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PubMed1995

The electrophysiologic effects of endothelin. A patch clamp study in guinea pig ventricular myocytes.

To study the electrophysiologic effects of endothelin-1 (ET-1), we used patch clamp and glass microelectrode techniques to investigate the effects of ET-1 on cardiac L-Ica, Ik and Ik2 in guinea pig ventricular myocytes. The prolongation of APD50 was induced and EADs was triggered by 50 nM ET-1 perfusion. L-Ica and Ik were enhanced by various ET-1 concentration from 1 to 50 nM with dose-dependence. Their steady-state activations of L-Ica and Ik shifted left with ET-1 concentration increments. ET-1 elicited a kind of GTP-dependent inward rectifier K+ current having a mean conductance of 82.36 +/- 1.27 pS. The open time and close time (both interburst intervals and burst durations) abbreviated with ET-1 concentration increase. The results suggested that EADs -ET evoked was ascribed to the prolongation on the plateau level, which resulted from L-Ica enhancement. The ET- evoked inward rectifier K+ current should be further studied.

Action Potentials↗

PubMed1998

Modalities of distortion of physiological voltage signals by patch-clamp amplifiers: a modeling study.

An extensive evaluation of the possible alterations affecting physiological voltage signals recorded with patch-clamp amplifiers (PCAs) working in the current-clamp (CC) mode was carried out by following a modeling approach. The PCA output voltage and current signals obtained during CC recordings performed under simplified experimental conditions were exploited to determine the equations describing the generation of error currents and voltage distortions by PCAs. The functions thus obtained were used to construct models of PCAs working in the CC mode, which were coupled to numerical simulations of neuronal bioelectrical behavior; this allowed us to evaluate the effects of the same PCAs on different physiological membrane-voltage events. The models revealed that rapid signals such as fast action potentials are preferentially affected, whereas slower events, such as low-threshold spikes, are less altered. Prominent effects of model PCAs on fast action potentials were alterations of their amplitude, duration, depolarization and repolarization speeds, and, most notably, the generation of spurious afterhyperpolarizations. Processes like regular firing and burst firing could also be altered, under particular conditions, by the model PCAs. When a cell consisting of more than one single intracellular compartment was considered, the model PCAs distorted fast equalization transients. Furthermore, the effects of different experimental and cellular parameters (series resistance, cell capacitance, temperature) on PCA-generated artifacts were analyzed. Finally, the simulations indicated that no off-line correction based on manipulations of the error-current signals returned by the PCAs can be successfully performed in the attempt to recover unperturbed voltage signals, because of alterations of the overall current flowing through the cell-PCA system.

Action Potentials↗

PubMed1991

The effects of muscarine and adrenaline on patch-clamped frog cardiac parasympathetic neurones.

1. The whole-cell patch-clamp technique was used to record membrane currents from neurones which were acutely dissociated from the intra-atrial parasympathetic ganglia of Rana pipiens. The effects of muscarine and adrenaline were observed at a holding potential of -30 mV. Extracellular potassium concentration ([K+]o) was 2, 6 or 20 mM. 2. Muscarine (10 microM) produced inward current in thirteen cells, outward current in eighteen cells and seven cells were unaffected. Inward currents were observed in six out of ten neurones in which the intracellular solution contained adenosine triphosphate (ATP; 100 microM) and outward currents were seen in eleven out of fourteen neurones which contained adenosine 3',5'-cyclic monophosphate (cyclic AMP; 100 microM). 3. In five out of nine cells tested, the inward current produced by muscarine was attributable to a 30% depression of a voltage-dependent current which resembled the M-current (IM). Muscarine-induced inward current in the other four cells involved a steady-state conductance increase that reached a null potential at -10 mV. Modest IM suppression also contributed to the response in three of these four cells. 4. Adrenaline (10 or 100 microM) produced inward currents in twelve cells, outward current in ten cells and three cells were unaffected. Outward currents were only seen in cells which contained ATP or cyclic AMP (ten out of sixteen cells) whereas inward currents were seen in eight out of nine cells which did not contain adenosine nucleotides. These inward currents were always attributable to IM suppression. 5. The outward currents induced by muscarine and adrenaline resulted from an increase in a potassium conductance (GK) that exhibited inward rectification.

Adenosine Triphosphate↗

PubMed1997

An operational amplifier B1404UD1A-1 in the patch-clamp current-to-voltage converter.

The applicability of the home-made operational amplifier B1404UD1A-1 in a patch-clamp current-to-voltage converter was analyzed. Its parameters (background noise, input bias current, and gain-bandwidth product) were estimated. Schematic solutions and practical recommendations for the use of this amplifier in a current-to-voltage converter were given. Based on the background noise and frequency parameters of the converter, we found that this device can be used for measuring ion channel currents with a high sensitivity and within a broad frequency range (0.055 pA, to 1 kHz; 0.4 pA, to 10 kHz). An example of the converter application in experiments is given.

Amplifiers, Electronic↗

PubMed2000

Effects of extracellular Ca2+ on membrane and seal resistance in patch-clamped rat thalamic and sensory ganglion neurons.

We studied the effects of [Ca2+]ext changes on seal resistance in patch-clamp experiments. Recordings were made on rat peripheral and thalamic neurons. Increasing [Ca2+]ext from 0.5 to 4.5 mM, reduced the ionic currents evoked at potentials from -100 to +50 mV, in cell-attached recordings, in all the neurons tested. The effect was greater at negative potentials. The change in seal conductance (deltaG) decreased with higher resistance seals and became very low over 1 Gohm (<0.5 nS). However, the ratio deltaG/G(0.5 Ca2+) rose from close to 0 up to 0.6, indicating that Ca2+ has a stronger effect when the microelectrode and the membrane are sealed more tightly. These findings suggest that changes in seal resistance may be misleading in experiments in which extracellular Ca2+ changes are used.

Animals↗

PubMed2003

Influence of the whole-cell patch-clamp configuration on electrophysiological properties of the voltage-dependent sodium current expressed in MDA-MB-231 breast cancer cells.

Voltage-gated ionic channels are known to be involved in oncogenesis. However, only a few studies describe the functional characteristics of these channels or the mechanisms by which they are involved in the proliferation and invasive processes. Breast cancer cells proliferate and migrate under the constant activation of growth factors, hormones, extracellular matrix interactions, etc. It would thus not be surprising if the activity of the ionic channels was modulated by intracellular regulation pathways such as kinases or phosphatases, which in turn can affect oncogenic properties. In the present study, we investigated some of the electrophysiological properties of the fast inward sodium current found in the breast cancer cell line MDA-MB-231 with two configurations of the patch-clamp technique. With perforated patch, a configuration which allows to keep the cytoplam intact, the mean current amplitude was lower, the relative conductance-voltage relationship was shifted to more positive potentials and the recovery from inactivation was accelerated when compared to ruptured patch, where the cytoplasm is dialysed by the intrapipette solution. There was no difference in availability-voltage (pseudo-steady-state inactivation) relationship and in time to peak of the current. These results suggest that regulation mechanisms, possibly involving kinases or phosphatases, are switched off when the cytoplasm is diluted. We propose that such a regulation can modulate the functioning of the channels even in the absence of membrane voltage changes, which in turn can affect oncogenic properties. This finding is of importance when evaluating the physiopathological role of ionic channel in cancer development.

Animals↗

PubMed1993

GTP gamma S stimulates exocytosis in patch-clamped rat melanotrophs.

We investigated the molecular mechanisms regulating exocytosis in patch-clamped melanotrophs by measuring the membrane capacitance. Ca(2+)-dependent exocytosis could be induced by membrane depolarization or by including solutions containing 2 microM free Ca2+ in the patch pipette. Ca(2+)-dependent exocytosis was inhibited by GDP beta S, suggesting involvement of a GTP-binding protein. The hydrolysis-resistant GTP analogs, GTP gamma S and GppNHp, were able to stimulate exocytosis at low free Ca2+ concentrations. The stimulatory response to GTP gamma S was abolished by both GDP beta S and GTP. The latter suggests that a sustained activation of a GTP-binding protein is necessary for exocytosis. This behavior is similar to the stimulation of exocytosis by guanine nucleotides in mast cells and other nonexcitable cells and suggests a common regulatory mechanism.

Animals↗

PubMed2004

Retinal bipolar cell input mechanisms in giant danio. II. Patch-clamp analysis of on bipolar cells.

Glutamate receptors on giant danio retinal on bipolar cells were studied with whole cell patch clamping using a slice preparation. Cone-driven on bipolars (Cbs) and mixed-input on bipolars (Mbs) were identified morphologically. Most Cbs responded to the excitatory amino acid transporter (EAAT) substrate d-aspartate but not to the group III metabotropic glutamate receptor (mGluR) agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) or the AMPA/kainate receptor agonist kainate, suggesting EAATs are the primary glutamate receptors on Cbs. The EAAT inhibitor dl-threo-beta-benzyloxyasparate (TBOA) blocked all light-evoked responses of Cbs, suggesting these responses are mediated exclusively by EAATs. Conversely, all Mbs responded to d-aspartate and l-AP4 but not to kainate, indicating they have both EAATs and group III mGluRs (presumably mGluR6). The light responses of Mbs involve both receptors because they could be blocked by TBOA plus (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III mGluR antagonist) but not by either alone. Under dark-adapted conditions, the responses of Mbs to green (rod-selective) stimuli were reduced by CPPG but enhanced by TBOA. In contrast, both antagonists reduced the responses to red (cone-selective) stimuli, although TBOA was more effective. Furthermore, under photopic conditions, TBOA failed to eliminate light-evoked responses of Mbs. Thus on Mbs, rod inputs are mediated predominantly by mGluR6, whereas cone inputs are mediated mainly by EAATs but also by mGluR6 to some extent. Finally, we explored the interactions between EAATs and mGluR6 in Mbs. Responses to d-aspartate were reduced by l-AP4 and vice versa. Therefore mGluR6 and EAATs suppress each other, and this might underlie mutual suppression between rod and cone signals in Mbs.

Aminobutyrates↗

PubMed1992

Synergistic effect of light and fusicoccin on stomatal opening : epidermal peel and patch clamp experiments.

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K(+) channel blocker, tetraethyl-ammonium(+) (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (-60 millivolts) at which K(+) channels are inactivated. These results are indicative of FC activation of a H(+) pump. FC effects on the magnitude of inward and outward K(+) currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H(+) ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.

Journal Article↗

PubMed1990

A patch clamp study of muscarinic excitation of the rat adrenal chromaffin cells.

A patch clamp study was performed to determine the ionic mechanisms underlying the muscarinic excitation of rat adrenal chromaffin cells. Burst-like, single-channel currents with brief openings were recorded during cell-attached or cell-free patch recording when the pipette solution contained high K+ media (70-140 mM), and the currents were recorded at potentials near the resting membrane potential. The amplitude and frequency of channel openings were dependent on the membrane potential and the K+ concentration of the external medium. Moreover, the single-channel currents observed with high K+ outside the membrane during inside-out recording were suppressed by the addition of a K+ channel blocker, tetraethylammonium, inside the membrane. These results suggest that the single K+ currents recorded in the present study had properties similar to those of the resting or muscarine-activated K+ currents in atrioventricular cells of the rabbit heart. During cell-attached patch recording with the high K+ medium in the recording pipette, muscarine at concentrations of 10(-5) to 10(-4) M dose dependently decreased the frequency of the channel openings but did not affect the current-voltage relationship or the time constants of open and close time histograms. These results indicate that muscarinic-induced suppression of K+ currents is caused by a decrease in the number of active K+ channels at the resting membrane potential.

Animals↗

PubMed1993

Multiple levels of native cardiac Na+ channels at elevated temperature measured with high-bandwidth/low-noise patch clamp.

Currents through single Na+ channels were studied in cell-attached patches of enzymatically dispersed heart cells of the mouse with a low-noise patch-clamp technique that allows evaluation of current levels at temperatures of up to 35 degrees C with bandwidths of up to 13 kHz. Noise arising from the pipette and the holder was reduced by the use of short (total length 8 mm) patch pipettes, which were sealed at their end with oil and inserted for only 1.5 mm into an appropriately tipped holder. At 9 degrees C (filter 5 kHz), channel openings were regularly dominated by one open level, and amplitude histograms could be fitted with high accuracy with a sum of Gaussian curves. Above 24 degrees C (filter 10 or 13 kHz), however, channel-open levels were heterogeneous with maximum levels of up to 4.5 pA at -50 mV. Amplitude histograms with improved resolution, based on variance calculation with window widths of 75 microseconds or 195 microseconds, confirmed the observed heterogeneity of open levels. Regular level patterns were not found. The frequency of the largest levels strongly varied from patch to patch and intermediate levels were always the most frequent. A corresponding dissociation of amplitudes was also observed at 35 degrees C. Averaged currents, formed from trace ensembles including only levels below arbitrarily set borders, obeyed equal kinetics. It is concluded that at low temperature the conductance of single Na+ channel currents is much more homogeneous than at 24 degrees C and above, where the same channels have multiple open states with different conductance.

Animals↗

PubMed1995

Patch-clamp analysis of glycine-induced currents in chick ciliary ganglion neurons.

1. The whole-cell configuration of the patch-clamp technique was used to analyse currents induced by glycine in chick ciliary ganglion neurons freshly dissociated from 14- to 15-day-old embryos. 2. Application of glycine to cells voltage-clamped at -60 mV induced inward currents in all neurons tested. Dose-response curves yielded an EC50 of about 50 microM. Similar responses were elicited by beta-alanine and taurine though higher concentrations were required. 3. Strychnine reversibly inhibited the glycine-induced responses. The effect was dose dependent with a half-maximal effect being obtained with 20 nM strychnine. 4. Glycine-induced currents were inhibited by 100 microM Zn2+. The inhibition had slow rates of onset and recovery, in contrast to Zn2+ inhibition of GABA responses. Both bicuculline and d-tubocurarine inhibited glycine responses in a dose-dependent manner. 5. The steady-state I-V curve for glycine-induced currents was linear over the range -60 to +60 mV, but showed an outward rectification at very hyperpolarized membrane potentials. The reversal potential of glycine-induced currents shifted with changes in intracellular chloride concentration in a manner expected for chloride-selective channels. 6. Expression of functional glycine receptors during development was examined at embryonic day 8 (E8), 11, 14, and 18. The mean peak current was about 60-fold larger at E14 than at E8 and vastly exceeded changes in cell size. During the same period, responses to GABA increased only 2-fold in amplitude and correlated with changes in cell size.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Antagonism of nicotinic receptors of rat chromaffin cells by N,N, N-trimethyl-1-(4-trans-stilbenoxy)-2-propylammonium iodide: a patch clamp and ligand binding study.

The effect of the oxystilbene derivative F3 was tested on nAChRs of whole-cell patch-clamped rat chromaffin cells in vitro and of rat adrenal gland membranes using (125)I-epibatidine. F3 (30 nM) rapidly and reversibly blocked inward currents generated by pulse applications of nicotine, shifting the dose-response curve to the right in a parallel fashion without changing the maximum response. The action of F3 was voltage insensitive and not due to altered current reversal potential. The R isomer of F3 was more potent (IC(50) = 350+/-30 nM) than its S-enantiomer (IC(50) = 1.5+/-0.3 microM). Nicotine-evoked currents were insensitive to 10 microM alpha-bungarotoxin. Equi-amplitude currents evoked by nicotine or epibatidine were similarly antagonized by R-F3 in a reversible fashion. Epibatidine-evoked currents readily produced receptor desensitization. Adrenal membranes specifically bound (125)I-epibatidine with a single population of binding sites endowed with high affinity (K(D) = 159 pM) and B(max) of 6.5+/-1.3 fmol mg(-1) of protein. (125)I-epibatidine binding was specifically displaced by cytisine (K(i) = 68 nM) or ACh (K(i) = 348 nM). F3 specifically displaced (125)I-epibatidine binding although with lower affinity (K(i) = 29.6 microM) than in electrophysiological experiments. (125)I-epibatidine binding to rat adrenal tissue was insensitive to alpha-bungarotoxin which readily antagonized (125)I-epibatidine binding to bovine adrenal tissue. The present results suggest that F3 is a relatively potent and apparently competitive antagonist of nAChRs on rat chromaffin cells. Since previous studies have indicated that F3 targets different subtypes on chick neuronal tissue, it appears that nAChRs display interspecies differences to be considered for drug development studies.

Adrenal Glands↗

PubMed1996

Noradrenaline inhibition of Ca2+ channels and secretion in single patch-clamped insulinoma cells.

Noradrenaline effects on voltage-operated calcium channels and exocytosis were studied, for the first time, in single patch-clamped RINm5F insulin-secreting cells. Noradrenaline, despite small and variable inhibition of calcium currents, strongly inhibited the increase in membrane capacitance (a measure of exocytosis) stimulated by both step depolarizations and the calcium ionophore, ionomycin. Noradrenaline similarly inhibited KCl- and ionomycin-induced [3H]serotonin release from RINm5F cell populations. Noradrenaline effects were mediated by PTX-sensitive G proteins. Noradrenaline inhibitory effects on secretion are, therefore, mainly exerted downstream from Ca2+ influx.

Animals↗

PubMed2002

[Intracellular dialysis with a microcatheter inserted into the patch-clamp pipette].

In this paper we present an easily available method of intracellular dialysis via a microcatheter inserted into glass pipette during patch clamp experiment. An oblique hole through the glass pipette holder (above the lateral hole for cell-seal suction) is drilled, through which a microcatheter (O.D.=0.1 mm) made from the universal pipetter tip by hand-drawing passes and sticks out of the holder mouth in parallel with the Ag-AgCl electrode. With a syringe connected to the microcatheter, substitution of intracellular solution and intracellular dialysis of drugs can be achieved easily. Compared with repatch technique and intracellular solution substitution techniques used abroad, this method operates more easily and can produce more reliable results.

Dialysis↗

PubMed1991

Potassium currents of isolated Necturus enterocytes: a whole-cell patch-clamp study.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the ionic currents of enterocytes isolated from the small intestine epithelium of Necturus maculosus. 2. When enterocytes were bathed in a physiological Ringer solution and dialysed with a K(+)-rich intracellular solution containing 1.5 x 10(-7) M intracellular Ca2+, strongly outwardly rectifying currents were observed. These currents were absent from enterocytes internally dialysed with K(+)-free solutions. 3. The kinetics of activation of the outwardly rectifying current was monoexponential with rate constants decreasing with depolarization from 160 ms at 20 mV to 40 ms at 60 mV. Similar voltage dependence of the relaxations after activation were observed. 4. Strongly buffering intracellular Ca2+ with EGTA inhibited outward currents, while increasing Ca2+ increased both their magnitude and rate of activation. 5. Bath application of the K+ channel inhibitors Ba2+ and TEA greatly attenuated outwardly rectifying currents. This observation plus the fact that tail currents reverse near EK points to K+ as the charge carrier in these currents. 6. Outside-out patches showed maxi K+ channels and lower conductance K+ channels. Averaging fluctuations of the maxi K+ channels gave a kinetic behaviour similar to the whole-cell currents.

Animals↗

PubMed1984

Channel currents during spontaneous action potentials in embryonic chick heart cells. The action potential patch clamp.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.

Action Potentials↗

PubMed1994

Paired-pulse facilitation in the dentate gyrus: a patch-clamp study in rat hippocampus in vitro.

1. Whole-cell patch-clamp recordings were used to study paired-pulse facilitation (PPF) of the lateral perforant path input to the dentate gyrus in thin hippocampal slices. 2. Orthodromic stimulation of the lateral perforant pathway evoked a excitatory postsynaptic current (EPSC) with a latency of 3.3 +/- 0.1 ms (mean +/- SE) that fluctuated in amplitude. The EPSC had a rise time (10-90%) of 2.79 +/- 0.06 ms (n = 35) and decayed with a single exponential time course with a time-constant of 9.14 +/- 0.24 ms (n = 35). No correlation was found between the amplitude of the EPSC and the rise time or decay time-constant. The non-N-methyl-D-aspartate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione completely blocked the EPSC whereas the NMDA antagonist D-aminophosphonovaleric acid (APV) had modest effects. 3. When a test (T-)EPSC was preceded at an interval of 100 ms by a conditioning (C-)EPSC, a significant increase in the amplitude of the T-EPSC was seen in 38 out of 44 trials analyzed from a total of 27 granule cells. The average amount of PPF was 35.7 +/- 2.1%. There was no apparent correlation between the amount of PPF and the stimulation intensity or mean amplitude of the C-EPSC. The time course of the facilitated T-EPSC was not significantly different from that of the C-EPSC. 4. No correlation was found between the amplitude of the C-EPSC and that of the T-EPSC. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared averaged EPSC amplitude (from 48 responses) to the variance of these responses (M2/sigma 2) whereas quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSC amplitude (sigma 2/M). PPF was found to be associated with an average increase in mcv of 64.8 +/- 7.2% (n = 38) whereas qcv was decreased by 12.1 +/- 3.8%. 5. The time course of PPF was studied by varying the interval between the C- and T-pulse from 10 to 400 ms while keeping the stimulation intensity constant. Maximal facilitation of the T-EPSC was obtained with interpulse intervals < or = 25 ms where the average facilitation amounted to approximately 70% (n = 6). The decline of facilitation was nearly exponential and was no longer evident with intervals > 350 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗