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Results for “patch clamp”

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PubMed2000

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart.

OBJECTIVE: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. METHODS: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. RESULTS: The membrane potential of the cells was -48.9+/-9.6 mV (mean+/-S.D.) with 5 mM and -23.2+/-2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2+/-123.2 pF. The current-voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 microM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9+/-2.8 nS. Inward rectification of whole-cell currents was steep (slope factor = 6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant = 12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor = 4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant = 0.1 mM). The maximum slope conductance at depolarized potentials was 16.6+/-15.6 nS. CONCLUSION: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.

Animals↗

PubMed2003

Inhibition of human TREK-1 channels by bupivacaine.

UNLABELLED: Human TWIK-related K(+) channels (TREK-1) stabilize the membrane potential (mp) of neurons and have a major role in the regulation of membrane excitability. In view of their physiological significance, interaction of bupivacaine with TREK-1 channels may be clinically important. Our aim was to characterize with the patch-clamp technique the properties of human TREK-1 channels and the effects of bupivacaine on these channels expressed in Chinese hamster ovary (CHO) cells. Transfection of CHO cells with TREK-1 channels (CHO(TREK-1) cells) hyperpolarized the mp from -33 +/- 13 to -78 +/- 4 mV. The channels were stimulated by intracellular acidosis. Inhibition of TREK-1 channels by bupivacaine was reversible, concentration-dependent, voltage-independent, and increased with intracellular acidosis. Bupivacaine depolarized the mp of CHO(TREK-1) cells in a reversible and concentration-dependent manner. Concentrations for channel inhibition and membrane depolarization were not linearly related (50% inhibitory concentration value for channel inhibition 370 +/- 20 micro M, Hill coefficient 1.8 +/- 0.1, n = 51; 50% inhibitory concentration value for membrane depolarization 856 +/- 14 micro M, Hill coefficient 2.4 +/- 0.1, mean +/- SEM, n = 27). The results suggest that protonated bupivacaine elicits the observed effects via a site of interaction accessible from the intracellular space. Inhibition of TREK-1 channels and consecutive depolarization of the cell membrane by bupivacaine may contribute to blockade of neuronal signal conduction during regional anesthesia. IMPLICATIONS: The interaction of bupivacaine with human TREK-1 channels was studied with the patch-clamp technique. Bupivacaine inhibited TREK-1 channels and depolarized the membrane potential of cells expressing TREK-1 channels in a concentration-dependent and reversible manner. Both effects may contribute to conductance block caused by bupivacaine.

Acidosis↗

PubMed2002

The actions of propofol on gamma-aminobutyric acid-A and glycine receptors in acutely dissociated spinal dorsal horn neurons of the rat.

UNLABELLED: The spinal cord plays an important role in modulating anesthetic-induced suppression of nociceptive transmission. To gain some insight into the anesthetic mechanisms of propofol at the spinal level, we investigated the direct action of propofol and its modulation on the gamma-aminobutyric acid-A receptor (GABA(A)R) and the glycine receptor (GlyR) in acutely dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp electrophysiology. Propofol induced Cl(-) currents (I(Cl)), which were sensitive to bicuculline and, to a lesser extent, to strychnine. The activation, desensitization, and deactivation of propofol-induced I(Cl) were slower than those of GABA- and glycine-induced I(Cl). In addition, this study revealed similar modulatory actions of propofol on GABA(A)R and GlyR. Propofol potentiated both GABA- and glycine-induced I(Cl) at small con-centrations and inhibited both GABA- and glycine-induced I(Cl) at large concentrations. The potentiation of propofol on I(Cl) was caused by slowing current desensitization and deactivation, whereas the inhibition actions might be involved in the cross-desensitization between GABA- and propofol-induced I(Cl) and the cross-inhibition between the GABA(A)R and GlyR. The results suggest that propofol facilitation of GABA(A)R and GlyR at the spinal level could contribute significantly to general anesthetic-induced analgesia and anesthesia. IMPLICATIONS: The actions of propofol on the gamma-aminobutyric acid-A receptor (GABA(A)R) and the glycine receptor (GlyR) were investigated in acutely dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp electrophysiology. Propofol was found to potentiate the functions of GABA(A)R and GlyR at the spinal level, which might contribute to propofol-induced analgesia and anesthesia.

Anesthetics, Intravenous↗

PubMed1999

Actin filaments and the opposing actions of CaM kinase II and calcineurin in regulating alpha7-containing nicotinic receptors on chick ciliary ganglion neurons.

Nicotinic acetylcholine receptors containing alpha7 subunits have a high relative permeability to calcium and influence numerous calcium-dependent cellular events. On chick ciliary ganglion neurons the receptors are concentrated on somatic spines containing actin filaments. Using conventional whole-cell patch-clamp recording from dissociated ciliary ganglion neurons, we show that responses from alpha7-containing receptors undergo substantial rundown when the receptors are repeatedly challenged with nicotine. Stabilization of actin filaments with phalloidin partially prevents the rundown, whereas collapse of actin filaments with latrunculin A exacerbates it. The rundown depends on calcium influx through the receptors because it requires receptor activation and can be prevented by replacing extracellular calcium with barium or by intracellular dialysis with BAPTA. Thapsigargin and ryanodine each inhibit the rundown, demonstrating further a requirement for calcium release from internal stores. Blockade of calmodulin by calmidazolium or blockade of CaM kinase II with either KN93 or autocamtide-2-related inhibitory peptide each prevents the rundown; blockade of the phosphatase calcineurin with either cyclosporin A or deltamethrin increases the rundown. The results indicate a balance of calcium-dependent kinase and phosphatase activities in regulating the function of alpha7-containing receptors. Manifestation of the rundown depends in part on the loss of intracellular components via dialysis because little rundown is seen if perforated patch-clamp recording is used to monitor receptor responses even in latrunculin A-treated cells. A membrane-permeable calcineurin inhibitor, however, still decreases the nicotinic response in a calcium-dependent manner, confirming that calcium-dependent phosphoregulation of alpha7-containing receptors occurs in the intact cell.

Actins↗

PubMed2003

P2 receptors in satellite glial cells in trigeminal ganglia of mice.

There is strong evidence for the presence of nucleotide (P2) receptors in sensory neurons, which might play a role in the transmission of pain signals. In contrast, virtually nothing is known about P2 receptors in satellite glial cells (SGCs), which are the main glial cells in sensory ganglia. We investigated the possibility that P2 receptors exist in SGCs in murine trigeminal ganglia, using Ca(2+) imaging, patch-clamp recordings, and immunohistochemistry. We found that ATP caused an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in SGCs. As adenosine had no effect on [Ca(2+)](i), and the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid largely blocked the response to ATP we conclude that P1 receptors did not contribute to the responses. We obtained the following evidence that the responses to ATP were mediated by metabotropic P2Y receptors: (i) persistence of the responses in Ca(2+)-free solution, (ii) inhibition of the response by cyclopiazonic acid, (iii) [Ca(2+)](i) increases in response to the P2Y agonists uridine triphosphate, adenosine thiodiphosphate, and 2-methylthio ADP, and (iv) failure of the P2X agonist alpha,beta-methylene ATP to elicit a response. Agonists of P2Y(1) receptors and uridine triphosphate, an agonist at P2Y(2) and P2Y(4) receptors, induced [Ca(2+)](i) increases suggesting that at least these P2Y receptor subtypes are present on SGCs. Using an antibody against the P2Y(4) receptor, we found immunopositive SGCs. Patch-clamp recordings of SGCs did not reveal any inward current due to ATP. Therefore, there was no evidence for the activation of ionotropic P2X receptors under the present conditions. The results indicate the presence of functional nucleotide (P2Y) receptors in SGCs.

Adenosine Triphosphate↗

PubMed1995

Involvement of the Na,K-ATPase in the induction of ion channels by palytoxin.

The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 microM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch-clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH4+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 microM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na,K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin.

Acrylamides↗

PubMed1998

Non-specificity of chloride channel blockers in rat cerebral arteries: block of the L-type calcium channel.

1. The effects of chloride channel blockers on pressure-induced constriction, K(+)-induced force, and whole-cell calcium channel currents were tested in rat cerebral arteries using isobaric and isometric myography, and patch clamp. 2. Under isobaric conditions at 75 mmHg, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a chloride channel blocker, reversibly depressed the myogenic constriction with an IC50 of 32.8 +/- 0.52 microM (mean +/- S.E.M., n = 5). Blockers of Ca(2+)-activated chloride channels, flufenamic acid (100 microM) and 9-anthracene chloride (9-AC; 1 mM), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel blocker, glibenclamide (100 microM), were without effect in this tissue (n = 3). 3. Under isobaric conditions at 20 mmHg, 37 degrees C, raising [K+]o to 45 mM induced a constriction which was unaffected by 100 microM NPPB (n = 4). In contrast, at 75 mmHg and 18-21 degrees C, 100 microM NPPB completely and reversibly blocked a 45 mM K(+)-induced constriction (n = 3). 4. Under isometric conditions, NPPB reversibly depressed a 45 mM K(+)-induced force with an IC50 of 10.0 +/- 0.76 microM (mean +/- S.E.M., n = 5). Indanyloxyacetic acid 94 (IAA-94), another chloride channel blocker, depressed the K(+)-induced force with an IC50 of 17.0 +/- 1.2 microM (mean +/- S.E.M., n = 4). 5. Using whole-cell patch clamp, 100 microM NPPB or 200 microM IAA-94 blocked calcium channel currents carried by 10 mM Ba2+ by 79.1 +/- 1.7 and 39.8 +/- 7.0%, respectively (mean +/- S.E.M., n = 6). 6. In summary, chloride channel blockers depress calcium channel currents in rat cerebral arteries, which could contribute to a reduction in myogenic contraction.

Animals↗

PubMed1998

Newborn rat brainstem preparation with the trigeminal nerve attached for pain study.

Studies using a brainstem-spinal cord preparation isolated from newborn rats have provided substantial information on neuro-physiology, -pharmacology and -anatomy of the respiratory center, such as mechanisms of respiratory rhythm generation, development of a respiratory center or respiratory reflex [T. Murakoshi, T. Suzue, S. Tamai, A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation from newborn rat, Br. J. Pharmac. 86 (1985) 95-104 [5]; H. Onimaru, A. Arata, I. Homma, Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparations from newborn rats, Brain Res. 445 (1988) 314-324 [6]; H. Onimaru, I. Homma, Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats, Pflügers Arch. 420 (1992) 399-406 [7]; J.C. Smith, K. Ballanyi, D.W. Richter, Whole-cell patch clamp recordings from respiratory neurons in neonatal rat brainstem in vitro, Neurosci. Lett. 314 (1992) 153-156 [10]; T. Suzue, Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation of the neonatal rat, J. Physiol. (London) 354 (1984) 173-183 [11], reviewed in H. Onimaru, A. Arata, I. Homma, Neuronal mechanisms of respiratory rhythm generation: An approach using in vitro preparation, Jpn. J. Physiol. 47 (1997) 385-403 [8]]. Recently, the dissecting method of the preparation was modified to introduce a brainstem preparation with the trigeminal primary afferents attached for pain studies [M. Hamba, Repetitive stimulation potentiated the stimulus-evoked firing in the trigeminal caudalis-in vitro study. Neurosci. Res. 20 (1996) s163 [2]; M. Hamba, Stimulation-induced responses of the trigeminal caudal neurons in the brainstem preparation isolated from newborn rats, Brain Res. 785 (1998) 66-74 [3]]. As reported previously [3], the activity-dependent change in the excitability of pain-processing neurons, wind-up phenomenon, was studied in the trigeminal caudalis by stimulating the mandibular nerve, using a modified brainstem preparation isolated from newborn rats. The caudalis, the medulla dorsal horn, is known as the center for processing pain and sensory information from the cranio-facial area. The results indicated that the brainstem preparation is applicable for studies on the neuroplasticity at the pain-processing synapses. Here, we describe the method for isolation of a brainstem preparation with the trigeminal mandibular nerve attached and for recording the synaptic response evoked in the caudal neurons, using a whole-cell patch clamp technique. In the present study, we demonstrated repetitive stimulation-induced responses of caudal neurons at postnatal day 1 as an example showing the feasibility of the preparation for pain studies.

Animals↗

PubMed2001

Renal proximal tubule function is preserved in Cftr(tm2cam) deltaF508 cystic fibrosis mice.

1. Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftr(tm2cam)deltaF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT-PCR and whole-cell patch clamping. 2. Renal Na(+) clearance was similar in wild-type (1.4 +/- 0.3 microl min(-1), number of animals, N = 12) and CF mice (1.6 +/- 0.4 microl min(-1), N = 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild-type (7.0 +/- 0.8 microl min(-1), N = 8) and CF mice (9.3 +/- 1.4 microl min(-1), N = 9); no difference between genotypes was observed. 3. In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild-type (2.2 +/- 0.4 nl mm(-1) min(-1), n = 10) and CF mice (1.9 +/- 0.3 nl mm(-1) min(-1), n = 11). Addition of a forskolin-dibutyryl cAMP (db-cAMP) cocktail to the perfusate caused no significant change in Jv in either wild-type (2.6 +/- 0.7 nl mm(-1) min(-1), n = 10) or Cftr(tm2cam)deltaF508 mice (2.0 +/- 0.5 nl mm(-1) min(-1), n = 10). 4. CFTR expression was confirmed in samples of outer cortex using RT-PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin-db-cAMP using whole-cell patch clamping. 5. In conclusion, no functional deficit in proximal tubule function was found in Cftr(tm2cam)deltaF508 mice. This may be a consequence of a lack of whole-cell cAMP-dependent Cl(-) conductance in mouse proximal tubule cells.

Animals↗

PubMed1999

Divalent cation block of inward currents and low-affinity K+ uptake in Saccharomyces cerevisiae.

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Delta trk2Delta strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K+ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K+ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K+ with Rb+, Cs+, or Na+ only slightly modulated the magnitude of the inward current, whereas replacement with Li+ reduced the inward current by approximately 50%, and tetraethylammonium (TEA+) and choline were relatively impermeant. The inward current was blocked by extracellular Ca2+ and Mg2+ with apparent Kis (at -140 mV) of 363 +/- 78 and 96 +/- 14 microM, respectively. Furthermore, decreasing cytosolic K+ increased the magnitude of the inward current independently of the electrochemical driving force for K+ influx, consistent with regulation of the inward current by cytosolic K+. Uptake of 86Rb+ by intact trk1Delta trk2Delta cells was inhibited by extracellular Ca2+ with a Ki within the range observed for the inward current. Furthermore, increasing extracellular Ca2+ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.

Calcium↗

PubMed1998

Enhancement of GABA-mediated inhibition of rat medial vestibular nucleus neurons by the neurosteroid 20-hydroxyecdysone.

In vivo electrophysiological and patch-clamp studies were performed to determine whether 20-hydroxyecdysone (20-HE), a neurosteroid, influenced neuronal activities of the medial vestibular nucleus (MVN) using chloral hydrate-anesthetized rats and dissociated MVN neurons, respectively. Single neuronal activities of MVN were extracellularly recorded with a glass-insulated silver wire microelectrode attached along a seven-barreled micropipette. Each micropipette was filled with 20-HE, glutamate, bicuculline or 2 M NaCl. These chemicals were applied microiontophoretically to the immediate vicinity of the target neurons. Microiontophoretically applied 20-HE (20-80 nA) dose-dependently decreased rotation-induced firings of both type I and II neurons, which were identified according to their responses to horizontal sinusoidal rotations. Microiontophoretically applied bicuculline, a GABAA receptor antagonist, inhibited 20-HE-induced decreases in neuronal firing of MVN. These findings suggest that 20-HE potentiates the action of GABA, probably by acting directly on the GABAA receptor of MVN neurons. In addition, microiontophoretically applied 20-HE decreased firings induced by glutamate in both type I and II neurons. This decrease by 20-HE was also antagonized with bicuculline. Furthermore, the effects of 20-HE on GABA-induced currents in acutely dissociated MVN neurons were investigated using the whole-cell patch-clamp technique. Under voltage-clamp conditions, GABA (10 microM)-induced currents were potentiated in the presence of 20-HE (100 microM). These findings suggest that 20-HE inhibits MVN neurons by acting on the modulatory site on GABA receptor-ion channel complexes to potentiate GABA inhibition.

Animals↗

PubMed1997

Lack of cystic fibrosis transmembrane regulator-type chloride current in pediatric human atrial myocytes.

Mature cardiomyocytes have been shown to possess a cyclic AMP-mediated chloride channel (I(Cl)) which is the product of the cystic fibrosis transmembrane regulator (CFTR) gene. Species variability has been demonstrated for other ion channels. This study was designed to evaluate human I(Cl) regulation using the whole-cell patch-clamp bioassay. Atrial tissue obtained from children undergoing congenital heart surgery was enzymatically dispersed into isolated myocytes. The patients ranged in age from 1 day to 11 years (mean 2 years). Isoproterenol was used to activate the cAMP second-messenger system in a potassium-free environment. Membrane calcium and sodium channels were pharmacologically blocked. Of 20 human atrial myocytes obtained from 13 pediatric patients, 80% had a small basal chloride current. The current could be inhibited by the anion transport blocker, 9-anthracene carboxylic acid. In 4 of 20 otherwise viable myocytes, no I(Cl) could be elicited, either at baseline or with beta-adrenergic stimulation. Of the 16 myocytes with a basal I(Cl), the current was unaffected by cAMP stimulation in 15 (94%) cells. There were no significant differences in age, gender or clinical status of patients whose cells conducted Cl- current compared with patients whose myocytes had no measurable I(Cl). Ten mature guinea pig ventricular myocytes were evaluated using the same whole-cell patch-clamp technique. Seven of 10 cells showed a reversible increase in I(Cl) with isoproterenol exposure. Despite presence of the CFTR gene in human cardiomyocytes, functional expression of the cAMP-activated I(Cl) does not appear evident in isolated pediatric atrial myocytes. Whether the pathophysiology of congenital heart disease may influence chloride current modulation via alterations in adrenergic tone, intracellular Ca2+ regulation, and cellular osmotic conditions remains to be established.

Adrenergic beta-Agonists↗

PubMed2005

Structural and functional properties of homologous electrical synapses between retinal amacrine cells.

Retinal amacrine cells regulate activities of retinal ganglion cells, the output neurons to higher visual centers, through cellular mechanism of lateral inhibition in the inner plexiform layer (IPL). Electrical properties of gap junction networks between amacrine cells in the IPL were investigated using combined techniques of intracellular recordings, Lucifer yellow and Neurobiotin injection, dual patch-clamp recordings and high voltage electron microscopy in isolated retinas of cyprinid fish. Six types of gap-junctionally connected amacrine cells were classified after their light-evoked responses to light flashes were recorded. Among them, gap junction networks of three types of amacrine cells were studied with structure-function correlation analysis. Cellular morphology of intercellular connections between three homologous cell classes was characterized. The interconnections between laterally extending dendrites in the IPL were localized at dendritic tip terminals. Three types of cells presented the dendrodendritic connections of tip-contact manner in the homologous cell population. High voltage as well as conventional electron microscopy revealed gap junctions between the dendritic tips of Neurobiotin-coupled cells. Receptive field properties of these amacrine cells were examined, displacing a slit of light along the distance from recording sites in the dorsal intermediate region of the retina. Receptive field size, space length constant, response latency and conduction velocity were measured. Spatial and temporal properties of receptive fields were symmetric along horizontally expanding dendrites in the dorsal retina. Simultaneous dual patch-clamp recordings revealed that the lateral gap junction connections between homologous amacrine cells expressed bidirectional electrical synapses passing Na(+) spikes. These results demonstrate that bidirectional electrical transmission in gap junction networks of these amacrine cells is symmetric along the lateral gap junction connections between horizontally extending dendrites. Lateral inhibition regulated by amacrine cells in the IPL appears to be associated with the directional extension of the dendrites and the orientation of dendrodendritic gap junctions.

Action Potentials↗

PubMed2004

Dendrodendritic electrical synapses between mammalian retinal ganglion cells.

Electrical synapses between alpha-type ganglion cells were detected using combined techniques of dual patch-clamp recordings, intracellular labeling, electron microscopy, and channel subunit connexin immunocytochemistry in the albino rat retina. After intracellular injection of Neurobiotin into alpha-cells of inner (ON-center) and outer (OFF-center) ramifying types, measurement of tracer coupling resulted in a preferentially homologous occurrence among cells of the same morphological type (n = 19 of 24). In high-voltage as well as conventional electron microscopic analysis, direct dendrodendritic gap junctions (average size, 0.86 mum long) were present in contact sites between tracer-coupled alpha-cells. In simultaneous dual whole-cell recordings from pairs of neighboring alpha-cells, these cells generated TTX-sensitive sustained spiking against extrinsic current injection, and bidirectional electrical synapses (maximum coupling coefficient, 0.32) with symmetrical junction conductance (average, 1.35 nS) were observed in pairs with cells of the same morphological type. Precise temporal synchronization of spike activity (average time delay, 2.7 msec) was detected when depolarizing currents were simultaneously injected into the pairs. To address whether physiologically identified electrical synapses constitute gap junctional connectivity between cell pairs, identified neuronal connexin36 immunoreactivity was undertaken in Lucifer yellow-labeled cell pairs after patch-clamp recordings. All alpha-cells expressed connexin36, and confocal laser-scanning imaging demonstrated that connexin36 is primarily located at dendritic crossings between electrically coupled cells (seven sites in a pair, on average). These results give conclusive evidence for electrical synapses via dendrodendritic gap junctions involving connexin36 in alpha retinal ganglion cells of the same physiological type.

Action Potentials↗

PubMed1996

Cellular and ionic mechanisms underlying erythromycin-induced long QT intervals and torsade de pointes.

OBJECTIVES: This study sought to elucidate the cellular and ionic basts for erythromycin-induced long QT syndrome. BACKGROUND: Erythromycin is known to produce long QTU intervals on the electrocardiogram (ECG) and to be associated with the development of torsade de pointes (TdP). The mechanisms responsible for the adverse effects of this widely used antibiotic are not well defined. METHODS: The present study used microelectrode and whole-cell patch-clamp techniques to assess the effects of erythromycin on epicardial, endocardial and M cells in transmural strips, arterially perfused wedges and single myocytes isolated from the canine left ventricle. RESULTS: In isolated strips, erythromycin (10 to 100 micrograms/ml) produced a much more pronounced prolongation of the action potential duration (APD) in M cells than in endocardial and epicardial cells, resulting in the development of a large dispersion of repolarization across the ventricular wall at slow stimulation rates. Erythromycin (50 to 100 micrograms/ml) induced early after depolarizations (EADs) in cells in the M (20%) but not epicardial or endocardial regions in transmural strips of ventricular free wall. Erythromycin (100 micrograms/ml) also caused APD prolongation and a transmural dispersion of repolarization, but not EADs, in intact arterially perfused wedges of canine left ventricle. These changes were attended by the development of a long QT interval on the transmural ECG. A polymorphic ventricular tachycardia closely resembling TdP was readily and reproducibly induced after erythromycin but not before. Whole-cell patch-clamp techniques, used to examine the effects of erythromycin on myocytes isolated from the M region, showed a potent effect of the drug to inhibit the rapidly activating component (IK(r)) but not the slowly activating component (IKs) of the delayed rectifier potassium current (IK). The inward rectifier current (IK1) was unaffected. CONCLUSIONS: Our data demonstrate a preferential response of M cells to the class III actions of erythromycin, due principally to the effect of the drug to inhibit IK(r) in a population of cells largely devoid of IKs. Our findings indicate that erythromycin thus produces long QT intervals as well as a prominent dispersion of repolarization across the ventricular wall, setting the stage for induction of TdP-like tachyarrhythmias displaying characteristics typical of reentry.

Action Potentials↗

PubMed2000

Differential effects of NMDA-receptor antagonists on long-term potentiation and hypoxic/hypoglycaemic excitotoxicity in hippocampal slices.

Whole-cell patch clamp recording from cultured hippocampal neurones was used to investigate the NMDA antagonistic effects of the glycineB antagonist 5,7-DCKA and the competitive antagonist CGP 37849. Extracellular field potential recording from area CA1 of hippocampal slices was used to investigate their effects on the induction of LTP and hypoxia/hypoglycaemia-induced suppression of fEPSPs. Additionally, memantine and (+)MK-801 were tested in the later model. 5,7-DCKA inhibited NMDA-induced plateau currents (IC50=0.24+/-0.02 microM) with around nine times higher potency than against peak (IC50=2.14+/-0.17 microM). In contrast, CGP 37849 slowed the onset of NMDA-induced currents considerably and antagonized currents at the time point when the peak component occurred in control responses (IC50=0.18+/-0.01 microM) with around seven times higher potency than against plateau (IC50=1.26+/-0.19 microM). Both 5,7-DCKA and CGP 37849 inhibited the induction of LTP (IC50s=2.53+/-0.13 and 0.37+/-0.04 microM respectively) with potencies close to those inhibiting peak currents in patch clamp studies. 5,7-DCKA and CGP 37849 also blocked the hypoxia/hypoglycaemia-induced suppression of fEPSPs but CGP 37849 (EC50=4.3+/-0.33 microM) was far less potent than against the induction of LTP whilst 5,7-DCKA (EC50=1.47+/-0.04 microM) had similar potency in these two models. Memantine and (+)MK-801 also blocked hypoxia/hypoglycaemia-induced suppression of fEPSPs with EC50s of 14.1+/-0.52 and 0.53+/-0.02 microM respectively. Whereas memantine blocked this effect with similar potency as we previously reported for LTP, (+)MK-801 was four time less potent in this model. The calculated relative therapeutic indices (IC50 LTP over EC50 hypoxia/hypoglycaemia) for 5,7-DCKA, CGP 37849, memantine and (+)MK-801 were 1.72, 0.09, 0.82 and 0.24 respectively. These results show that even in a severe model of hypoxia/hypoglycaemia, glycineB site antagonists and moderate affinity channel blockers exhibit a better therapeutic index than competitive antagonists and high affinity channel blockers. It is likely that in milder forms of pathology the observed differences in therapeutic indices remain the same but the absolute values are expected to be higher.

2-Amino-5-phosphonovalerate↗

PubMed2005

Bradykinin potentiates 5-HT3 receptor-mediated current in rat trigeminal ganglion neurons.

AIM: To explore the modulatory effect of bradykinin (BK) on 5-HT(3 )receptor-mediated current in trigeminal ganglion (TG) neurons in rats. METHODS: The whole-cell patch-clamp technique was used to record 5-HT-activated currents (I(5-HT)) in neurons freshly dissociated from rat TG. Drugs were applied by rapid solution exchange. RESULTS: The majority of the neurons examined responded to 5-HT applied externally with an inward current (76.3%, 74/97) that could be blocked by the 5-HT(3 )receptor antagonist, ICS-205,930 (10(-6) mol/L). In 66 of the 74 cells sensitive to 5-HT (89.2%), pretreatment for 30 s with BK (10(-6)-10(-10) mol/L) could potentiate I(5-HT) with the maximal modulatory effect occurring at 10(-7) mol/L BK (71.6%+/-4.9%). BK shifted the 5-HT concentration-response curve upwards with an increase of 68.9%+/-7.2% in the maximal current response, but with no significant change in the EC(50) value (19.1+/-3.2 mumol/L vs 20.9+/-3.5 micromol/L; t-test, P>0.05; n=8). BK potentiated I(5-HT) in a holding potential-independent manner and did not alter the reverse potential of I(5-HT). This BK-induced potentiation of I(5-HT) was almost completely blocked by Hoe 140 (5*10(-7) mol/L), a selective B2 BK receptor antagonist, and was removed after intracellular dialysis of GF-109203X (2 micromol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. CONCLUSION: Pre-application of BK exerts an enhancing effect on I(5-HT) via a PKC-dependent pathway in rat TG neurons, which may explain the peripheral mechanism of pain and hyperalgesia caused by, for example, tissue damage and inflammation.

Animals↗

PubMedDate not supplied

Serotonin and norepinephrine transporters: possible relationship between oligomeric structure and channel modes of conduction.

In the past several years there has been significant progress made on the biophysics of neurotransmitter transporters, leading to the proposal of new models of substrate and ion permeation across membranes. Questions arising from these studies are as follows: How are substrate uptake and substrate-induced current related? Where and how does substrate-ion coupling occur? What is the functional significance of the coupled and uncoupled currents? Because of a long-standing interest and collaboration, and because of their importance for normal function and disease, the authors have focused on the properties of human norepinephrine and serotonin transporters, using other clones and mutations as specific needs arise. It has been know for decades that hNETs (human norepinephrine transporters) clear NE+ (norepinephrine) following its release in peripheral sympathetic and central noradrenergic synapses. Neuronal activity influences NE+ uptake, so one is also interested in the acute regulation of hNET. To study these problems, hNET-expressing cells have been developed that are suitable for patch clamp, radioligand uptake, biochemistry, and transiently expressed clones for structure-function analysis, and new protocols have been designed combining patch-clamp, microamperometry, Ca2+ imaging, and native catecholamine transporter preparations to study transporters in whole cells and isolated patches. Using these methods, Na-dependent, NE+-induced hNET currents that are blocked by cocaine and antidepressants, channel modes of NE+ conduction, voltage-dependent uptake coupled to NE+-induced ion channel activity, PKC (phosphokinase C) regulation of NE+ uptake, and transporter modulation by [Ca2+]i have all been discovered. There is also provocative new data on other transporters in this family, such as Li/Na mole fraction experiments in the Drosophila serotonin transporters and sided enkephalin block in proline transporters. These studies have led one to postulate the existence of a narrow pore within transporters through which the substrate (NE+ or serotonin, 5HT+) and other ions (principally Na+) pass. It is hypothesized that the pore resides in an oligomeric structure and that separate gene products of hNET or hSERT (human serotonin transporters) come together to form a channel.

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