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At least 649 records · Page 36Linked to original sources

PubMed1988

A new oil-gate concentration jump technique applied to inside-out patch-clamp recording.

A new method was developed to instantaneously replace the solution on the inner side of an inside-out membrane patch in order to measure time courses with which active substances acted on single ionic channels. Inside-out membrane patches were isolated from single ventricular cells of the guinea pig heart. The recording bath consisted of two chambers separated by a partition having a narrow slit. Mixing of two test solutions through this slit was prevented by filling it with paraffin oil. The pipette tip with a tightly sealed inside-out membrane patch was moved through the oil from one solution to the other so that the pipette tip was instantaneously exposed to a new solution. When the pipette tip was jumped between different K+ concentrations, the leak current through the membrane patch increased or decreased with a half time of 6.3 +/- 3.0 ms (n = 15). The amplitude of single K+ channel currents changed to a new steady level within approximately 20 ms. These time courses were well explained by diffusion of K+ in the dead space between the pipette tip opening and the membrane patch. An application of this method to the ATP-regulated K+ channel revealed a latent period of 1-2 s before the channel started its activity after the instantaneous removal of ATP, whereas no obvious latency was observed in the rapid suppression of the channel, which was completed in 100-300 ms after reapplying ATP.

Adenosine Triphosphate↗

PubMed1978

The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes.

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at mu-molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10--100ms. In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1--3 micrometer internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had be to found in which channels open at a relatively low frequency.

Animals↗

PubMed1984

Patch-clamp study of rubidium and potassium conductances in single cation channels from mammalian exocrine acini.

Single-channel current recordings were carried out on excised inside-out patches of baso-lateral plasma membrane from exocrine acinar cells. The mouse pancreas and submandibular gland as well as the pig pancreas were investigated. In the mouse pancreas the voltage-insensitive Ca2+-activated cation channel was studied. Single-channel current-voltage (i/v) relationships were studied in symmetrical Rb+-rich solutions and in asymmetrical Rb+/Na+ and Na+/Rb+ solutions. In all cases the i/v relations were linear and had the same slope representing a single-channel conductance of about 33 pS which is identical to that previously obtained with symmetrical Na+ solutions or asymmetrical Na+/K+ solutions. In the mouse submandibular gland and the pig pancreas the voltage and Ca2+-activated K+ channel was studied. The outward currents observed after depolarization in the presence of quasi-physiological Na+/K+ gradients were immediately abolished when all the K+ in the bath fluid was replaced by Rb+ (bath fluid in contact with inside of plasma membrane). This effect was immediately and fully reversible upon return to the high K+ solution. The voltage and Ca2+-activated K+ channel was also studied in asymmetrical K+/Rb+ and Rb+/K+ solutions. In the first case inward (K+) currents could be observed but not outward (Rb+) currents, while in the other case inward (Rb+) currents could not be seen whereas outward (K+) currents were measured. The current-voltage relationships were approximately linear and the null potential was close to 0 mV in both situations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

A patch-clamp study of acetylcholine-activated ion channels in Ascaris suum muscle.

Acetylcholine-activated single-channel currents were recorded from cell-attached and inside-out patches of isolated muscle vesicles from Ascaris suum. Acetylcholine (1-10 mumols l-1) activated cation-selective channels of two amplitudes: 40-50 pS and 25-35 pS. Both channels had linear I/V relationships and mean open durations independent of voltage. The larger conductance was analysed in detail to determine its open-, closed- and burst-time kinetics; the open and burst durations were composed of two components (short and long), while closed durations had at least three components (short, intermediate and long). The data were then corrected to allow for missing short events in order to estimate various parameters including corrected mean open time (1.26 + 0.11 ms, mean +/- S.E.). Values were also derived for the efficacy (beta/alpha = 4.9) and affinity [1/KD = 147 x 10(3) (mol l-1) -1] of acetylcholine at this receptor. Larger concentrations of acetylcholine (25-100 mumols l-1) were shown to produce desensitization and caused single-channel currents to occur in clusters with long closed times (mean 171 s) between clusters. It was concluded that the extrasynaptic muscle of Ascaris suum contains two types of acetylcholine-activated ion channel and these are possible sites of action of various anthelmintic drugs. This paper is the first to describe acetylcholine-activated single-channel currents in invertebrate muscle.

Acetylcholine↗

PubMed1983

Properties of the CA2+-activated K+ conductance of human red cells as revealed by the patch-clamp technique.

Application of Ca2+ to the inner surface of red-cell membranes activates unitary currents that can be measured in cell-attached and cell-free membrane patches. Ca2+ can be replaced by Pb2+ to activate the single channels. In addition to internal Ca2+ external K+ has to be present. The channels are preferentially permeable to K+ with a selectivity ratio PK:PNa of about 15:1 as estimated from measurement of reversal potentials. The dependence of channel activity on Ca2+ is compatible with the conception that the binding of two Ca2+ is necessary to open a single channel. Both the channel activity and the single-channel conductance exhibit inward rectification. External and internal Na+ inhibit the K+ currents. The reported results suggest that the unitary current events are responsible for the Ca2+-dependent K+ permeability known from measurement on cell suspensions. Therefore, comparison of the two techniques allows calculation of the number of K+ channels per red cell, which on average is about 10.

Calcium↗

PubMed1992

Schistosoma mansoni: patch-clamp study of a nonselective cation channel in the outer tegumental membrane of females.

An apparent ion channel with a conductance of 295 pS is present in isolated inside-out patches of outer tegumental membrane taken from female Schistosoma mansoni. With positive voltages applied to the intracellular face of the patch, percentage open time for the channel was 0 to 50; with negative voltages applied, percentage open time was greater than 99. Step changes in applied voltage characteristically induced opening-closing activity. However, there was no maintained applied voltage at which there was a high level of sustained opening-closing activity. The 295 pS conductance was by far the most commonly occurring conductance but it appears to result from cooperativity among several channels, the unitary conductance for the channel averaging 95 pS. Alterations in the Na+ or K+ concentration ratios changed the reversal potential for this conductance but alterations in the Cl- concentration did not. From this it is concluded that this channel is selective for Na+ or K+ over Cl- and it appears to be a nonselective cation channel.

Animals↗

PubMed1994

Comparison of large-conductance Ca(2+)-activated K+ channels in artificial bilayer and patch-clamp experiments.

We compared the gating, ion conduction, and pharmacology of large-conductance Ca(2+)-activated K+ channels (BK channels) from canine colon in artificial lipid bilayers and in excised patches. Both protocols identified 270-pS K(+)-selective channels activated by depolarization and Ca2+ (approximately 130-mV shift of half-activation voltage per 10-fold change in Ca2+) that were inhibited by extracellular tetraethylammonium (TEA) and charybdotoxin. These similarities suggest that the same BK channels are studied in the two techniques. However, we found three quantitative differences between channels in artificial bilayers and patches. 1) Channels in artificial bilayers required fivefold higher free Ca2+ or 80-mV stronger depolarization for activation. 2) The voltage dependence of TEA block was smaller for channels in artificial bilayers. The apparent distance across the membrane field for the TEA binding site was 0.031 for channels in artificial bilayers and 0.23 for channels in patches. 3) ATP (2 mM) decreased open probability (Po) of channels in artificial bilayers, whereas channels in patches were unaffected. Neither GTP nor UTP reduced Po of channels in artificial bilayers. It is possible that these differences may be due to a lack of molecular identity between the channels studied in the two protocols. Alternatively, they may be attributed to alterations in channel properties during reconstitution or to influences of the artificial lipid environment.

Adenosine Triphosphate↗

PubMed1987

A novel holder allowing internal perfusion of patch-clamp pipettes.

We describe a simple pipette holder which allows, within a single experiment, multiple exchanges of the solution inside "gigaseal" glass pipettes commonly used for electrical studies of single cells or isolated membrane patches. The design minimizes electrostatic and mechanical perturbations associated with perfusion by integrating into the holder a reservoir which is connected to a perfusion pipette fabricated from flexible, resilient quartz tubing. The tip of the perfusion pipette can be pulled to any diameter and positioned precisely within the main patch-pipette by sliding the reservoir along a guide in the holder. An open reservoir for suction driven solution exchange, and a closed reservoir for pressure driven solution exchange were developed. For the open system, the speed of solution exchange was studied as a function of the tip diameter of the perfusion pipette (approximately 22 s for a 40 micron tip diameter). Both systems were characterized using atrial myocytes (a) by examining the effects of intracellular applications of cAMP or of the catalytic subunit of protein kinase A on calcium currents in the whole cell recording mode and (b) by studying the effects of local applications of acetylcholine (ACh) on single channel currents in the isolated membrane patch mode.

Animals↗

PubMed1989

Patch clamp recordings from membranes which contain gap junction channels.

The septal membranes of the median and lateral giant axons of earthworm, which contain gap junctions, were exposed by cutting one segment of the cord. Patch recordings were obtained from the exposed cytoplasmic side of the septum. Seal resistances ranged from 2 to 15 G omega. The patch could be excised (detached) or left attached to the whole cell. Two types of channels were observed. One type was blocked by tetraethylammonium (TEA) or Cs+ and had a unitary conductance of 30-40 pS. It appears to be a K+ channel. The other channel type had a unitary conductance of 90-110 pS and was unaffected by TEA+ or Cs+. In the detached configuration the channel was shown to conduct Cs+, K+, Na+, TMA+, Cl- and TEA+ even in the presence of 2 mM Zn2+, 1 mM Ni2+, 1 mM Co2+, and 4 mM 4-aminopyridine. The conductance ratios relative to K+ were 1.0 for Cs+, 0.84 for Na+, 0.64 for TMA+, 0.52 for Cl- and 0.2 for TEA+. The channel appears to be voltage insensitive whether monitored in detached or attached recording mode. Both H+ and Ca2+ reduce the probability of opening. Thus, the 100 pS channel has many of the properties expected of a gap junction channel.

Animals↗

PubMed1989

A patch-clamp study of effects of dihydroavermectin on Ascaris muscle.

1. Effects of bath-application of the anti-parasitic agent 22,23-dihydroavermectin B1a (DHAVM 20 fM to 0.2 microM) on outside-out and inside-out patches from collagenase-treated muscle membranes of Ascaris suum were examined. 2. DHAVM was tested as a gamma-aminobutyric acid (GABA) agonist by application to outside-out patches. DHAVM failed to open characteristic GABA channels even when high concentrations (20 nM to 0.2 microM) were used. These high concentrations of DHAVM also failed to potentiate opening of channels activated by application of 3-4 microM GABA; instead they acted to depress GABA-activated channel currents by reducing mean conductances and P open. 3. In outside-out patches, low concentrations of DHAVM (1 pM to 100 pM) produced opening of 0-13 channels, the number depending on the patch. The progressive opening of channels gave rise to a 'Staircase' effect. The conductance of these channels was 9-15 pS and open times were long (greater than 100 mS). Ion substitution experiments showed these channels to be permeable to Cl. The channels were not blocked by 20 microM picrotoxin. There was a long delay (greater than 15 s) between DHAVM application and channel opening; this delay and lipophilic nature of DHAVM suggested a site of action in the lipid phase of the membrane. 4. The effect of bath-application of low concentrations of DHAVM on inside-out patches was investigated under low-Ca conditions (to avoid Ca-activated Cl channels). DHAVM (20 fM to 1 nM) did not produce opening of Cl channels but produced opening after a long delay (mean 2.5 minutes) of noisy cation-selective channels which had conductances of 5-30 pS. 5. The actions of DHAVM are discussed.

Animals↗

PubMed1985

Patch-clamp recordings of the light-sensitive dark noise in retinal rods from the lizard and frog.

In cell-attached recordings from rods in the intact lizard retina, light decreased a standing inward membrane current with a reversal potential approximately 60 mV more positive than the resting potential. The peak amplitude of saturating responses depended upon the area of recorded membrane and varied from cell to cell over approximately 100-fold range. Small patches of membrane gave variable responses to identical moderately intense flashes. Whole-cell voltage-clamp recordings were obtained on isolated frog rods with intact ellipsoids. Peak whole-cell photocurrent was related to flash intensity by a Michaelis equation with saturating response amplitudes ranging up to 30 pA in 0.1 mM-Ca2+ Ringer solution. In darkness the steady-state current-voltage relation, determined with whole-cell voltage clamp, showed outward rectification. Photocurrent had nearly constant amplitude between -80 and -10 mV, a mean reversal potential of +8 mV and recovered from flashes more slowly at positive holding potentials. Although it was not possible to resolve light-sensitive single-channel current events, power spectral analysis revealed both low- and high-frequency components of the light-sensitive noise in both cell-attached and whole-cell recordings. The low-frequency component was described by the product of two Lorentzians using time constants derived from the kinetics of the dim flash response. The high-frequency component of the light-sensitive noise was described by a single Lorentzian with a half-power frequency of 62 Hz in lizard and 212 Hz in frog. The half-power frequency was not appreciably affected by steady illumination. The Lorentzian nature of the noise suggests that the light-sensitive channel is a pore rather than a shuttle-type carrier. In cell-attached recordings the high-frequency component declined monotonically with increasing light intensity, suggesting that less than one-half of the channels are open in darkness. Furthermore, the ratio of the variance of the high-frequency noise to the mean photocurrent was independent of light intensity. Changing external Ca2+ from 0.1 to 0.5 mM reduced the ratio from 19.7 to 9.0 fA without a significant effect on the cut-off frequency of the noise. The results support the conclusion that the light-sensitive pore is opened by an internal transmitter that acts as an agonist and that both open and closed states of the pore may be blocked by external Ca2+. The conductance of the light-sensitive pore in the absence of external Ca2+ is estimated to be 1.25-2 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1989

Patch clamp of sarcolemmal spheres from stretched skeletal muscle fibers.

Stretching frog skeletal muscle fibers to the breaking point results in the rapid formation of numerous large spheres of membrane (5-80 microns diam). The surface of the spheres readily forms gigaohm (G omega) seals against patch pipettes, allowing low-noise single-channel recording. Currents recorded from patches isolated from these spheres indicate that they contain a variety of channels including 1) a small Na+-selective channel seen in the presence of veratridine, 2) a K+-selective channel which is blocked by millimolar Mg-ATP, and 3) a relatively large voltage-dependent Cl- channel which is blocked by Zn2+ and limited in selectivity over other anions [PCl/PMOPS = 3.7; MOPS, 3-(N-morpholino)propanesulfonic acid]. These channels have been described previously and have been identified as markers for sarcolemmal (SL) membrane. Accordingly, this method allows rapid and direct recording of channels in the SL membrane without first having to pretreat fibers with proteolytic enzymes to render the SL accessible to patch pipettes.

Animals↗

PubMed1985

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode.

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.

Acrosome↗

PubMed1987

In situ patch-clamp recording of calcium-activated potassium channels from an identified leech neuron.

The paired anterior lateral giant cells of the leech Haementeria have only two active voltage gated ionic currents. We took advantage of this simple complement of ionic currents to investigate the single channel properties of this cells' calcium-activated K+ current (I(K,Ca) in situ. Cell-attached patch recordings showed large, bursting events with a conductance of approximately 90-100 pS which had extrapolated reversal potential consistent with K+ events. The channel open time distribution was well described by a single exponential process while the channel closed times were bi-exponentially distributed. The results show that the single channel properties of I(K,Ca) in annelids closely resemble those of similar currents described in vertebrates.

Animals↗

PubMed1992

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores.

The effects of agents known to interfere with Ca2+ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca(2+)-activated potassium channels [K(Ca2+) channels]. In cell-attached patch experiments, the external application of caffeine (1 mM) caused a brief activation of K(Ca2+) channels in Ca(2+)-free and Ca(2+)-containing external solutions. The application of BK (10 nM) during cell stimulation by caffeine (1-20 mM) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mM) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomitant presence of both agents was due to their synergic action on internal stores and/or the external Ca2+ entry pathway resulting in an increased [Ca2+]i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca2+) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca2+ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca(2+)-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca(2+)-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP3)-dependent and a caffeine-induced Ca(2+)-release process.

Animals↗

PubMed1987

Patch-clamp study of isolated taste receptor cells of the frog.

Taste discs were dissected from the tongue of R. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110 mM KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was -65.2 mV and the slope resistance 150 to 750 M omega. Pulse-depolarization from a holding voltage of -80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at -25 mV and was half-maximal at -8 mV. Steady-state inactivation was half-maximal at -67 mV and complete at -50 mV. Peak Na current averaged -0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5 mM) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5 mM) and 4-amino-pyridine (1 mM) did not block, but 5 mM Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted the IK(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10 mM Ca caused a reversible 5- to 10-mV depolarization in the current-clamp mode. Quinine (0.1 mM, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5 mM in the external solution or 0.5 microM in the pipette) caused reversible depolarization (to -40 to -20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1992

A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.

Acetylcholine↗

PubMed1989

Patch-clamp study of cultured human sweat duct cells: amiloride-blockable Na+ channel.

The reabsorptive duct of the eccrine sweat gland has a large transepithelial conductance consisting mainly of a high conductance to Cl- and a smaller, amiloride-blockable Na+ conductance (Bijman and Frömter 1986; Quinton 1986). Cells have been cultured from sweat ducts and their properties previously studied in Ussing chambers (Pedersen 1988) and with microelectrodes (Jones et al. 1988). We have now studied the ion channels present in excised, inside-out patches of human cultured sweat duct cells, and find a marked predominance of linear, 15 pS, amiloride-blockable, low selectivity, Na+ channels. Such channels were seen in 54/92 (59%) of the patches, with up to 7 channels recorded in a single patch. Other channel types were seen at much lower densities. The prevalence of an amiloride-blockable Na+ channel in cultured duct cells clearly distinguishes these cells from cultured sweat gland secretory cells, which lack such a channel.

Amiloride↗