SEARCH · PubMed Health

Results for “patch clamp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43Linked to original sources

PubMed2005

Melanotrope cells of Xenopus laevis express multiple types of high-voltage-activated Ca2+ channels.

Pituitary melanotrope cells are neuroendocrine signal transducing cells that translate physiological stimuli into adaptive hormonal responses. In this translation process, Ca2+ channels play essential roles. We have characterised which types of Ca2+ current are present in melanotropes of the amphibian Xenopus laevis, using whole-cell, voltage-clamp, patch-clamp experiments and specific blockers of the various current types. Running an activation current-voltage relationship protocol from a holding potential (HP) of -80 mV/or -110 mV, shows that Xenopus melanotropes possess only high-voltage activated (HVA) Ca2+ currents. Steady-state inactivation protocols reveal that no inactivation occurs at -80 mV, whereas 30% of the current is inactivated at -30 mV. We determined the contribution of individual channel types to the total HVA Ca2+ current, examining the effect of each channel blocker at an HP of -80 mV and -30 mV. At -80 mV, omega-conotoxin GVIA, omega-agatoxin IVA, nifedipine and SNX-482 inhibit Ca2+ currents by 21.8 +/- 4.1%, 26.1 +/- 3.1%, 24.2 +/- 2.4% and 17.9 +/- 4.7%, respectively. At -30 mV, omega-conotoxin GVIA, nifedipine and omega-agatoxin IVA inhibit Ca2+ currents by 33.8 +/- 3.0, 24.2 +/- 2.6 and 16.0 +/- 2.8%, respectively, demonstrating that these blockers substantially inhibit part of the Ca2+ current, independently from the HP. We have previously demonstrated that omega-conotoxin GVIA can block Ca2+ oscillations and steps. We now show that nifedipine and omega-agatoxin IVA do not affect the intracellular Ca2+ dynamics, whereas SNX-482 reduces the Ca2+ step amplitude. We conclude that Xenopus melanotrope cells express all four major types of HVA Ca2+ channel, as well as the resulting currents, but no low-voltage activated channels. The results provide the basis for future studies on the complex regulation of channel-mediated Ca2+ influxes into this neuroendocrine cell type as a function of its role in the animal's adaptation to external challenges.

Animals↗

PubMed2001

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter.

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.

Animals↗

PubMed1995

Altered beta-adrenergic and muscarinic response of CFTR Cl- current in dialyzed cardiac myocytes.

Autonomic regulation of the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current was studied in isolated guinea pig ventricular myocytes using various configurations of the whole cell patch-clamp technique. When currents were recorded using the conventional patch-clamp technique, it was possible to continue to activate the Cl- current on repeated exposure to isoproterenol (Iso) for up to 60 min after initiating dialysis. However, there was significant rundown of the magnitude of the Cl- current response to the maximally stimulating concentrations of Iso. In addition, the concentration of Iso that produced half-maximal activation of the Cl- current (K1/2) increased with time. Conversely, the K1/2 for acetylcholine inhibition of the Iso-activated current decreased with time. When currents were recorded using the perforated patch-clamp technique, the sensitivity to both beta-adrenergic- and muscarinic-receptor stimulation was stable. Immediately after initiation of dialysis with the conventional patch-clamp technique, the sensitivity to Iso was nearly identical to that determined using the perforated patch-clamp technique. However, the initial sensitivity to muscarinic-receptor activation was significantly greater. These results indicate that cell dialysis associated with conventional patch-clamp techniques not only results in a time-dependent rundown of current amplitude, but it also significantly alters the concentration dependence of beta-adrenergic and muscarinic-receptor regulation of ion channel function.

Acetylcholine↗

PubMed1996

Na(+)-permeable channels induced by maitotoxin in guinea-pig single ventricular cells.

The characteristics of maitotoxin-induced single channel currents were studied in guinea-pig single ventricular cells using the cell-attached or inside-out configuration of the patch clamp. When the patch electrode was filled with normal Tyrode solution containing 10 nM maitotoxin, elementary currents flowing through the single channel were observed in the cell-attached patch. The amplitude of the single channel current at the resting potential was 1.6 +/- 0.1 pA. The current-voltage relation of the current was linear and the single channel conductance was 16.0 +/- 0.9 pS. The distribution of open times was fitted by a single exponential function (decay time constant: 27 ms), while that of closed times was fitted by the sum of two exponential functions (decay time constants: 1.6 and 34 ms). When the electrode solution was filled with the Ca(2+)-free Tyrode solution, maitotoxin also induced single channel currents with parameters similar to those in the normal Tyrode solution. Under inside-out patch clamp conditions and in 150 mM Na+ solution on both sides of the patch membrane, maitotoxin also induced single channel currents. Choline+ could not substitute for Na+. These results indicate that maitotoxin induces single ionic channels irrespective of the presence or absence of Ca2+ and that the charge carrier of the single channel current is Na+ rather than Ca2+. The increase in Na+ permeability through maitotoxin-induced channels may be possibly responsible for its biological actions.

Animals↗

PubMed1991

A localized zone of increased conductance progresses over the surface of the sea urchin egg during fertilization.

Although activation of a sea urchin egg by sperm leads to three phases of membrane conductance increase in the egg, the mechanism by which the sperm causes these conductance changes is not known. We used the loose patch clamp technique to localize the conductance changes in voltage clamped eggs. A patch of the egg's membrane was isolated from the bath by pressing the loose patch clamp pipette against the egg surface. Sperm added to the bath attached to the surface of the egg in a region other than at the isolated membrane patch. During phase 1 of the activation current, no changes of the membrane conductance were detected. At the time of, and subsequent to the onset of phase 2, large currents recorded between the interior of the patch pipette and the bath were attributed to changes of the seal resistance between the surface of the egg and the pipette. A local change of membrane conductance was observed during phase 2 despite the changes of seal resistance. During phase 2, the large amplitude and short duration of the local membrane conductance increase relative to the membrane, conductance increase for the whole egg during phase 2 indicated that the conductance increase occurred over the entire surface of the egg, but not simultaneously. The time when the peak conductance for the membrane patch occurred, relative to the time of onset for phase 2 in the whole egg, depended on the distance, measured in a straight line, between the site of sperm attachment and the tip of the pipette. These data indicate that the localized conductance increase progressed over the surface of the egg from the site of sperm attachment to the opposite pole of the egg. It is proposed that the local conductance increase, the cortical reaction, and the change of seal resistance are all evoked by a common cytoplasmic message that progresses throughout the cytoplasm of the egg from the site of sperm attachment to the opposite pole of the egg.

Animals↗

PubMed1993

K+ channels in the basolateral membrane of rat cortical collecting duct.

Impalement studies in isolated perfused cortical collecting ducts (CCD) of rats have shown that the basolateral membrane possesses a K+ conductive pathway. In the present study this pathway was investigated at the single-channel level using the patch-clamp technique. Patch-clamp recordings were obtained from enzymatically isolated CCD segments and freshly isolated CCD cells with the conventional cell-free, cell-attached and the cell-attached nystatin method. Two K+ channels were found which were highly active on the cell with a conductance of 67 +/- 5 pS (n = 18) and 148 +/- 4 pS (n = 21) with 145 mmol/l K+ in the pipette. In excised patches the first channel had a conductance of 28 +/- 2 pS (n = 15), whereas the second one had a conductance of 85 +/- 1 pS (n = 53) at 0 mV clamp voltage with 145 mmol/l K+ on one side and 3.6 mmol/l K+ on the other side of the membrane. So far it has not been possible to characterize the smaller channel further. Excised, and with symmetrical K+ concentrations of 145 mmol/l, the intermediate channel had a linear conductance of 198 +/- 19 pS (n = 5). After excision in the inside-out configuration the open probability (Po) of this channel was low (0.18 +/- 0.05, n = 13) whereas in the outside-out configuration this channel had a threefold higher Po (0.57 +/- 0.04, n = 12). Several inhibitors were tested in excised membranes. Ba2+ (1 mmol/l), tetraethylammonium (TEA+, 10 mmol/l) and verapamil (0.1 mmol/l) all blocked this channel reversibly.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed2003

Automated electrophysiology: high throughput of art.

Electrophysiological measurements, in particular, patch clamping, have long been regarded as the "gold standard" for assaying ion channels. Despite its high information content, the technique suffers from laborious, manual processing by highly skilled workers and extremely low throughput. Recently, a number of researchers have started to automate patch clamping by either automating conventional micropipette-based patch clamping or developing planar microelectrode arrays. This article reviews the brief history of these emerging technologies and discusses the technical details, advantages, and disadvantages of each approach and technique. As will be evident from the discussion, two types of automated patch-clamping technologies are emerging. The first places emphasis on data quality, comparable to conventional patch clamping, and the second has much higher throughput. Future developments will include sophisticated patch-clamping devices with both high-quality data and high throughput capabilities and further integration of patch clamping with other cell-based assays.

Animals↗

PubMed1995

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.

Amino Acid Sequence↗

PubMed1994

Cl- currents of unstimulated T84 intestinal epithelial cells studied by intracellular recording.

The ionic currents spontaneously present in T84 intestinal epithelial cells, a line of colonic carcinoma origin, have been studied using the whole-cell recording mode of the patch-clamp technique and the single-electrode voltage-clamp method. Patch-clamp experiments showed that nonstimulated T84 cells already possess large currents but that these tend to disappear during the course of the experiments, presumably through the dialysis of some essential cytoplasmic component against the micropipette solution. The main charge carrier in these experiments appears to be Cl- as judged from ion replacement. Microelectrode impalement of T84 cells gave a membrane potential of around -30 mV, similar to the equilibrium potential for Cl- estimated from previously published values for intracellular Cl- concentration. Voltage-clamp experiments with a single microelectrode revealed three kinetically distinguishable current patterns; currents decaying during hyperpolarizing voltage pulses, currents slowly activating during hyperpolarizing pulses and time-independent currents. The appearance of these distinct kinetic patterns was not predictable from cell to cell, and was not dependent on extracellular Ca2+. Ionic replacement experiments suggest that the charge carrier was always Cl-, regardless of the kinetic pattern observed. No K+ currents appear to be present in the non-stimulated T84 cells. Exposure of T84 cells to the muscarinic agonist carbachol induced a shift in the membrane potential towards more negative values, consistent with an activation of a K+ conductance. Thus, we suggest that the resting membrane potential in T84 cells is determined by the distribution of Cl-. This might imply that activation of K+ conductance could by itself support secretion by T84 monolayers through tonically active Cl- channels.

Carbachol↗

PubMed1993

Regulation and possible physiological role of the Ca(2+)-dependent K+ channel of cortical collecting ducts of the rat.

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca(2+)-dependent and a small Ca(2+)-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 mumol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mumol/l Ca2+ activity the open probability (Po) of this channel was pH-dependent. At pH 7.0 Po was decreased to 4 +/- 2% (n = 9) and at pH 8.5 it was increased to 425 +/- 52% (n = 9) of the control. At this low Ca2+ activity the Po of the channel was reduced by 1 mmol/l ATP to 8 +/- 4% (n = 6). Cell swelling activated the large-conductance K+ channel (n = 14) and hyperpolarized the membrane potential of the cells by 9 +/- 1 mV (n = 23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1991

In vitro denervation of frog skeletal muscle: expression of several conductance classes of nicotinic receptors.

The question arose as to whether the altered kinetic properties of the nicotinic acetylcholine receptor-ion channel complex (AChR) observed after muscle denervation could be due to post-translational modifications. Single AChR properties of in vitro denervated skeletal muscle were studied using the cell-attached patch-clamp technique. Patch-clamp recordings from junctional regions of fibers daily treated with cycloheximide, a protein synthesis inhibitor, and phenylmethylsulfonyl fluoride, a protease inhibitor, revealed single conductance class of acetylcholine (ACh)-activated currents after acute dissociation and several conductance classes 3-14 days after muscle denervation. alpha-Bungarotoxin blocked all conductance states induced by ACh. Biochemical assays carried out on the same experimental conditions disclosed that chronic treatment of the fibers with cycloheximide inhibited the incorporation of [3H]leucine into precipitable protein. The present findings provided evidence that the altered functional properties of nicotinic receptors after muscle denervation may occur without new transcription.

Acetylcholine↗

PubMed2003

High quality ion channel analysis on a chip with the NPC technology.

In evaluating ion channel function, electrophysiology, e.g., patch clamping, provides the highest information content. For the analysis of ion channel-modulating compounds, one variant of the patch-clamp technique, the whole-cell configuration, is particularly useful. We present here patch-clamp recordings in the whole-cell configuration and single channel recordings performed with planar patch-clamp chips, which are microstructured from borosilicate glass substrate. The chips are used in the Port-a-Patch, an ion channel research/screening instrument that enables automated patch-clamp experiments on a single cell. A software runs the experiment by executing user-determined protocols for cell positioning, as well as for electrical stimulation and current readout. In various electrophysiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated. Quantitative pharmacological experiments are performed on sodium channels expressed in HEK cells using solution volumes in the low microliter range. The exceptionally low volume consumption in the experiments make the system attractive for work on rare or expensive compounds. Due to the low volumes necessary, a rapid solution exchange is facilitated, which is shown on RBL cells. The patch-clamp chip enables a rapid and precise perfusion, allowing sophisticated investigations on ion channel function with the Port-a-Patch.

Algorithms↗

PubMed1997

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro.

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235-2245, 1997. The hyperpolarization-activated current (Ih) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21-30 days old) in 400 micro;m tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current (Ih) was voltage and time dependent. The current just was seen at a membrane potential of -70 mV and was activated fully at -140 mV. The voltage value of half-maximal activation of Ih was -78.0 +/- 6.0 (SE) mV. The rate of Ih activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 +/- 27 ms at -100 mV to 186 +/- 11 ms at -140 mV. Reversal potential (Eh) of Ih current was more positive than the resting potential. Raising the extracellular potassium concentration shifted Eh to a more depolarized value, whereas lowering the extracellular sodium concentration shifted Eh in a more negative direction. Ih was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about -140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about -60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than -70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. Ih in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.

Animals↗

PubMed1987

Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid.

1. The properties of fast transient Na channels induced in the Xenopus laevis oocyte plasma membrane after injection of the oocyte with foreign messenger ribonucleic acid (mRNA) were investigated with the whole-cell voltage clamp and with the patch-clamp technique. 2. The time course of expression and the effect of the metabolic inhibitors actinomycin D and tunicamycin were studied. The rate of channel insertion reached a maximum only about 3 days after injection with mRNA and corresponded to the incorporation of 20 active channels/s, into a single oocyte. When applied intracellularly tunicamycin blocked the appearance of active channels nearly completely while actinomycin D added to the medium had no effect. 3. The whole-cell currents showed activation and inactivation properties reminiscent of skeletal muscle Na+ currents. The maximal peak current amplitude was 6 microA. Tetrodotoxin blocked the observed transient inward current. 50% inhibition was observed at 10 nM concentration. Veratridine depressed inactivation of the current and led to prolonged tail currents. 4. After removal of the surrounding layers of the oocyte tight seals were obtained with a patch-clamp electrode pushed on the surface membrane. Single-channel currents endogenous to the oocyte and Na+-channel currents induced by injected mRNA could be recorded. The single-channel slope conductance of the latter was 12-15 pS. Two different types of kinetic behaviour were evident from an analysis of single-channel currents and ensemble average currents. One type showed fast inactivation (tau less than 1 ms) and brief channel openings (less than 1 ms) whereas the second type was characterized by slower inactivation and a bursting behaviour. 5. When veratridine (75 microM) was present in the pipette solution the single-channel behaviour was modified in a complex manner. In addition to the channel openings with normal conductance a second open state was observed with a slope conductance of 3.5 pS. This second type of channel opening could still be recorded after return to the holding potential. Its final closure followed an exponential time course with a constant time constant of 0.5 s at -100 mV. These events probably underlie the tail currents in the whole-cell configuration. 6. The Xenopus oocyte represents a useful system for the study of the expression of channels induced by foreign mRNA, for the characterization of their single-channel behaviour and for the investigation of the action of pharmacologically active substances on these channels. This system may prove useful for the study of channels that are not accessible to patch-clamp experiments 'in situ'.

Action Potentials↗

PubMed1996

Gene targeting of CFTR DNA in CF epithelial cells.

A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wt homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell patch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.

Cell Line↗

PubMed1996

The relationship between the resistance of a membrane patch and predicted changes in total patch circuit resistance secondary to spontaneous or induced alterations in patch geometry.

The objective of this study was to determine the relationship between the magnitude of the membrane resistance in the free area of a cell-attached patch-clamp recording and the change in total patch circuit resistance that would be produced by the introduction of a series resistance, such as would be observed upon acquiring a patch-clamped membrane vesicle. The results describe a method for determining the magnitude of the membrane resistance in the free area of a membrane patch, and demonstrate that: (a) at a given value of shunt resistance, areas of membrane with higher resistivity produce smaller proportional increases in total patch circuit resistance upon acquiring a membrane vesicle; and (b) a presumption of spherical vesicle formation provides a lower limit estimate of the membrane resistance. The described procedures and relationships are useful in developing new techniques for examining channel activity in membrane patches where individual events are below the present limits of detection, for examining changes in membrane resistivity and/or shunt resistance in patches undergoing cytoskeletal re-organization, and for assessing the potential influence of series resistance changes on single channel parameters in longer term cell-attached patch-clamp recordings.

Animals↗

PubMed1999

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane.

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Biodegradation, Environmental↗

PubMed1996

Attenuation of stimulated Ca2+ influx in colonic epithelial (HT29) cells by cAMP.

In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity ([Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3',5'-cyclic monophosphate (cAMP) (forskolin 10 micromol/l = FOR) on the Ca2+ transient in the presence of CCH (100 micromol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca2+ ([Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 +/- 0.12 to 1.8 +/- 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (Vm) of the patch-clamped cells significantly and reversibly from -54 +/- 7.4 to -27 +/- 5.9 mV (n = 6). In additional experiments Vm was depolarized by 15-54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in [Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in Vm and change in [Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.

Adenosine Triphosphate↗