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At least 217 records · Page 12Linked to original sources

PubMed1993

Effect of various diuretics on membrane voltage of macula densa cells. Whole-cell patch-clamp experiments.

The tubuloglomerular feedback mechanism is inhibited by diuretics such as furosemide. For the macula densa (MD) cells similar transport systems, as present in thick ascending limb (TAL) cells, have been suggested. To examine this further, membrane voltages (Vm) of MD cells were recorded with the fast or slow whole-cell patch-clamp method. The effects of diuretics on voltages and the conductance properties of these cells were examined. Vm of MD cells measured with the whole-cell patch-clamp method were as high as those in TAL cells: -72 +/- 1 mV (n = 21). An increase in the extracellular K+ concentration by 15 mmol/l depolarized Vm of MD cells by 11 +/- 1 mV (n = 18). Ba2+ (1 mmol/l) reversibly depolarized MD cells by 10 +/- 2 mV (n = 10). Thus, MD cells possess a K+ conductance that could allow for the recycling of K+ taken up by the Na(+)-2 Cl(-)-K+ cotransporter. MD cells hyperpolarized reversibly upon addition of the loop diuretics furosemide, piretanide and torasemide, whereas muzolimine and hydrochlorothiazide, neither one acting on this cotransport system in other preparations including the TAL, had no effect on Vm. MD cells most likely possess the same cotransport system as the TAL cells, which drives NaCl reabsorption in the TAL and serves as sensor for the tubular NaCl concentration in MD cells.

Animals↗

PubMed2003

A device for automated control of pipette internal pressure for patch-clamp recording.

Formation of a high-resistance seal between the tip of a glass recording pipette and the membrane of the recorded cell is the crucial step in patch clamping, or whole cell recording with patch pipettes. Formation of the seal, and subsequent rupture of the membrane for whole cell recording, requires a specific sequence of changes in pipette internal hydrostatic pressure. Generating this sequence of pressure changes adds to the complexity of setting up, gaining proficiency, and performing experiments. Automation of routine pipette pressure manipulations would simplify seal formation, and benefit productivity. Here we describe a device that automates control of patch pipette internal pressure. Solenoid valves sequentially operated by manual switching, or external electronic control, automatically provide the necessary sequence of connections to the pipette interior. This greatly simplifies the operations performed to obtain membrane seals and whole cell recordings and improves standardization and reproducibility in patch recording.

Electronics↗

PubMed1999

Evidence for norepinephrine-activated Ca2+ permeable channels in guinea-pig hepatocytes using a patch clamp technique.

To determine whether the hepatocyte plasma membrane possesses a Ca2+ channel. we applied a patch clamp technique to isolated guinea-pig hepatocytes. In a cell-attached configuration, using an internal pipette solution of 110 mM BaCl2 or CaCl2, we observed sporadic inward single channel currents (Po = 0.004 +/- 0.002, n = 6) at various membrane potentials. The unit amplitude was 0.60 +/- 0.15 pA (n = 6) at resting membrane potential. The single channel conductance was 20.4 +/- 4.6 pS (n = 6) and this channel showed no rectification and no voltage dependence. Bay K 8644, a dihydropyridine Ca2+ channel activator, did not affect this channel activity. Although norepinephrine in the pipette solution did not activate this channel, its external application increased channel activity. These observations suggest that guinea-pig hepatocytes possess Ca2+ permeable channels that differ from the voltage-operated Ca2+ channels found in excitable cells and that such channels are responsible for the agonist-stimulated Ca2+ entry in hepatocytes.

Animals↗

PubMed1990

Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration.

Calcium channel activity is crucial for secretion and synaptic transmission, but it has been difficult to study Ca2+ channel modulation because survival and regulation of some of these channels require cytoplasmic constituents that are lost with the formation of cell-free patches. Here we report a new patch clamp configuration in which activity and regulation of channels are maintained after removal from cells. A pipette containing the pore-forming agent nystatin is sealed onto a cell and withdrawn to form an enclosed vesicle. The resulting perforated vesicle, formed from pituitary tumour cells, contains Ca2+ and K+ channels. Ca2(+)-activated K+ channels in the vesicle are activated by cyclic AMP analogues, and by a neuropeptide (thyrotropin-releasing hormone) that stimulates phosphatidylinositol turnover and inositol trisphosphate-gated Ca2+ release from intracellular organelles. Thus, the perforated vesicle retains signal transduction systems necessary for ion channel modulation. Functional dihydropyridine-sensitive Ca2+ channels (L-type) are maintained in the vesicle, and their gating is inhibited by thyrotropin-releasing hormone. Hence, this new patch clamp configuration has allowed a direct detection of the single-channel basis of transmitter-induced inhibition of L-type Ca2+ channels. The modulation of Ca2(+)-channel gating may be an important mechanism for regulating hormone secretion from pituitary cells.

Calcium↗

PubMed1993

A patch-clamp investigation of the Streptococcus faecalis cell membrane.

The patch-clamp technique was used to study the membrane of giant protoplasts from the gram-positive bacterium Streptococcus faecalis, demonstrating the presence of ion-conducting pores in the cytoplasmic membrane of procaryotes. The single channel recordings were characterized by a variety of conductances, ranging up to a few nanoSiemens, arising from stretch-activated, voltage-modulated, cooperative channels. Activation by stretch and voltage took place via both a decrease of the mean closed time and an increase of the mean open time of the channels, which are strictly controlled in intact cells, where they might constitute parts of a membrane apparatus or transport system.

Cell Membrane↗

PubMed1987

Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique.

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach of Bufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K+ selective. The probability of the channel being open (Po) increased when [Ca2+]i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca2+-activated K+ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot of Po versus membrane potential followed the Boltzmann relationship. Increasing [Ca2+]i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity of Po residing in the mean closed time. These results were verified with a series of experiments carried out at low Po (less than 0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NPo) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.

Animals↗

PubMed1990

Patch clamp studies of lymphocyte activation.

In both T- and B-lymphocyte activation, antigen receptor or mitogen stimulation results in phosphoinositide turnover, generation of InsP3 and diacylglycerol, and a sustained rise in intracellular Ca2+, from both intracellular Ca2+ stores release and enhanced transmembrane Ca2+ influx. There is also an associated K+ efflux and membrane hyperpolarization. Patch clamp studies in T and B cells have revealed the presence of several types of ion channels that apparently contribute to the ion fluxes and to the membrane potential changes associated with lymphocyte activation. Three types of T-cell channels are described in this review. First, patch clamp studies have revealed the presence of a nonvoltage-gated, Ca2+ permeable channel, the probability of whose opening increases upon exposure of the T cell to activating ligands. Enhanced opening probability appears to be mediated by the second messenger InsP3, implying that InsP3 is responsible for both intracellular Ca2+ stores release and enhanced transmembrane Ca2+ influx. Thus, the control of [Ca2+]i remains coupled to TCR/CD3 function. The Ca2+ permeable channel also undergoes a Ca2(+)-dependent inactivation process in an autoregulatory fashion. In addition, voltage-gated K+ channels, which closely resemble the delayed rectifier K+ channel of nerve and muscle, can be classified into three subtypes, according to their voltage dependence of activation, inactivation kinetics, and pharmacological sensitivity. The expression of the three K+ channel subtypes varies with the cell's developmental state and functional class. The voltage-activated K+ channel is postulated to have a role in mitogenesis, based on studies that demonstrate an increase in K+ channel amplitude in the 24-48 hr following mitogen stimulation, and on studies that demonstrate that K+ channel blockers inhibit mitogenesis in a dose-dependent manner with the same potency sequence for ion channel block. The precise functional role of the voltage-activated K+ channel remains to be determined. Finally, a Ca2(+)-activated K+ channel in T cells has recently been described. This channel presumably underlies the K+ efflux and membrane hyperpolarization that accompany the mitogen-induced increase in [Ca2+]i. Three channel types that may contribute to activation have also been described in B lymphocytes. In murine myeloma and hybridoma cells, a voltage-gated Ca2+ channel similar to Ca2+ channels in nerve, heart, and muscle is present. It is unclear whether or not this type of Ca2+ channel is present in straight B-cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗

PubMed2004

A technical consideration concerning the removal of oocyte vitelline membranes for patch clamp recording.

We have developed an efficient method for removing the vitelline membrane of Xenopus oocytes for patch clamp recording. Functional studies using oocytes as models provide insights into the biological profiles and physiological properties of ion channels. A methodological modification is described in this paper. The important feature of this modification is that protease treatment is used to remove the oocyte's vitelline membrane. This method is simple and the oocytes produced remain in a healthy state during the recording process.

Animals↗

PubMed2003

Whole-cell patch-clamp recordings from identified spinal neurons in the zebrafish embryo.

We describe a preparation for obtaining patch-clamp recordings from identified embryonic spinal cord interneurons, motoneurons and sensory neurons in an in vivo zebrafish preparation. This preparation is used to study the spatial and temporal patterns of spontaneous and touch-evoked electrical activity during the initial development of circuitry in the spinal cord. The combination of these physiological techniques with the powerful genetic and molecular tools available in the zebrafish has the potential to increase our understanding of the complex interactions between genes and electrical activity during the development of the vertebrate nervous system.

Animals↗

PubMed1996

Patch-clamp study on T-lymphocyte potassium conductance in patients with chronic renal failure.

Using the patch-clamp technique, we studied the differences in whole-cell potassium conductance (g(K)+) in T lymphocytes (TL) from three groups of patients suffering from renal failure: not dialyzed patients, dialyzed patients, and dialyzed patients treated with human recombinant erythropoietin (rHuEPO). The differences in g(K+) values in the group of not dialyzed patients in comparison with controls was not significant (p > 0.05). In the group of dialyzed patients, after roughly 6 years of the hemodialysis therapy, the g(K+) value was significantly higher than in controls. In dialyzed patients treated with rHuEPO, g(K+) value was significantly lower in comparison with control. Moreover, in dialyzed patients treated with rHuEPO, the time duration of dialysis therapy did not significantly affect the TL whole-cell conductance. We conclude that the g(K+) is changed in TL in renal failure patients and that the time duration of hemodialysis therapy as well as the use of rHuEPO affect the g(K+) value. Possible mechanisms underlying the observed changes in g(K+) values, as well as medical implications of obtained results are discussed.

Anemia↗

PubMed2005

A subsequent fit of time series and amplitude histogram of patch-clamp records reveals rate constants up to 1 per microsecond.

Fast gating in time series of patch-clamp current demands powerful tools to reveal the rate constants of the adequate Hidden Markov model. Here, two approaches are presented to improve the temporal resolution of the direct fit of the time series. First, the prediction algorithm is extended to include intermediate currents between the nominal levels as caused by the anti-aliasing filter. This approach can reveal rate constants that are about 4 times higher than the corner frequency of the anti-aliasing filter. However, this approach is restricted to time series with very low noise. Second, the direct fit of the time series is combined with a beta fit, i.e., a fit of the deviations of the amplitude histogram from the Gaussian distribution. Since the "theoretical" amplitude histograms for higher-order Bessel filters cannot be calculated by analytical tools, they are generated from simulated time series. In a first approach, a simultaneous fit of the time series and of the Beta fit is tested. This simultaneous fit, however, inherits the drawbacks of both approaches, not the benefits. More successful is a subsequent fit: The fit of the time series yields a set of rate constants. The subsequent Beta fit uses the slow rate constants of the fit of the time series as fixed parameters and the optimization algorithm is restricted to the fast ones. The efficiency of this approach is illustrated by means of time series obtained from simulation and from the dominant K+ channel in Chara. This shows that temporal resolution can reach the microsecond range.

Algorithms↗

PubMedDate not supplied

K channels in T lymphocytes: a patch clamp study using monoclonal antibody adhesion.

Ionic fluxes are thought to be involved in mediating the proliferation of peripheral blood lymphocytes (PBLs) in response to mitogenic substances. Among the earliest events occurring after the addition of mitogen to cultured lymphocytes are changes in rates of cation transport. We were interested, therefore, in the possible role of ion channels in mediating the lymphocyte proliferative response. The development of patch clamp techniques by Neher and colleagues has made it possible to study membrane conductances in a variety of small cell types. We have developed a method which uses monoclonal antibodies to make cells adhere to solid surfaces for two major reasons: (1) it is much easier to patch clamp stationary cells, and (2) the method can be used to selectively adhere a particular cell type from a heterogeneous population. We have used these techniques here to identify whole-cell potassium currents, in lymphocytes recognized by OKT11 monoclonal antibody, which are increased 1.9-fold by mitogenic stimulation.

Antibodies, Monoclonal↗

PubMed2004

The voltage-dependent nonselective cation current in human red blood cells studied by means of whole-cell and nystatin-perforated patch-clamp techniques.

Human red blood cells (RBC) can be studied by means of whole-cell and nystatin-perforated patch-clamp techniques. In 85% of the whole-cell experiments (n=86) and 69% of the perforated-patch recordings (n=13), steps to positive potentials, from a holding potential of 0 mV, induced a slow-activating non-inactivating persistent outward current which reverted at about 0 mV. The current activation phase fitted well with a two-component exponential curve. Half-maximal conductance was reached at about 42 mV. Na+ and K+ carried this current, which was not affected by 20 nM charybdotoxin or 20 mM TEA, but was reduced following a partial substitution of extracellular Cl- by tartrate. This current has characteristics similar to the single-channel currents already described in RBC and may be involved in the rapid adaptations of these cells in the circulation.

Chlorides↗

PubMedDate not supplied

Patch-clamping of the inner mitochondrial membrane reveals a voltage-dependent ion channel.

The prime function of mitochondria is to provide the cell with adenosine triphosphate (ATP). ATP synthesis is driven by the protonmotive force (delta p), which is generated and maintained across the inner mitochondrial membrane (IMM) by the activity of the respiratory chain. It is widely believed that the IMM is unlikely to contain ion channels like those present in the plasma membrane, because the high rates of ion transport characteristic of open channels would be expected to dissipate the delta p. Although the small size of the organelle has prevented the use of classical electrophysiological methods, the recent introduction of the patch-clamp technique, which allows currents to be recorded from very small cells, has enabled us to test this hypothesis. By patch-clamping the IMM, we have identified a slightly anion-selective channel, which is voltage-dependent and has a mean conductance of 107 pS in the presence of symmetrical 150 mM KCl.

Adenosine Triphosphate↗

PubMed1986

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.

Cell Membrane↗

PubMed1990

Patch-clamp profile of ion channels in resting murine B lymphocytes.

Patch-clamp studies of single ion channel currents in freshly isolated murine B lymphocytes are characterized here according to their respective unitary conductances, ion selectivities, regulatory factors, distributions and kinetic behavior. The most prevalent ion channel in murine B lymphocytes is a large conductance (348 pS) nonselective anion channel. This report characterizes additional conductances including: two chloride channels (40 and 128 pS), a calcium-activated potassium channel (93 pS), and an outwardly rectifying potassium channel which displays two distinct conductances (18 and 30 pS). Like the anion channel, both chloride channels exhibit little activity in the cell-attached patch configuration. The kinetic behavior of all of these channels is complex, with variable periods of bursting and flickering activity interspersed between prolonged closed/open intervals (dwell times). It is likely that some of these channels play an important role in the signal transduction of B cell activation.

Animals↗

PubMed1997

Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria.

After heterologous expression in E. coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments. Single channel currents across excised patches revealed an anion channel function of the PIC protein. Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC. The PIC channel activity was sensitive towards phosphate as its physiological substrate. Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode. Furthermore, the current through the channel and its gating activity were affected by divalent cations. In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25 +/- 5 pS whereas 40 +/- 10 pS was observed in the absence of divalent cations. Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+. The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane. Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found.

Carrier Proteins↗

PubMed2004

The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl- channel.

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.

Cystic Fibrosis Transmembrane Conductance Regulato↗