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PubMedDate not supplied

Patch-clamp capacitance measurements: new insights into the endocytic uptake of transferrin.

Since its introduction by Neher and Sakmann in 1976 the patch-clamp technique has been extensively used to study processes such as signalling and synaptic transmission, but also for monitoring endo- and exocytosis. Since biological membranes behave like electrical capacitors high-resolution measurements of membrane capacitance allow detection of small changes in membrane surface area that accompany exocytosis and endocytosis. We here describe our recent work on patch-clamp capacitance measurements in stably transfected HeLa cells expressing HFE, the hereditary hemochromatosis gene product, under the control of a tetracycline-sensitive promotor. By means of whole-cell and cell-attached techniques we were able to reveal transferrin-induced decreases in membrane capacitance reflecting increased endocytosis at the single cell level. Moreover, cell-attached recordings revealed significant alterations in the formation of single endocytic vesicles. Time-resolved measurements of cell membrane capacitance provide a new methodological approach to study the endocytic uptake of transferrin and its regulation by HFE, the hereditary hemochromatosis protein.

Cytoplasmic Vesicles↗
PubMed1996

Laser microsurgery of higher plant cell walls permits patch-clamp access.

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Cell Membrane↗
PubMed1983

Phospholipid bilayers made from monolayers on patch-clamp pipettes.

Phospholipid bilayers were made from phospholipid monolayers at the air/water interface on patch-clamp pipettes. Lipid bilayers were characterized using the K+ carrier nonactin and the channel formers gramicidin and alamethicin. Bilayers were also formed from monolayers spontaneously assembled in a suspension of native vesicles from cardiac sarcolemma and lobster axonal membranes and an excess of lipids. In these types of bilayers we observed several different channels including one contained in the axonal membrane that shows delayed rectifier behavior. This technique permits the study of reconstituted channels on a time scale and noise comparable to cellular patch-clamp standards.

Alamethicin↗
PubMed1994

Configuration of light responses in isolated retinal rods. A patch-clamp study.

The whole-cell patch-clamp technique was employed to investigate the light responses of single retinal rods of the frog (Rana esculenta and R. temporaria). In the majority of experiments, completely isolated cells were studied. Coupling with neighboring cells gave rise to a more complex response configuration. Responses were recorded under voltage-clamp and under current-clamp conditions. Stimulus response curves were measured in experiments with local stimuli illuminating only parts of the outer segment. Metabolic factors such as cGMP, GTP and ATP were also tested and were found to have specific and different influences on the response configurations. When the recording pipette was filled with an intracellular medium devoid of nucleotides, a retardation in the recovery of the light responses was observed during the course of an experiment. Addition of 1 mM ATP to the pipette medium prevented the larger part of the retardation, while 1 mM GTP accelerated the response recovery at the beginning of an experiment but did not prevent a subsequent retardation. Micromolar concentrations of cGMP were sufficient to elicit both a depolarization of the photoreceptor membrane and an increase in the response duration. These results show that, in single photoreceptors, the configuration of light responses not only depends on the stimulus parameters but also on those properties of the cells that are directly controlled by their nucleotide metabolism.

Adenosine Triphosphate↗
PubMed1992

A patch-clamp study of Bacillus subtilis.

In patch-clamp experiments on giant protoplasts of the Gram-positive bacterium Bacillus subtilis, membrane stretch resulted in an initial transient collapse of the membrane resistance, after which stretch-activated, voltage modulated, high-conductance channels could be observed. The channel open probability increased exponentially with applied suction and positive voltage, as a result of variations of both the mean open and the mean closed times. The substate structure and other characteristics of the electrical activity suggested the presence of a family of pores exhibiting cooperative behavior. A role in osmotic protection is suggested. In the intact bacteria, the pores may be part of an unidentified envelope apparatus, having other functions as well.

Bacillus subtilis↗
PubMed1996

Patch-clamp capacitance analysis of the effects of alpha-SNAP on exocytosis in adrenal chromaffin cells.

We have examined the effect of alpha-SNAP on exocytosis in adrenal chromaffin cells by direct assay of exocytosis using patch-clamp capacitance analysis. Cells were recorded using the whole cell patch-clamp configuration and the cells dialysed with control pipette solution or with a pipette solution containing alpha-SNAP or the deletion mutant alpha-SNAP(41-295). The deletion mutant was found to be unable to bind to syntaxin allowing a test of the requirement for syntaxin-binding for any effect of alpha-SNAP on exocytosis. Following cell dialysis for 10 minutes, cells were depolarised five times at 2 minute intervals. At each depolarisation step cells dialysed with alpha-SNAP showed a significant increase in both the initial rate and extent of exocytosis which was seen as a rise in membrane capacitance. This increase in exocytosis was not observed with alpha-SNAP(41-295) which instead produced some inhibition of the extent but had no effect on the initial rate of exocytosis. These results show directly that alpha-SNAP has a specific and marked stimulatory effect on exocytosis in chromaffin cells.

Animals↗
PubMed2004

Inward rectifying currents stabilize the membrane potential in dendrites of mouse amacrine cells: patch-clamp recordings and single-cell RT-PCR.

PURPOSE: To explore the possible existence of inward rectifying currents in the distal dendrites of amacrine cells. METHODS: Patch-clamp recordings were made from amacrine cells in a new horizontal slice preparation of mouse retina. Single-cell RT-PCR studies were performed after the patch-clamp recordings. RESULTS: In contrast to results from vertical slices or dissociated cells, all amacrine cells tested demonstrated inward rectifying currents, IIR. Within the limits of our sample, this current did not depend on the morphological and physiological type of the amacrine cell. Amacrine cells from which the dendrites had been removed did not possess detectable amounts of IIR. Pharmacological experiments with ZD7288 (100 microM) and single-cell RT-PCR from recorded cells revealed that IIR includes an h-current (I(H)) carried by hyperpolarization-activated cyclic nucleotide gated channels (HCN), HCN1 and/or HCN2 subtypes. In the presence of extracellular Cs+ (5 mM), which greatly suppressed IIR, the resting membrane conductance was reduced. IIR suppressed the generation of oscillatory potentials. Intracellular cAMP (8-cpt-cAMP, 1 mM) activated IIR. CONCLUSIONS: IIR appears to occur within dendrites of many amacrine cells, where it tends to stabilize the resting membrane potential. HCN1 and/or HCN2 channels contribute to IIR in amacrine cells. Dendritic IIR would be expected to contribute to functional independence of the distal dendrites of amacrine cells that express it.

Amacrine Cells↗
PubMed2003

Patch-clamp experiments with porins extracted from a marine bacterium (Photobacterium profundum strain SS9) and reconstituted in liposomes.

The reconstitution of bacterial porins in liposome bilayers for patch-clamp recording is well established. However, the solutions used in the dehydration, rehydration, and osmotic swelling of the liposomes have been developed for porins from enteric bacteria. Porins from marine bacteria normally function in contact with seawater whose ionic composition and osmotic pressure would appear to be incompatible with the established methods. Here, we show that, contrary to expectation, an established reconstitution and patch-clamp method works well with porins, mainly OmpH and OmpL, extracted from the deep-sea marine bacterium Photobacterium profundum strain SS9 and that seawater can be introduced at a supplementary stage.

Bacterial Outer Membrane Proteins↗
PubMed1998

Two distinct K+ channels in lamprey (Lampetra fluviatilis) erythrocyte membrane characterized by single channel patch clamp.

Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane of lamprey erythrocytes. A small-conductance, inwardly rectifying K(+)-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel conductance was 26 +/- 3 pS in isotonic (132 mM K+) solutions and 24 +/- 2 pS in hypotonic (63 mM K+) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium. Cell swelling activated another inwardly rectifying K+ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this channel, rectification was due to the block of outward currents by Mg2+ and Ca2+ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence of barium in the pipette. Neither type of channel was dependent on the presence of Ca2+ ions on the cytosolic side for activity.

Animals↗
PubMed1989

Action potentials of cultured human oat cells: whole-cell measurements with the patch-clamp technique.

Oat cells (of the small cell carcinoma of the lung) have been reported to generate calcium action potentials. The calcium channels have further been suggested to play a crucial role in the relation between oat cell carcinoma and the often associated myasthenic syndrome. We have examined cultured human oat cells (U-1690) under voltage-clamp conditions, using the patch-clamp technique. We found, contrary to previous reports, that the action potential was caused by sodium and potassium currents. No calcium current was detected under these conditions, which indicates that calcium channels, if present, are very rare. The findings restrict, but do not rule out, the hypothesis that calcium plays a key role in the carcinoma/myasthenic syndrome relation.

Action Potentials↗
PubMed1987

A patch-clamp study of mammalian platelets and their voltage-gated potassium current.

1. Mammalian platelets were freshly isolated from human, rabbit, or rat blood. The whole-cell and cell-attached voltage-clamp variations of the patch-clamp technique were employed to study the passive electrical properties and ion channels of unstimulated platelets. 2. The input capacitance of a platelet measured by the phase-sensitive detection method was about 128 fF, the input resistance of a platelet was about 59 G omega and the resting membrane potential was about -50 mV which was directly measured by a whole-cell recording in the current-clamp mode. 3. The predominant ion channel was a voltage-gated K+ channel resembling the delayed rectifier K+ channel of nerve, muscle and T-lymphocyte. There was no indication of any inward current in the platelet membrane. The activation of the K+ current could be fitted by n4 kinetics, and was half-maximal at about -35 mV. 4. The time constant of K+ current inactivation was virtually independent of voltage and varied from cell to cell. Recovery from inactivation was slow and dependent on the size and duration of the preceding conditional voltage step. Steady-state inactivation was half-maximal at about -50 mV and was complete at positive potentials. 5. The predominant single-K+-channel conductance was 9 pS and the estimated number of K+ channels per platelet was about 325, corresponding to a density of 25/micron2 apparent membrane area.

Action Potentials↗
PubMed1989

Curvature-electric effects in artificial and natural membranes studied using patch-clamp techniques.

Methods for applying sound pressure to membrane patches formed at the tips of patch-clamp pipettes have been developed. Artificial membrane patches were formed from diphytanoyl phosphatidylcholine using a pipette dipping technique. Natural membrane patches were excised (inside-out mode) from collagenase-treated locust muscle membrane. Curvature-electric signals were registered under both voltage clamp and current clamp conditions. The phenomenon of flexoelectricity in membranes has previously been attributed to curvature-induced polarization originating from the liquid crystalline properties of membranes. The estimated magnitude (2 x 10(-18) C) of the flexoelectric coefficient of the artificial lipid bilayers is consistent with previous findings while that of the muscle membrane was in certain cases several times larger. The present study is the first to report on flexoelectricity in a natural membrane and raises the question of the biological significance of this phenomenon.

Animals↗
PubMed1998

A method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels.

The aim of this project was to develop a method to enable routine application of all patch-clamp configurations to smooth muscle cells while they remain embedded in blood vessels. Small blood vessels were isolated from rabbit brain using an enzymatic and mechanical procedure. Vessels were identified under a microscope and the majority were small arterioles with a mean external diameter, in Ca2+-containing (1.5 mM) solution, of 29 microm and variable lengths of 100 microm or more. Arterioles excluded trypan blue, constricted in response to 60 mM K+ and dilated in response to levcromakalim. Patch-clamp gigaOhm seals were made regularly on smooth muscle cells embedded in arterioles. The membrane potential recorded using amphotericin-B-containing patch pipettes averaged -72 mV. Short arteriolar segments could be voltage-clamped. Injection of depolarising current or bath application of 10 mM Ba2+ induced constriction of the entire arteriolar segment. Cell-attached patch, inside-out patch and outside-out patch recordings were made readily and K+ channel unitary currents were studied. The method is readily applied and has several advantages over previous methods for the study of ion channels in smooth muscle cells. Notably, avoidance of single-cell isolation means that enzymatic treatment is minimised and cells can be studied within their normal environment of the blood vessel wall.

Animals↗
PubMed1999

Common components of patch-clamp internal recording solutions can significantly affect protein kinase A activity.

Common components of whole-cell internal recording solutions were tested both in vitro and in patch-clamp experiments for their effects on the activity of cAMP-dependent protein kinase. Potassium fluoride (KF), 440 mM trimethylamine chloride and exclusion of bovine serum albumin (BSA) decreased the activity of the enzyme, while ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and the potassium salts of aspartate, gluconate, methylsulfate and monobasic phosphate increased its activity. Addition of KF to the internal solution produced a hyperpolarizing shift in the V1/2 of Ih channel activation, consistent with the KF-induced reduction of protein kinase A activity. Therefore, consideration of the composition of internal solutions is warranted when studying channel physiology by patch-clamp techniques.

Animals↗
PubMed2005

Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp.

The authors used the PatchXpress 7000A system to measure compound activity at the hERG channel using procedures that mimicked the "gold-standard" conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress should help accelerate secondary screening for ion channel modulators and the drug discovery process.

Cell Line↗
PubMed2025

Ion-selective channels in K562 cells: a patch-clamp analysis.

Four types of ion-selective channels were found by the patch-clamp technique in the human erythroleukemia K562 cells. I) in cell-attached configuration at potentials less negative than -40 mV an 8 ps channel was detected. The potential dependence of channel activity suggests that this is the TTX-sensitive Na+ channel. II) A cation-selective channel was observed with equal permeability for Na+ and K+ and a potential-independent single-channel conductance of 19 pS. The channel is activated by intracellular Ca2+ and inhibited by TEA, III) A predominantly anion-selective channel was identified with the selectivity sequence NO3- > J- > Cl- = Br- >> SO4(2-). The single-channel conductance shows outward rectification, and is in symmetrical NaCl solution 19 pS at -60 mV and 54pS at +50 mV. The open- and closed-time distributions suggest one open and at least four closed states. At submicromolar concentrations, the open state is blocked by H2DIDS leading to channel flicker between open and blocked channel; higher concentrations (apparent KI = 6.8 uM) lead to a longer-lasting blocked state. Both components of inhibition are reversible. IV) In addition, an 8 pS, Na(+)- and K(+)- selective channel could be induced by application of palytoxin. For channel activity, the presence of extracellular Na+ is essential. It is assumed that the Na+, K(+)-pump molecule is involved in the channel formation. Similarly, it is discussed whether the anion-selective channel represents a pore conformation of an electrically silent anion exchanger.

Acrylamides↗
PubMed2001

Whole-cell patch-clamp: true perforated or spontaneous conventional recordings?

Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.

Animals↗