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Results for “patch clamp”

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PubMed1997

The two intracellular Ca2+ release channels, ryanodine receptor and inositol 1,4,5-trisphosphate receptor, play different roles during fertilization in ascidians.

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs of Phallusia mammillata using whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3 receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3 (10 microM perfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3 perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3 causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15-20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.

Animals↗

PubMed2005

Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl channel activity in Xenopus oocytes.

Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl(-) channel activity in Xenopus oocytes. Background. Cyst expansion in autosomal-dominant polycystic kidney disease (ADPKD) is characterized by active Cl(-) secretion in excess of solute reabsorption. However, the connections between elevated epithelial Cl(-) secretion and loss-of-function or dysregulation of either ADPKD gene polycystin-1 (PC1) or polycystin-2 (PC2) remain little understood. Methods. Cl(-) transport in Xenopus oocytes expressing the CD16.7-PKD1 (115-226) fusion protein containing the final 112 amino acid (aa) of the PC1 C-terminal cytoplasmic tail, or in oocytes expressing related PC1 fusion protein mutants, was studied by isotopic flux, two-electrode voltage clamp, and outside-out patch clamp recording. Results. Expression in oocytes of CD16.7-PKD1 (115-226) increased rates of both influx and efflux of (36)Cl(-), whereas CD16.7-PKD1 (1-92) containing the initial 92 aa of the PC1 C-terminal cytoplasmic tail was inactive. The increased Cl(-) transport resembled CD16.7-PKD1 (115-226)-stimulated cation current in its sensitivity to ADPKD-associated missense mutations, to mutations in phosphorylation sites, and to mutations within or encroaching upon the PC1 coiled-coil domain, as well as in its partial suppression by coexpressed PC2. The NS3623- and 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive (36)Cl(-) flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La(3+), suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for (36)CI(-) flux activation. Outside-out patches from oocytes expressing CD16.7-PKD1 (115-226) also exhibited increased NS3623-sensitive Cl(-) current. Conclusion. These data show that CD16.7-PKD1 (115-226) activates Cl(-) channels in the Xenopus oocyte plasma membrane in parallel with, but not secondary to, activation of Ca(2+)-permeable cation channels.

Animals↗

PubMed1992

Voltage activation of heart inner mitochondrial membrane channels.

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above +/- 60 mV [Kinnally et al., Biochem. Biophys. Res. Commun. 176, 1183-1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin A are likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.

Animals↗

PubMed2001

Human myofibroblastic hepatic stellate cells express Ca(2+)-activated K(+) channels that modulate the effects of endothelin-1 and nitric oxide.

BACKGROUND/AIMS: High-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the effects of vasoactive factors in contractile cells. It is unknown whether hepatic stellate cells (HSCs) contain BK(Ca) channels and what their role in the regulation of HSCs contractility is. METHODS: The presence of BK(Ca) channels in HSCs was assessed by the patch-clamp technique. The functional role of BK(Ca) channels was investigated by measuring intracellular calcium concentration ([Ca(2+)](i)) and cell contraction in individual cells after stimulation with endothelin-1 in the presence or absence of specific modulators of BK(Ca) channels. RESULTS: BK(Ca) channels were detected by patch-clamp in most of the activated HSCs studied. Incubation of cells with iberiotoxin, a BK(Ca) channel blocker, increased both the sustained phase of [Ca(2+)](i) elicited by endothelin-1 and the number of cells undergoing contraction, while the use of NS1619, a BK(Ca) channel opener, induced opposite effects. Stimulation of HSCs with S-nitroso-N-acetyl-penicillamine (SNAP), a nitric oxide (NO)-donor, increased the opening of BK(Ca) channels and reduced the effects of endothelin-1. Conversely, iberiotoxin abolished the inhibitory effect of SNAP on endothelin-induced [Ca(2+)](i) increase and cell contraction. CONCLUSIONS: Activated human HSCs contain BK(Ca) channels that modulate the contractile effect of endothelin-1 and mediate the inhibitory action of NO.

Benzimidazoles↗

PubMed1998

Direct participation of starburst amacrine cells in spontaneous rhythmic activities in the developing mammalian retina.

Spontaneous, rhythmic waves of excitation in the developing mammalian retina play a critical role in the formation of precise neuronal connectivity in the visual system. However, it is not known what circuits in the retina are responsible for the production of these waves. Using patch-clamp recordings in the whole-mount neonatal rabbit retina, this study reports that the displaced starburst amacrine cell, a unique cholinergic interneuron in the ganglion cell layer of the retina, undergoes rhythmic bursts of membrane depolarization with a frequency and duration similar to those of spontaneous retinal waves. Simultaneous patch-clamp recordings from pairs of neighboring starburst and ganglion cells show that the rhythmic activity in starburst cells is closely correlated with that in ganglion cells, and that the excitation in both cell types is most likely driven by synaptic input. However, in contrast to ganglion cells, displaced starburst cells usually do not generate spontaneous somatic action potentials. Instead, they seem to use subthreshold potentials (at least at the soma) to mediate the rhythmic excitation. The results suggest that acetylcholine is likely released rhythmically in the developing retina. Thus, starburst amacrine cells form the first identified network of retinal interneurons that directly participate in spontaneous rhythmic activities in the developing retina.

Action Potentials↗

PubMed1997

[Etomidate inhibits neuronal potassium channels in humans].

INTRODUCTION: There is no data available on the action of etomidate on ion currents in human neuronal cells. Therefore the effects of etomidate on a human neuronal delayed rectifier potassium current were investigated. METHOD: Outward rectifying potassium currents of human neuroblastoma SH-SY5Y cells were measured using the whole cell patch-clamp technique. Cells were grown in RPMI-medium (+Pen/Strep and FCS) at 37 degrees C and 5% CO2. The holding potential was -80 mV. Potassium currents were evoked by depolarizing the membrane potential to values from -50 mV to +90 mV in 10 mV steps using an EPC-7 patch-clamp amplifier (List medical electronics) and pclamp version 5.71 (Axon instruments). RESULTS: Etomidate differentially inhibited steady-state and peak potassium current with IC50-values of 170 microM for peak current suppression and 120 microM for steady-state current respectively. Etomidate induced inactivation-like behaviour of the potassium current and changed the voltage dependence of potassium current activation. CONCLUSION: The results demonstrate that etomidate has more than one effect on the potassium current, indicating the complexity of general anaesthetic actions on neuronal targets. The actions of etomidate on human neuronal potassium currents may potentially contribute to the myocloni observed with this general anaesthetic agent during induction of anaesthesia.

Anesthetics, Intravenous↗

PubMed1997

Comparative effects of nonsedating histamine H1 receptor antagonists, ebastine and terfenadine, on human Kv1.5 channels.

The effects of ebastine and terfenadine, long-acting nonsedating histamine H1 receptor antagonists, were studied on hKv1.5 channels using the whole-cell voltage-clamp configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding the hKv1.5 channel. Upon depolarization to +60 mV, terfenadine, 1 microM and 3 microM, inhibited the hKv1.5 current by 42.4 +/- 6.4% and 69.3 +/- 4.2% (P < 0.01). In contrast, at the same range of concentrations, ebastine-induced inhibition of this K+ current averaged 6.5 +/- 2.0% and 13.0 +/- 2.0 (P < 0.05). At the highest concentration tested (3 microM) neither terfenadine carboxylate nor carebastine significantly modified hKv1.5 current. All these results suggest that ebastine could represent a safer alternative to terfenadine in the clinical practice.

Animals↗

PubMed2001

Tetrandrine inhibits Ca2+ release-activated Ca2+ channels in vascular endothelial cells.

The effects of tetrandrine (TET), a Ca2+ antagonist of bis-benzylisoquinoline alkaloid origin, on cultured single bovine pulmonary artery endothelial cells were examined using fluorescence ratio imaging and whole-cell attached patch-clamp techniques. Thapsigargin (TSG, 100 nM), a selective endoplasmic reticulum Ca2+-ATPase pump inhibitor known to induce the release of nitric oxide (NO) from vascular endothelial cells via a Ca2+-dependent manner, caused a rapid elevation of cytosolic Ca2+ concentration, which was inhibited by 30 microM TET. In whole-cell patch-clamp study using the same vascular endothelial cells, addition of 100 nM TSG caused a significant enhancement of depolarization-evoked Ca2+-dependent, outward K+ currents, which could also be abolished by 30 microM TET. The present results demonstrate directly that TET, in addition to its known inhibitory effects on vascular smooth muscle by virtue of its Ca2+ antagonistic actions, also inhibits NO production by the endothelial cells through blockade of Ca2+ release-activated Ca2+ channels.

Alkaloids↗

PubMed1998

Histamine H1-receptor-mediated increase in the Ca2+ transient without a change in the Ca2+ current in electrically stimulated guinea-pig atrial myocytes.

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.

Action Potentials↗

PubMed1995

Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells.

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole-cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl >> Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume-sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue, Brown↗

PubMed1994

Inhibition of ATP-sensitive K+ channel by a non-sulfonylurea compound KAD-1229 in a pancreatic beta-cell line, MIN 6 cell.

We studied the mechanism of action of KAD-1229, a non-sulfonylurea compound shown to stimulate insulin secretion, in a glucose responsive insulinoma cell line, MIN 6 cells. In microsomal fraction of MIN 6 cells, KAD-1229 displaced binding of [3H]glibenclamide in a concentration-dependent manner. The dissociation constant and the maximum binding capacity were 0.61 nM and 8.70 pmol/mg.protein, respectively. In inside out configuration of patch-clamp technique, KAD-1229 attenuated the opening of ATP-sensitive K+ channels. The effect of KAD-1229 was detected at 10(-8) M, and 10(-5) M KAD-1229 almost completely blocked the activity of ATP-sensitive K+ channel. When membrane potential was monitored by a perforated mode of patch clamp, KAD-1229 induced depolarization of plasma membrane, which was followed by a burst of action potentials. These action potentials were blocked by cobalt. In a fura-2-loaded single MIN 6 cell, KAD evoked an elevation of intracellular free Ca2+ concentration, [Ca2+]i. The KAD-1229-mediated elevation of [Ca2+]i was attenuated by either removal of extracellular Ca2+ or an addition of nifedipine. Finally, KAD-1229 augmented insulin secretion in MIN 6 cells in a concentration-dependent manner. KAD-1229 also enhanced the effect of glucose and nifedipine inhibited the action of KAD-1229 on insulin secretion. These results indicate that KAD-1229 stimulates insulin secretion by stimulating Ca2+ influx and that, despite the lack of sulfonylurea structure, KAD-1229 binds to sulfonylurea receptors and inhibits the activity of ATP-sensitive K+ channel in MIN 6 cells.

Action Potentials↗

PubMed2001

In situ model for studying potassium currents in various growth plate chondrocyte subpopulations.

A new in situ model of partially digested growth plate cartilage suitable for patch clamp study of membrane currents of chondrocytes from various differentiation stages was developed. Thin sections of growth plate were enzyme digested to expose intact membranes of chondrocytes previously covered by extracellular matrix. This treatment dramatically increased the success rate of tight-seal formation from virtually 0% up to 40%. Whole-cell patch clamp recording revealed a delayed outward rectifying current as the major macroscopic current in chondrocytes of all differentiation stages. This current was sensitive to tetraethylammonium chloride and reversed polarity at a membrane potential close to the equilibrium potential of K+. Chondrocytes at resting stage expressed a much smaller K+ current than the proliferative and hypertrophic chondrocytes. When the current amplitudes were normalized for the cell membrane area, proliferative cells expressed a significantly higher outward current density.

Animals↗

PubMed2001

Down-regulation of L-type calcium channels in inflamed circular smooth muscle cells of the canine colon.

BACKGROUND & AIMS: Circular smooth muscle phasic contractions and tone are suppressed during colonic inflammation, but the contributing factors are poorly understood. This study investigated if the expression level of voltage-gated long-lasting (L-type) Ca(2+) channel protein and functional Ca(2+) channel current are down-regulated in the circular muscle cells of the inflamed canine colon. METHODS: L-type Ca(2+) channel expression was compared between normal and inflamed smooth muscle cells by Western immunoblots using an antibody directed against the pore-forming alpha 1C-subunit, and patch-clamp methods were used to evaluate Ca(2+) channel current density. RESULTS: The expression of the L-type Ca(2+) channel protein was significantly reduced in inflamed compared with normal circular smooth muscle cell membranes, and this finding was associated with suppressed levels of Ca(2+) channel current in patch-clamped cells. The L-type Ca(2+) channel current in normal and inflamed cells increased proportionately in response to Bay K 8644, but the maximal current density was still lower in the inflamed cells. Acetylcholine increased the L-type Ca(2+) channel current in normal but not in inflamed cells. CONCLUSIONS: The expression level of L-type Ca(2+) channels is down-regulated in the circular smooth muscle cell membranes of the inflamed colon, which may result in reduced Ca(2+) influx. The functional and pharmacologic properties of the channels seem normal. Although some Ca(2+) channels are still present in the inflamed cells, acetylcholine does not activate these channels, which may be caused by additional upstream defects in the receptor signaling cascade. The down-regulation of L-type Ca(2+) channel expression may suppress circular smooth muscle contractions in the inflamed colon and contribute to the abnormalities in motility and digestion observed during inflammatory disorders.

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

PubMed2006

Effect of simulated I(to) on guinea pig and canine ventricular action potential morphology.

The transient outward current (I(to)) is a major repolarizing current in the heart. Marked reduction of I(to) density occurs in heart failure and is accompanied by significant action potential duration (APD) prolongation. To understand the species-dependent role of I(to) in regulating the ventricular action potential morphology and duration, we introduced simulated I(to) conductance in guinea pig and canine endocardial ventricular myocytes using the dynamic clamp technique and perforated patch-clamp recordings. The effects of simulated I(to) in both types of cells were complex and biphasic, separated by a clear density threshold of approximately 40 pA/pF. Below this threshold, simulated I(to) resulted in a distinct phase 1 notch and had little effect on or moderately prolonged the APD. I(to) above the threshold resulted in all-or-none repolarization and precipitously reduced the APD. Qualitatively, these results agreed with our previous studies in canine ventricular cells using whole cell recordings. We conclude that 1) contrary to previous gene transfer studies involving the Kv4.3 current, the response of guinea pig ventricular myocytes to a fully inactivating I(to) is similar to that of canine ventricular cells and 2) in animals such as dogs that have a broad cardiac action potential, I(to) does not play a major role in setting the APD.

Action Potentials↗

PubMed2002

Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene.

1 Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA) and glycine receptors at concentrations that do not affect non-NMDA receptors. 2 We utilized two-electrode voltage-clamp and whole cell patch-clamp techniques to assess the effects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 micro M to 10 mM) produced a reversible, concentration-dependent inhibition of acetylcholine-induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The alpha4beta2 and alpha3beta2 subunit combinations were significantly more sensitive to toluene inhibition than the alpha4beta4, alpha3beta4 and alpha7 receptors. 3 Receptors composed of alpha4 and beta2(V253F) subunits showed alpha4beta4-like toluene sensitivity while those containing alpha4 and beta4(F255V) subunits showed alpha4beta2-like sensitivity. 4 In hippocampal neurons, toluene (50 micro M to 10 mM) dose-dependently inhibited ACh-mediated responses with an IC(50) of 1.1 mM. 5 Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit-dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural effects of toluene.

Acetylcholine↗

PubMed1995

The ion channel behavior of the nuclear pore complex.

Macromolecule-conducting pores have been recently recognized as a distinct class of ion channels. The poor role of macromolecules as electrical charge carriers can be used to detect their movement along electrolyte-filled pores. Because of their negligible contribution to electrical ion currents, translocating macromolecules reduce the net conductivity of the medium inside the pore, thus decreasing the measured pore ion conductance. In the extreme case, a large translocating macromolecule can interrupt ion flow along the pore lumen, reflected as a negligible pore conductance. Therefore, ion conductance serves as a measurement of macromolecular transport, with lesser values indicating greater macromolecular translocation (in size and/or number). Such is the principle of operation of the widely used Coulter counter, an instrument for counting and sizing particles. It has long been known that macromolecules translocate across the central channel of nuclear pore complexes (NPCs). Recently, large conductance ion channel activity (100-1000 pS) was recorded from the nuclear envelope (NE) of various preparations and it was suggested that NPCs may be the source of this activity. Despite its significance to understanding the regulation of transcription, replication, mRNA export, and thus gene expression of normal and pathological states, no report has appeared demonstrating that this channel activity corresponds to ion flow along the central channel of the NPC. Here we present such a demonstration in adult mouse cardiac myocyte nuclei. In agreement with concepts introduced for macromolecule-conducting channels, our patch clamp experiments showed that ion conductance is reduced, and thus that ion flow is restricted during translocation of macromolecules containing nuclear targeting signals. Ion flow was blocked by mAb414, a monoclonal antibody raised against a major NPC glycoprotein and known to localize on the NPC channel where it blocks macromolecular transport. These results also establish patch clamp as a useful technique for the measurement of macromolecular translocation along the large central channel of the NPC and provide a basis for the design of future investigations of nuclear signaling for control of gene activity, mRNA export for gene expression, as well as other processes subservient to NPC-mediated nucleocytoplasmic exchange.

Animals↗

PubMed2002

Sodium current in human jejunal circular smooth muscle cells.

BACKGROUND & AIMS: Sodium channels are key regulators of neuronal and muscle excitability. However, sodium channels have not been definitively identified in gastrointestinal smooth muscle. The aim of the present study was to determine if a Na(+) current is present in human jejunal circular smooth muscle cells. METHODS: Currents were recorded from freshly dissociated cells using patch-clamp techniques. Complementary DNA (cDNA) libraries constructed from the dissociated cells were screened to determine if a message for alpha subunits of Na(+) channels was expressed. Smooth muscle cells were also collected using laser-capture microdissection and screened. RESULTS: A tetrodotoxin-insensitive Na(+) channel was present in 80% of cells patch-clamped. Initial activation was at -65 mV with peak inward current at -30 mV. Steady-state inactivation and activation curves revealed a window current between -75 and -60 mV. The Na(+) current was blocked by lidocaine and internal and external QX314. A cDNA highly homologous to SCN5A, the alpha subunit of the cardiac Na(+) channel, was present in the cDNA libraries constructed from dissociated cells and from smooth muscle cells collected using laser-capture microdissection. CONCLUSIONS: Human jejunal circular smooth muscle cells express a tetrodotoxin-insensitive Na(+) channel, probably SCN5A. Whether SCN5A plays a role in the pathophysiology of human gut dysmotilities remains to be determined.

Anesthetics, Local↗

PubMed1998

Modulation of GABAA receptor-mediated inhibition by postsynaptic calcium in epileptic hippocampal neurons.

Visualization of neurons during patch clamp recordings from slices provides concurrent neuroanatomical information for physiological studies. Although, the technique becomes increasingly popular in immature brains, it has not been fully utilized in aged/adult and diseased brains including post-surgical human specimen. In the present study, glutamatergic modulation of GABAA receptor-mediated inhibition was investigated by whole-cell patch clamp recordings from visualized hippocampal dentate granule cells (DGCs) in slices that were prepared from surgically-removed human medial temporal lobe specimens and the rat pilocarpine model of temporal lobe epilepsy. GABAA receptor-mediated synaptic inhibition was recorded by isolating inhibitory postsynaptic currents (IPSCs) at a membrane potential of 0 mV where glutamatergic excitatory postsynaptic currents are near equilibrium. Peak amplitude of GABAA IPSC was not different between epileptic DGCs of both human and pilocarpine-treated rat hippocampi and those in the control rat DGCs. However, when high frequency stimulation (30 Hz for 10 s) preceded immediately before the generation of a GABAA IPSC, its peak amplitude was significantly reduced in epileptic DGCs. The application of an NMDA receptor antagonist prevented this decrease indicating that the high frequency stimulation activated the NMDA receptor and that this activation is involved in the induction of response-decrement of GABAA IPSCs in epileptic DGCs. In addition, intracellular application of a calcium chelator, BAPTA through a patch pipette was found effective in preventing the response-decrement of GABAA IPSCs suggesting that postsynaptic calcium-increase is also involved in this process. It is proposed that activation of the NMDA receptor in epileptic DGC may trigger an epileptogenic increase of intracellular free calcium, and this calcium-increase plays a crucial role for the induction of the response-decrement of GABAA IPSCs in epileptic hippocampus, which possibly leads to the initiation of epileptic seizures and ictal events.

Animals↗