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PubMed1998

Inhibition of calcium currents in rat colon sensory neurons by K- but not mu- or delta-opioids.

Inhibition of calcium currents in rat colon sensory neurons by kappa- but not mu- or delta-opioids. J. Neurophysiol. 80: 3112-3119, 1998. We previously reported that kappa-, but not mu- or delta-opioid receptor agonists (ORAs) have selective, potentially useful peripheral analgesic effects in visceral pain. To evaluate one potential site and mechanism by which these effects are produced, we studied opioid effects on high-voltage activated (HVA) Ca2+ currents in identified (Di-I) pelvic nerve sensory neurons from the S1 dorsal root ganglion (DRG). Results were compared with opioid effects on cutaneous neurons from L5 or L6 DRG. Di-I-labeled DRG cells were voltage clamped (perforated whole cell patch clamp), and HVA Ca2+ currents were evoked by depolarizing 240-ms test pulses to +10 mV from a holding potential of -60 mV. Neither mu-ORAs (morphine, 10(-6 )M, n = 16; [D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin, 10(-6 )M, n = 12) nor delta-ORAs ([D-Pen2, D-Pen5] enkephalin, 10(-7 )M, n = 16; SNC-80, 10(-7 )M, n = 7) affected HVA Ca2+ currents in colon sensory neurons. In contrast, the kappa-ORAs U50, 488 (10(-6 )M), bremazocine (10(-6)M), and nalBzoH (10(-6 )M) significantly attenuated HVA Ca2+ currents in colon sensory neurons; effects on cutaneous sensory neurons were variable. A nonreceptor selective concentration of naloxone (10(-5 )M) and nor-BNI (10(-6 )M), a selective kappa-opioid receptor antagonist, reversed the inhibitory effect of kappa-ORAs. In the presence of N-, P-, or Q-, but not L-type Ca2+ channel antagonists, the effect of U50,488 on HVA Ca2+ currents was significantly reduced. Pretreatment with pertussis toxin (PTX) prevented the inhibition by U50,488. These results suggest that kappa-opioid receptors are coupled to multiple HVA Ca2+ channels in colon sensory neurons by a PTX-sensitive G protein pathway. We conclude that inhibition of Ca2+ channel function likely contributes in part to the peripheral analgesic action of kappa-ORAs in visceral nociception.

Animals↗

PubMed2000

Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels.

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.

Animals↗

PubMed1997

Comparison between functional characteristics of healthy and pathological human retinal Müller glial cells.

Enzymatically isolated, noncultured Müller glial cells from human organ donor and patient eyes were studied using the whole-cell-voltage-clamp and the patch-clamp technique. The patients suffered mainly from choroidal melanomas, retinal detachment due to proliferative vitreorentinopathy, glaucomas, and perforating eye injuries. The organ donor eyes were used as a source of corneas for corneal transplantation. Use of the human retinal tissue was approved by the Ethics Committee of the School of Medicine, University of Leipzig. Most of the patient Müller cells showed a marked or complete loss of inwardly rectifying K+ currents, causing a dramatic increase in the input resistance. The zero current potential of the patient Müller cells, which is equivalent to the membrane potential, was significantly reduced (depolarized) as compared with the donor Müller cells. In contrast to the K+ current loss, the Na+ current density was significantly higher in patient Müller cells than in donor Müller cells; the number of Müller cells depicting Na+ currents increased from 33% (3 pA/pF) in donor Müller cells to 85% (about 12pA/pF) in patient Müller cells. Application of glutamate to the Müller cells generated a glutamate-transporter-mediated current, such as that seen in other species. A highly significant increase was noted for the high-affinity Na+-dependent glutamate-transporter-current density in patient Müller cells compared with donor cells. The application of gamma-aminobutyric acid (GABA) evoked, in addition to the GABA transporter currents already known from Müller cells of other mammalian species, GABA(A)-receptor mediated currents in human Müller cells. We found that GABA(A) receptors are expressed in human Müller cells, but not in other nonprimate mammals. Whether a difference exists between the GABA(A) current density in donor and patient Müller cells remains to be seen. The results concerning the disappearance of K+ currents and diminution of the membrane potential may demonstrate early glial changes that may possibly precede pathological neuronal changes, at least in retinas from eyes with choroidal melanomas. In later stages of the diseases, the glial changes may be deleterious for the neurons, because they could diminish glutamate uptake due to the depolarized membrane potential. However, increased extracellular glutamate concentration is toxic for most neurons.

Adolescent↗

PubMed2000

Calpastatin domain L is involved in the regulation of L-type Ca2+ channels in guinea pig cardiac myocytes.

We have found previously that L-type Ca2+ channel run-down in cell-free patches is partially (10-28%) reversed by calpastatin (CS) and have suggested that CS, an endogenous inhibitor of calpain, has a Ca2+-channel-regulating function. CS is composed of repetitive domains 1-4 (calpain-inhibitory domain) and domain L (a domain whose function is unknown). We therefore investigated which domain of CS was involved in the regulation of Ca2+ channel activity in guinea pig cardiac myocytes using the patch-clamp technique. After the patches were excised into inside-out mode in basic internal solution, the Ca2+ channel activity ran down to 0.45% of the control level recorded in the cell-attached mode. Application of human recombinant full-length CS (25 microM) and domain L (25 microM) restored the Ca2+ channel activity to 13 and 19% of the control level, respectively, while the channel activity was not restored by CS domain 1 (25 microM) (0.66%). Mouse CS domain XLL (25 microM), a complex of domain XL and domain L, restored the calcium channel activity to 11% of the control level. These results suggested that the Ca2+ channel-regulating function of CS is located in domain L. This study is the first description of the function of CS domain L.

Animals↗

PubMed1998

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres.

We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)2 the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of -0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca2+)/P(K+)=2.4, P(Ca2+)/ P(Na+)=2.7, P(Ca2+)/P(Tris+)=3.1, P(Ca2+)/P(Mg2+)=1.0 and P(Ca2+)/P(Ba2+)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)2 solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)2 solution. The channel activity in symmetrical 50 mM Ca(gluconate)2 solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 microM). The results are in agreement with the conclusion that the sarcoball Ca2+ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca2+-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel.

Animals↗

PubMed2002

Antimalarial drugs inhibit the acetylcholine-receptor-operated potassium current in atrial myocytes.

BACKGROUND: It has been reported that halofantrine, an antimalarial drug, was associated with electrocardiographic prolongation of the QT interval and ventricular arrhythmias. Inhibition of the delayed rectifier potassium channel, a voltage-gated potassium channel, by halofantrine was the likely underlying cellular mechanism for this cardiotoxicity. However, influences of anti-malarial drugs on the ligand-gated potassium channels have not been well-documented. The influences of three different antimalarial drugs, chloroquine, primaquine and pyrimethamine, on the acetylcholine-receptor-operated potassium current (I(K.ACh)), a ligand-gated potassium current, were compared with the effect of quinidine in isolated guinea pig atrial myocytes using patch-clamp techniques. METHODS: The whole-cell patch-clamp method was used in the present studies he I(K.ACh) was induced by extracellular application of carbachol (1 micromol/L) or intracellular loading of guanosine 5'-O-(3-thiotriphosphate) GTPgammaS (100 micromol/L) in acutely isolated guinea pig atrial myocytes. RESULTS: The I(K.ACh) induced by carbachol was inhibited by chloroquine, primaquine, pyrimethamine and quinidine in a concentration-dependent manner, and the concentrations required to produce 50% of the maximal inhibitory effect (IC(50) values) were 0.7, 2.5, 12 and 1.8 micromol/L, respectively. These drugs also inhibited the intracellular GTPgammaS-activated I(K.ACh), and the IC(50) values were 0.8,13,19 and 21 micromol/L, respectively. CONCLUSIONS: Chloroquine and pyrimethamine may inhibit I(K.ACh) by interacting with the muscarinic potassium channel itself and/or associated guanosine 5'-triphosphate-binding proteins, whereas primaquine and quinidine may mainly inhibit the current by the blockade of the muscarinic receptors. These results indicate that antimalarial drugs exert anticholinergic effects via different molecular mechanisms.

Journal Article↗

PubMed1999

Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels.

1. In order to investigate the mechanism underlying MgATP-dependent recovery of ATP-sensitive potassium (KATP) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K+ channel subunit/sulfonylurea receptor) or C-terminal-truncated Kir6.2 (Kir6.2DeltaC26) in COS7 cells (Green monkey kidney cells), and carried out inside-out patch clamp experiments. 2. After patch excision in ATP-free internal solution, the activity of Kir6.2/SUR2A channels could be maximally recovered by the application of 5 mM MgATP. Subsequent application of 100 microM Ca2+ induced a rapid decay of Kir6.2/SUR2A activity to 11.6 +/- 1.1 % (mean +/- s.e.m.) of the control level (Ca2+-induced run-down; n = 64). 3. MgATP (5 mM) recovered 99.4 +/- 4.2 % (n = 13) of the Ca2+-induced run-down. Protein kinase inhibitors such as W-7, H-7, H-8 and genistein did not inhibit this reaction. However, wortmannin, an inhibitor of phosphatidylinositol 3- and 4-kinases, blocked the MgATP-dependent recovery in a concentration-dependent manner; the magnitudes of recovery were 35.7 +/- 7.2 % (10 microM) and 4.3 +/- 2.5 % (100 microM) of the Ca2+-induced run-down. 4. MgUDP (10 mM) reversed the Ca2+-induced run-down of Kir6.2/SUR2A channels by 60.4 +/- 7.6 % (n = 5). Wortmannin failed to modify this reaction. 5. Kir6.2DeltaC26 channels, which opened in the absence of SUR2A, were less sensitive to Ca2+; Kir6.2DeltaC26 channels were inactivated to 44.8 +/- 4.4 % (n = 14) by 100 microM Ca2+. MgATP recovered the Ca2+-induced run-down of Kir6.2DeltaC26 by 89.8 +/- 7. 7 % (n = 9), and 100 microM wortmannin inhibited this reaction (1.8 +/- 2 %, n = 7). 6. Application of 10 microM phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) recovered the activity of Kir6.2/SUR2A channels after Ca2+-induced run-down (104.3 +/- 6.4 %, n = 10). Even after the MgATP-dependent recovery was blocked by 100 microM wortmannin, PI-4,5-P2 reactivated the channels (102.3 +/- 8.6 %, n = 5). Similar results were obtained with Kir6.2DeltaC26. 7. These results suggest that the entity of MgATP-dependent recovery may be membrane lipid phosphorylation rather than protein phosphorylation, and that synthesis of PI-4,5-P2 or phosphatidylinositol-3,4, 5-trisphosphate may upregulate Kir6.2 channels.

ATP-Binding Cassette Transporters↗

PubMed2006

Relationships between calcium and pH in the regulation of the slow afterhyperpolarization in cultured rat hippocampal neurons.

The Ca(2+)-dependent slow afterhyperpolarization (AHP) is an important determinant of neuronal excitability. Although it is established that modest changes in extracellular pH (pH(o)) modulate the slow AHP, the relative contributions of changes in the priming Ca(2+) signal and intracellular pH (pH(i)) to this effect remain poorly defined. To gain a better understanding of the modulation of the slow AHP by changes in pH(o), we performed simultaneous recordings of intracellular free calcium concentration ([Ca(2+)](i)), pH(i), and the slow AHP in cultured rat hippocampal neurons coloaded with the Ca(2+)- and pH-sensitive fluorophores fura-2 and SNARF-5F, respectively, and whole cell patch-clamped using the perforated patch technique. Decreasing pH(o) from 7.2 to 6.5 lowered pH(i), reduced the magnitude of depolarization-evoked [Ca(2+)](i) transients, and inhibited the subsequent slow AHP; opposite effects were observed when pH(o) was increased from 7.2 to 7.5. Although decreases and increases in pH(i) (at a constant pH(o)) reduced and augmented, respectively, the slow AHP in the absence of marked changes in preceding [Ca(2+)](i) transients, the inhibition of the slow AHP by decreases in pH(o) was correlated with low pH(o)-dependent reductions in [Ca(2+)](i) transients rather than the decreases in pH(i) that accompanied the decreases in pH(o). In contrast, high pH(o)-induced increases in the slow AHP were correlated with the accompanying increases in pH(i) rather than high pH(o)-dependent increases in [Ca(2+)](i) transients. The results indicate that changes in pH(o) modulate the slow AHP in a manner that depends on the direction of the pH(o) change and substantiate a role for changes in pH(i) in modulating the slow AHP during changes in pH(o).

Action Potentials↗

PubMed1998

GABAA currents in immature dentate gyrus granule cells.

We used whole cell patch clamp and gramicidin perforated patch recordings in hippocampal slices to study gamma-aminobutyric acid (GABA) currents in granule cells (GCs) from juvenile rat dentate gyrus (DG). GCs are generated postnatally and asynchronously such that they can be detected at different stages of their maturation in DG within the first month. In contrast, inhibitory interneurons are generated embryonically, and their circuitry is well developed even as their target GCs and GC excitatory connections are still being formed. In this study, two GABA currents evoked in GCs by medial perforant path stimulation are compared. The first, pharmacologically isolated by glutamate receptor blockade, is the product of direct activation of GABA interneurons with monosynaptic input to the recorded GC (monosynaptic GABAA). Monosynaptic GABAA displays slight outward rectification of its current-voltage relation, is 97% eliminated by 10 microM bicuculline and coincides temporally with the excitatory components of GC postsynaptic currents as has been described for GABAA currents in other brain regions. The second is a novel GABA response that is detectable in 10 microM bicuculline and is present on GCs only at the earliest stages of their maturation. Unlike monosynaptic GABAA, this transient GABA is eliminated by glutamate receptor blockade and hence is likely to be generated by interneurons activated via an intervening glutamatergic synapse (polysynaptically). It is predominantly chloride mediated, has a relative bicarbonate/chloride permeability ratio of 26%, and is unchanged by bath-applied saclofen and strychnine or by intracellular calcium chelation. It is 97% antagonized by 100 microM picrotoxin and 99% antagonized by 100 microM bicuculline. This current is thus a relatively bicuculline (BMI)-resistant GABAA current (BMIR-GABAA). Compared with monosynaptic GABAA, BMIR-GABAA has a later peak, slower time course of decay, and marked outward rectification. Its reversal potential is 7-8 mV depolarized to that of monosynaptic GABAA whether recorded in whole cell or with gramicidin perforated patch to preserve native internal chloride concentration. Together these data may suggest that BMIR-GABAA is evoked by an anatomically segregated population of interneurons activating a unique, developmentally regulated GABAA receptor. Further, the transient nature of this current coupled with its temporal characteristics that preclude overlap with the excitatory components of the synaptic response are consistent with a role that is trophic or signaling rather than primarily inhibitory.

Animals↗

PubMed2005

Intracellular and extracellular concentrations of Na+ modulate Mg2+ transport in rat ventricular myocytes.

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25 degrees C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24 mM Mg2+ either in the presence of 106 mM Na+ plus 1 mM ouabain (Na+ loading) or in the presence of only 1.6 mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140 mM Na+ and 1 mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31 mM and 47 mM after Na+ loading for 1 and 3 h, respectively, and decreased to approximately 0 mM after 3 h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at approximately 40 mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130 mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.

Amphotericin B↗

PubMed1997

Enzymatic isolation and characterization of single vascular smooth muscle cells from cremasteric arterioles.

OBJECTIVE: The goal of the present study was to develop a method to isolate viable arteriolar muscle cells from single cremasteric arterioles, which retain the contractile and electrophysiological phenotype of the donor microvessels. METHODS: Arterioles were hand-dissected from rat and hamster cremaster muscles and dissociated by incubation in papain and dithioerythritol for 35 min followed by incubation in collagenase, elastase, and soybean trypsin inhibitor for 10 to 25 min in solutions containing 100 microM Ca2+. 10 microM sodium nitroprusside, and 1 mg/ml albumin at 37 degrees C. RESULTS: Populations of single smooth muscle cells enzymatically isolated from cremasteric arterioles showed elongated fusiform morphology and intact plasmalemmal membranes as indicated by retention of calcein, by exclusion of ethidium homodimer-1 and by high membrane resistances (11 +/- 0.8 C omega, n = 36 for rat cells; 8 +/- 0.6 C omega, n = 21 for hamster cells: p < 0.05). Muscle cells contracted in a concentration-dependent fashion in response to pipette application of norepinephrine (10 nM-100 microM). Cell shortening in response to 1 microM norepinephrine was inhibited by 10 microM phentolamine, 1 microM sodium nitroprusside, and 1 microM nifedipine or nominally Ca(2+)-free media. Resting membrane potential recorded in patch-clamped cells by perforated patch methods was -48 +/- 1 mV (n = 47) for rat cells and -44 +/- 2.8 mV (n = 14) for hamster cells (p > 0.05). Families of voltage-dependent K+ currents were observed during stepwise depolarizing pulses from -60 mV to more positive potentials. Blockers of voltage-gated and ATP-sensitive K+ channels (4-aminopyridine [3 mM] and glibenclamide [1 microM], respectively) inhibited membrane K+ conductance, increased membrane resistance, and depolarized cells by 20 +/- 4 mV (n = 8) and 14 +/- 3 mV (n = 6), respectively. CONCLUSIONS: The present method permits isolation of smooth muscle cells from a single cremasteric arteriole. These cells seem to retain the contractile phenotype, alpha-adrenergic signaling cascade, membrane potential, and K+ conductances described for the donor arteriole. Correlating the functional and electrophysiological properties of these smooth muscle cells to in situ and in vitro studies of their donor arterioles should provide a useful extension for understanding the physiology, pathophysiology, biophysics, and cell biology of the microcirculation in skeletal muscle.

Animals↗

PubMed2000

Large-conductance calcium-activated anion channel characteristics in neuroblastoma cells.

Large-conductance anion channel characteristics were investigated in neuroblastoma cells (N2A) by using different configurations of the patch-clamp technique. In excised patches, the channel was induced by depolarising potentials in 90% of experiments, had a conductance of 340 pS in symmetrical 135 mmol/l NaCl and exhibited the typical bell-shape activity. Neither the channel induction nor the channel activity was affected by rising the Ca2+ concentration on the cytopasmic side of membranes. In cell-attached configuration the maximal channel activity was shifted towards more positive potentials in comparison to that of excised patches and an increase in intracellular Ca2+, obtained by extracellular application of the Ca2+-ionophore A23187 in the presence of 0.2 micromol/l Ca2+, induced single-channel currents in 80% of patches compared to 31% of cell-attached experiments showing channel activity in normal conditions. In turn, application of 2 micromol/l Ca2+ induced channel activity in 100% of patches. The reversal potential of the channel in cell-attached patches was around -10 mV as the resting potential of cells eliciting channel activity. For cells where channel activity was not detected in cell-attached mode, the resting potential was around -45 mV. Channel activity could be restored in most whole-cell recordings in the presence of 2 micromol/l or more intracellular Ca2+ concentrations. The Ca2+-induction and the relation between channel activity and cell resting potential seem to suggest a role of the large-conductance anion channel in resting potential modulation during some basic functions of the neuroblastoma cell proliferation.

Animals↗

PubMed2002

[Low conductivity cationic channel in intact endothelium of the rat aorta].

A new low-conductance cationic channel has been recorded with the patch-clamp technique in excised patches of the luminal membrane of intact endothelial cells. The channel had conductance of 5.4 pS and the lifetime of 46 ms. It was equally permeable for Na+ and K+, but impermeable to Cl-. The lifetime and probability of the open state of the channel were independent of the membrane potential.

Animals↗

PubMed2003

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

Animals↗

PubMed2006

Inhibition by alpha-tetrahydrodeoxycorticosterone (THDOC) of pre-sympathetic parvocellular neurones in the paraventricular nucleus of rat hypothalamus.

BACKGROUND AND PURPOSE: alpha-tetrahydrodeoxycorticosterone (THDOC) is an endogenous neuroactive steroid which increases in plasma and brain concentration during stress. It has both positive and negative modulatory effects on GABA activated GABAA currents, dependent upon the dose. We investigated the effects of THDOC on spinally-projecting "pre-sympathetic" neurones in the parvocellular subnucleus of the hypothalamic paraventricular nucleus (PVN), to determine whether it activates or inhibits these neurones, and by what mechanism. EXPERIMENTAL APPROACH: Rat spinally-projecting (parvocellular) PVN neurones were identified by retrograde labelling and the action of THDOC investigated with three modes of patch-clamp: cell-attached action current, whole-cell voltage-clamp and cell-attached single-channel recording. KEY RESULTS: In cell-attached patch mode, parvocellular neurones fired action potentials spontaneously with an average frequency of 3.6 +/- 1.1 Hz. Bath application of THDOC reduced this with an EC50 of 67 nM (95% confidence limits: 54 to 84 nM), Hill coefficient 0.8 +/- 0.04, n = 5. In whole-cell patch-clamp mode, pressure ejection of GABA evoked inward currents. These were clearly GABAA currents, since they were inhibited by the GABAA receptor antagonist bicuculline, and reversed near the chloride equilibrium potential. THDOC significantly potentiated GABAA currents (1 microM THDOC: 148 +/- 15% of control, n = 5, p < or = 0.05, ANOVA). Single-channel analysis showed no differences in conductance or corrected mean open times in the presence of 1 microM THDOC. CONCLUSIONS AND IMPLICATIONS: THDOC inhibited parvocellular neuronal activity without showing any evidence of the bidirectional activity demonstrated previously with cultured hypothalamic neurones. Our data are consistent with the hypothesis that THDOC acts by potentiating the post-synaptic activity of endogenously released GABA.

Action Potentials↗

PubMed1999

ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation.

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

Action potential duration restitution and alternans in rabbit ventricular myocytes: the key role of intracellular calcium cycling.

Action potential duration (APD) restitution properties and repolarization alternans are thought to be important arrhythmogenic factors. We investigated the role of intracellular calcium (Ca2+i) cycling in regulating APD restitution slope and repolarization (APD) alternans in patch-clamped rabbit ventricular myocytes at 34 to 36 degrees C, using the perforated or ruptured patch clamp techniques with Fura-2-AM to record Ca2+i. When APD restitution was measured by either the standard extrastimulus (S1S2) method or the dynamic rapid pacing method, the maximum APD restitution slope exceeded 1 by both methods, but was more shallow with the dynamic method. These differences were associated with greater Ca2+i accumulation during dynamic pacing. The onset of APD alternans occurred at diastolic intervals at which the APD restitution slope was significantly <1 and was abolished by suppressing sarcoplasmic reticulum (SR) Ca2+i cycling with thapsigargin and ryanodine, or buffering the global Ca2+i transient with BAPTA-AM or BAPTA. Thapsigargin and ryanodine flattened APD restitution slope to <1 when measured by the dynamic method, but not by the S1S2 method. BAPTA-AM or BAPTA failed to flatten APD restitution slope to <1 by either method. In conclusion, APD alternans requires intact Ca2+i cycling and is not reliably predicted by APD restitution slope when Ca2+i cycling is suppressed. Ca2+i cycling may contribute to differences between APD restitution curves measured by S1S2 versus dynamic pacing protocols by inducing short-term memory effects related to pacing-dependent Ca2+i accumulation.

Action Potentials↗

PubMed2006

Effects of ethanol on tonic GABA currents in cerebellar granule cells and mammalian cells recombinantly expressing GABA(A) receptors.

The effects of ethanol on the GABA(A) receptors, which are regarded as one of the most important target sites of ethanol, are very controversial, ranging from potentiation to no effect. The delta subunit-containing GABA(A) receptors expressed in Xenopus oocytes were recently reported to be potently augmented by ethanol. We performed patch-clamp experiments using the cerebellar granule cells and mammalian cells expressing recombinant GABA(A) receptors. In granule cells, the sensitivity to GABA increased from 7 to 11 days in vitro. Furosemide, an antagonist of alpha6-containing GABA(A) receptors, inhibited GABA-induced currents more potently at 11 to 14 days than at 7 days. Ethanol at 30 mM had either no effect or an inhibitory effect on currents induced by low concentrations of GABA in granule cells. On alpha4beta2delta, alpha6beta2delta, or alpha6beta3deltaGABA(A) receptors expressed in Chinese hamster ovary cells, ethanol at 10, 30, and 100 mM had either no effect or an inhibitory effect on GABA currents. Ethanol inhibition of GABA(A) receptor was observed in all of the subunit combinations examined. In contrast, the perforated patch-clamp method to record the GABA currents revealed ethanol effects on the alpha6beta2delta subunits ranging from slight potentiation to slight inhibition. Ethanol seems to exert a dual action on the GABA(A) receptors and the potentiating action may depend on intracellular milieu. Thus, the differences between the GABA(A) receptors expressed in mammalian host cells and those in Xenopus oocytes in the response to ethanol might be due to changes in intracellular components under patch-clamp conditions.

Animals↗