voltage-independent time constants. The two components were similar in amplitude. IA was activated above -60 mV, reaching a maximal conductance of 3.6 +/- 0.4 nS at above 20 mV. Steady-state inactivation was complete above -10 mV. Rates of onset of inactivation (at -40 mV) and recovery from inactivation (at -100 mV) were similar with time constants of 39 +/- 5 and 41 +/- 9 ms, respectively. 6. IK(FR) and IA were found in every neurone tested in the SCN and in all locations throughout the nucleus. The possible function of these currents is discussed, particularly in relation to the circadian rhythm of firing rate in the SCN.

4-Aminopyridine↗

PubMed1992

A Ca(2+)-dependent K+ current is enhanced in arterial membranes of hypertensive rats.

This study was designed to investigate the role and regulation of arterial membrane K+ channels in hypertension. Aortic segments from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were suspended for isometric tension recording. In other experiments, proximal aortic segments (PS) (exposed to high pressure) and distal aortic segments (DS) (exposed to lower pressure) were removed from surgically coarcted Sprague-Dawley rats and similarly prepared. Aortas from SHR and PS dose-dependently contracted to the K+ channel blocker tetraethylammonium (TEA) (0.1-10 mM), and this contraction was abolished by preincubation with 0.1 microM nifedipine. In contrast, the same concentrations of TEA did not contract either WKY or DS aortas. Since block of K+ channels by TEA had a different effect on aortic segments exposed to high versus low blood pressure, we compared whole-cell K+ currents in isolated vascular cells from the same aortas. The reversal potentials of depolarization-induced outward currents in WKY, SHR, DS, and PS aortic cells showed a Nernst relation to external K+ concentration indicative of selective K+ permeability. TEA (1 and 10 mM) was equipotent in blocking these K+ currents in patch-clamped cells from all aortic preparations, suggesting that the lack of TEA-induced contractions in WKY and DS aortas was not due to an absence of TEA-sensitive K+ channels in these arterial membranes. However, when the Ca2+ ionophore A23187 (10 microM) was used to increase the level of cytosolic Ca2+ in patch-clamped cells, the K+ current density in SHR and PS aortic cells was twofold or more higher than in WKY and DS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Functional differentiation of ganglion cells from multipotent progenitor cells in sliced retina of adult goldfish.

Multipotent progenitor cells at the retinal margin of adult goldfish give rise to all cell types in the rest of the retina. We took advantage of this spatial arrangement of progenitor and mature cells in slices of peripheral retina, to investigate the appearance and maturation of voltage-activated Na(+) current. We divided the peripheral retina into three broad regions (marginal, intermediate, and mature) on the basis of their morphological development. Whole-cell patch-clamp recordings were performed in ruptured-patch mode, so that cells from which currents were recorded could be identified by Lucifer Yellow fills. No voltage-activated Na(+) current was detected in the slender, peripherally located marginal cells. Voltage-activated Na(+) currents were detected in rounded cells found alongside or near marginal cells, facing the vitreal side of the retina. Some of these "intermediate cells" had a long axon-like process which ran along the vitreal surface. Intermediate cells adjacent to the marginal region tended to have smaller Na(+) currents than intermediate cells closer to the mature region. On average, the maximum Na(+) current amplitude recorded from intermediate cells was roughly 6-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current in intermediate cells was nearly 14 mV more positive than that of mature ganglion cells. The results indicate that voltage-activated Na(+) current, as a possible marker of retinal ganglion cells, begins to develop well before these cells migrate to their adult position within the retina.

Animals↗