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At least 451 records · Page 25Linked to original sources
PubMed1993

Receptor-activated Ca influx in human airway smooth muscle: use of Ca imaging and perforated patch-clamp techniques.

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchi↗
PubMed1984

Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique.

The effect of cholecystokinin (CCK) and internal Ca2+ on outward K+ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 X 10(-10) M) applied to the bath evoked a marked increase in the outward K+ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca2+. When strongly buffered Ca2+-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca2+ concentration [( Ca2+]i) from 5 X 10(-8) M to 10(-7) and 5 X 10(-7) M mimicked the effect of CCK. It would appear therefore that CCK controls K+ conductance in the acinar cells via changes in the internal free ionized Ca2+ concentration.

Animals↗
PubMed1988

Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes.

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposomes could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential. Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.

Animals↗
PubMed1984

A patch-clamp study of the partial agonist actions of tubocurarine on rat myotubes.

Single channels activated by (+)-tubocurarine (curare) were recorded from rat myotubes using the patch-clamp technique. The agonist-like action of curare does not result from a contaminant molecule, as the same effects were observed with curare purified by high-performance liquid chromatography. A curare-activated channel can adopt two levels of conductance: full (F) or partial (P). The F state has a slope conductance of 40 pS (identical to that of the acetylcholine (ACh)-activated channel) and the P state has a conductance of 13 pS. At low concentrations of agonist (ACh or curare), the distribution of channel open times is biphasic. The briefer channels may result from the binding of a single agonist molecule whereas the longer-lived channels probably occur following the binding of two agonist molecules. The mean open time of the F state decreases with increasing curare concentration. It is shown that band-width limitations are likely to account for only a very small part of this observed reduction. In contrast, the mean open time of the P state is independent of the concentration of curare. A simple interpretation is that the F state is susceptible to channel blockade by curare, whereas the P state is not. The P state preceded the F state almost as often as it followed the F state; it can also be observed separately from the F state. The fraction of events including a P state increases from about 4% in the presence of 1 microM-curare to 30% at 100 microM-curare. This fraction is also increased by hyperpolarization. When the curare concentration is increased, the F-state frequency first increases and then decreases at higher concentration. This frequency is also decreased by hyperpolarization. The decrease in F-state frequency is probably related to channel blockade by curare; it cannot be wholly accounted for by problems associated with limited time resolution. A synthetic analogue of curare, (+)- tubocurine dimethiodide presents an agonist activity similar to that of curare but with a faster closing rate for both F and P states. The various actions of curare are summarized in two possible models where the P state is interpreted as either a partially open channel or a channel which is partially blocked.

Acetylcholine↗
PubMed1991

A patch-clamp study of K(+)-channel activity in bovine isolated tracheal smooth muscle cells.

1. Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K(+)-channels in these cells were studied by the patch-clamp recording technique. 2. Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 microM. Large unitary currents of both Ca(2+)-dependent and -independent types were observed. Measured between + 20 and + 40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3. Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to much greater than + 40 mV, suggesting that both currents were carried by K(+)-channels. 4. The Ca(2+)-dependent and -independent K(+)-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100 microM) applied to the cytosolic surface of the patches. 5. Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 microM. Ca(2+)-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS. 6. Activity of the Ca2+-dependent K+-channel detected in outside-out patches could be inhibited by application of TEA (1 mM), Cs+ (10mM), Ba2(+210mM) or quinidine (100 microM) to the external surface of the patch. 4-Aminopyridine (4-AP; 1 mM) was ineffective as an inhibitor. 7. The activity of the Ca2+-dependent K+-channel recorded from outside-out patches was reversibly inhibited by charybdotoxin (100 nM). 8. When whole-cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2 +] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nM-I microM) applied to the external surface of the cell.9. We conclude that bovine trachealis cells are richly endowed with charybdotoxin-sensitive, large conductance, Ca2 +-dependent K+-channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2 + -independent K+ -channels remains unclear.

Animals↗
PubMed1988

Patch-clamp study of single-channel and whole-cell K+ currents in guinea pig pancreatic acinar cells.

K+ channels in the plasma membrane of isolated guinea pig pancreatic acini were studied by patch-clamp single-channel and whole-cell current recording techniques. Three types of K+-permeable pores were found in excised patch experiments: Ca2+-activated nonselective cation channels with a unit conductance of approximately 25 pS that could be inhibited by ATP acting on the membrane inside, and two kinds of Ca2+- and voltage-activated K+-selective channels with unit conductances (in symmetrical K+-rich solutions) of about 200 and 30 pS, respectively. In intact cells, pentagastrin activation of currents through the 30 pS K+-selective pores was demonstrated. In these experiments pentagastrin was added to the bath solution and had no direct contact with the electrically isolated membrane area from which the single-channel currents were recorded, suggesting that the activation is mediated via an intracellular messenger system. Pentagastrin stimulation of voltage-gated K+ currents was also observed in whole-cell recording experiments. Results from these experiments suggest that in the stimulated condition the membrane electrical properties were dominated by the 30 pS K+-selective channels.

Animals↗
PubMed1984

Beta-adrenergic increase in the calcium conductance of cardiac myocytes studied with the patch clamp.

In bovine, cat and guinea pig myocytes the effect of bath application of adrenaline or isoprenaline and of injection of cAMP on the Ca channel was studied with the patch clamp (Hamill et al. 1981), and the following results were obtained. On beta-adrenergic stimulation more activity of the single channel on repeated depolarizations and less records without activity were observed. Correspondingly, the average currents were increased. When the patch contained only one channel as judged from the lack of superpositions during all depolarizations, beta-adrenergic stimulation never produced superpositions indicating that the total number of channels did not increase. Also, it was never possible to activate a channel in the mute patch. The single channel conductance was not changed by catecholamines or cAMP. Increase in probability during depolarization of the channel to be in the open state was proven by non-stationary fluctuation analysis. The kinetic analysis showed a prolongation of the open times and shortening of the shut times by catecholamines, indicating that the rate constants in a three state model C1 in equilibrium C2 in equilibrium O are changed in such a way that the equilibrium shifted towards the open state. In some patches clusters of channels were observed, and activity was greatly increased by adrenaline, isoprenaline and cAMP-injection. The decay of the mean current was either mono-exponential or, in most cases, double-exponential. When the decay was mono-exponential, fluctuation analysis showed an increase in open probability on beta-stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Agonist potency determination by patch clamp analysis of single glutamate receptors.

Agonists (L-quisqualate, L-glutamate and L-cysteine sulphinate) of the locust muscle glutamate receptor differ in potency. Patch clamp analysis of single glutamate receptors reveals that the relative potencies of agonists are determined both by the life-times of the channels that they gate and their probability of activating the glutamate receptor-ionophore complex. Furthermore, agonists which most readily activate the receptor channel complex gate channels with longer life-times than agonists which are less efficient in this respect.

Animals↗
PubMed1992

Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch-clamp.

E. coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration-rehydration and studied by patch-clamp. The porins could be closed by voltage pulses under -100 mV. The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl. Rapid fluctuations (in the millisecond range) of about one third (60-70 pS) of the large closure steps were also observed. The data are interpreted as follows: an increase in membrane potential favours the cooperation transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short-lived closed state.

Bacterial Outer Membrane Proteins↗
PubMed2006

Neutrophil secretion induced by an intracellular Ca2+ rise and followed by whole-cell patch-clamp recordings occurs without any selective mobilization of different granule populations.

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (deltaC(m)). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca(2+). A sustained secretion following a short lag phase was induced by high concentrations of Ca(2+) (100 microM and higher). A stable plateau was reached after 5-7 minutes at deltaC(m) values corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca(2+) concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca(2+)-sensitivity.

Journal Article↗
PubMed1985

Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording.

The effects of tetraethylammonium ions on currents through high-conductance voltage- and Ca2+-activated K+ channels have been studied with the help of patch-clamp single-channel and whole-cell current recording on pig pancreatic acinar cells. In excised outside-out membrane patches TEA (1 to 2 mM) added to the bath solution virtually abolishes unitary current activity except at very positive membrane potentials when unitary currents corresponding to a markedly reduced conductance are observed. TEA in a lower concentration (0.2 mM) markedly reduces the open-state probability and causes some reduction of the single-channel conductance. In inside-out membrane patches bath application of TEA in concentrations up to 2 mM has no effect on single-channel currents. At a higher concentration (10 mM) slight reductions in single-channel conductance occur. In whole-cell current recording experiments TEA (1 to 2 mM) added to the bath solution completely suppresses the outward currents associated with depolarizing voltage jumps to membrane potentials of 0 mV and blocks the major part (70 to 90%) of the outward currents even at very positive membrane potentials (30 to 40 mV). In contrast TEA (2 mM) added to the cell interior (pipette solution) has no effect on the outward K+ current. Our results demonstrate that TEA in low concentrations (1 to 2 mM) acts specifically on the outside of the plasma membrane to block current through the high-conductance Ca2+- and voltage-activated K+ channels.

Animals↗
PubMed1999

A patch-clamp study of ion channels in protoplasts prepared from the marine alga Valonia utricularis.

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant "mother cells" (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123-134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl(-) channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K(+) channel (VKC1, Valonia K(+) Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl(-) media, VAC1 was most frequently observed and had a single channel conductance of 36 +/- 7 pS (n = 4) in the outside-out and 33 +/- 5 pS (n = 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 +/- 4 mV (n = 4, outside-out) and 9 +/- 7 mV (n = 10, cell-attached) close to the Nernst potential of Cl(-) and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl(-) media (bath/pipette; E(Cl(-)) -20 +/- 7 mV (n = 4); Nernst potential -28 mV). Consistent with a selectivity for Cl(-), VAC1 was inhibited by 100 micronM DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl(-) conditions yielded a midpoint potential of -12 +/- 5 mV (n = 4, outside-out) and -3 +/- 6 mV (n = 9, cell-attached) and corresponding apparent minimum gating charges of 15 +/- 3 (n = 4) and 18 +/- 5 (n = 9). The midpoint potential shifted to more negative values in the presence of a Cl(-) gradient. VAC2 was activated by voltages more negative than E(Cl(-)) and was always observed together with VAC1, but less frequently. It showed a "flickering" gating. The single channel conductance was 99 +/- 10 pS (n = 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 +/- 5 pS (n = 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 +/- 27 pS (n = 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl(-) conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

A membrane fusion strategy for single-channel recordings of membranes usually non-accessible to patch-clamp pipette electrodes.

Membranes of cellular organelles and plasma membranes of some type of cells are not accessible to the high-resolution recordings that the conventional patch-clamp technique allows. However, when these purified membranes are dehydrated together with small lipid vesicles and hydrated again, cell-size vesicles (5-100 micron diameter) are obtained, on which single-channel recordings are possible. This approach, which has been proven successful with about ten different membrane preparations of varied origin, is further illustrated with two examples. First, a known conductivity of the sarcoplasmic reticulum membrane is compared with data obtained by using other techniques. Second, a new sodium current, present at purified postsynaptic membranes from the Torpedo electric organ, is described.

Animals↗
PubMed1989

A modified horizontal capillary puller for fabrication of patch-clamp pipettes.

A simple low-cost modification to a conventional horizontal two-stage micropipette puller is described. It allows the production of pipettes suitable for patch-clamp studies. It requires that the hard-pull stage of the fabrication process be disabled and two heating cycles, each with a different heating filament temperature, be employed. The modification is described specifically for an Industrial Science Associates, Inc. M-1 micropipette puller. However, in principle it should be applicable to any horizontal two-stage puller using a solenoid to generate the pull force.

Electronics↗
PubMed1986

A patch-clamp study of histamine-secreting cells.

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.

Animals↗
PubMed1988

Isoflurane causes "flickering" of the acetylcholine receptor channel: observations using the patch clamp.

The authors investigated the effects of a volatile anesthetic (isoflurane) on the functional properties of a well characterized membrane ion channel (the acetylcholine receptor channel), using the patch clamp electrophysiological method together with local microperfusion of isoflurane. They found that isoflurane causes single acetylcholine receptor channels to "flicker" rapidly between open and closed states, reminiscent of the kinetic pattern induced by local anesthetics. The mean amplitude of currents flowing through open channels was unchanged. Although these observations initially suggested that isoflurane blocks open ion channels, closer analysis of the "isoflurane pattern" revealed features inconsistent with classical open channel block: the duration of bursts of channel openings is shortened, and that of the brief closures within bursts lengthens as the concentration of isoflurane is increased. The authors suggest that isoflurane exerts its characteristic effects on the kinetic properties of acetylcholine receptor channel by an allosteric mechanism. In addition, and apart from their mechanistic significance, these effects may underlie the known potentiation by isoflurane of curariform neuromuscular blockade.

Acetylcholine↗
PubMed1993

Electrical and integrative properties of rabbit sympathetic neurones re-evaluated by patch clamping non-dissociated cells.

1. Voltage recordings were performed on non-dissociated sympathetic neurones from rabbit coeliac ganglia using the whole-cell configuration of the patch clamp technique. 2. Cells were classified depending on their firing pattern as silent cells (63%) producing either phasic (24%) or tonic (76%) spike discharge in response to depolarizing currents, and pacemaker cells (37%). 3. All the cells produced large overshooting spikes and prolonged postspike after-hyperpolarization. The peak-to-peak spike amplitude was 113.8 +/- 1 mV. Spikes were shortened and the after-hyperpolarization was suppressed when calcium channel blockers (Cd2+ and La3+) were added. 4. Silent cells have a resting potential of -58.8 +/- 1.5 mV. At potentials ranging from -50 to -90 mV, the input impedance was 490 +/- 27 M omega at 22-24 degrees C and 426 +/- 47 M omega at 35-36 degrees C. The time constant at voltages corresponding to the high input impedance region was 126 +/- 7 ms at 22-24 degrees C and 86 +/- 7 ms at 35-36 degrees C. 5. The firing frequency of the pacemaker cells was 3.2 +/- 0.5 Hz at 35-36 degrees C in the presence of nicotinic blockers. Evidence is given that the firing did not result from cell injury but was induced by an intrinsic pacemaker mechanism. Input impedance of pacemaker neurones was 580 +/- 47 M omega at 22-24 degrees C and 473 +/- 56 M omega at 35-36 degrees C. 6. Most of the pacemaker cells (63%) were motoneurones, since they were antidromically fired by stimulating post-ganglionic nerves. In addition, they received synaptic inputs from both preganglionic fibres (splanchnic nerves) and the periphery (postganglionic nerves). Long-lasting depolarizations were induced in either silent or pacemaker cells by single shocks applied to pre- and postganglionic nerves. 7. Slowly rising voltage ramps revealed the presence of an N-shaped current-voltage relationship in voltage clamped pacemaker cells. The negative slope was located in a subthreshold voltage range, between -83.4 +/- 1.4 and -59.0 +/- 1.8 mV. It was induced by the activation of a low threshold persistent inward current. Although it was tiny (22 +/- 3 pA at its peak level) this current brought the null-current voltage up to -41.0 +/- 1.4 mV, which resulted in continuous firing. 8. Due to the instability introduced by the N-shaped I-V relationship, pacemaker cells can display bistable behaviour characterized by hyperpolarizing responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗