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PubMed1991

The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy.

We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.

Animals↗

PubMed1995

Transient outward current in adult rat supraoptic neurones with slice patch-clamp technique: inhibition by angiotensin II.

1. Outward potassium currents were recorded from microscopically identified supraoptic neurones of adult Wistar male rats using the whole-cell patch-clamp technique in thin-slice preparations. The basic characteristics of transient outward current (IA or A-current) and the effects of angiotensin II (AII) on the currents were studied. 2. IA was isolated by subtracting outward currents elicited by stepping from two different holding potentials to a test potential or by applying 4-aminopyridine (4-AP) at 5 mM. The isolated IA had a threshold for activation between -55 and -65 mV and was characterized by fast activation and inactivation. Values of the time to peak and the inactivation time constants for current decay at different test potentials were voltage dependent. 3. Normalized currents for activation and steady-state inactivation of IA were fitted to the Boltzmann function. The mid-points and the slope factors were, respectively, -35.0 and -14.3 +/- 0.40 mV (n = 5) for the activation curve, and -72.0 and 7.0 +/- 0.68 mV (n = 5) for the inactivation curve. 4. The time course of recovery from inactivation was best fitted to a single exponential function with the time constant of 37.8 +/- 6.6 ms (n = 6). 5. The effects of AII on IA and delayed rectifier current (IK) were investigated. According to their responses to AII, cells were classified into two groups, sensitive and low-sensitive. Bolus injection of AII (10 microM, 100 microliters) decreased the IA amplitude by 25.1 +/- 2.4% in seven (53.8%) of the thirteen neurones tested (sensitive group), whereas the other six neurones (low-sensitive group) changed by only 2.2 +/- 0.8%. Perfusion of AII (0.1 microM) decreased the IA amplitude by 21.3 +/- 3.1% in six (54.5%) of eleven neurones tested (sensitive group), whereas the other five neurones (low-sensitive group) changed only by 1.7 +/- 0.8%. Bolus injection of AII (10 microM, 100 microliters) decreased the IK amplitude 9.6 +/-1.6% mV in five (45.5%) of the eleven neurones tested (sensitive group), whereas the other six neurones (low-sensitive group) changed only by 0.46 +/- 0.27%. In the sensitive groups, the reduction of IA by AII was significantly larger than that of IK (P < 0.05). 6. Application of saralasin at 1 microM, an AII antagonist, blocked the effects of AII on IA. 7. These results suggest that the excitatory action of AII on supraoptic neurosecretory cells is mediated at least in part through suppression of IA.

Angiotensin II↗

PubMed1998

Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.

An experimental method has been established to measure the electric properties of a cell membrane by combination of patch clamp and dual-wavelength ratio imaging of a fluorescent potentiometric dye, 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl ]pyridinium betaine (di-8-ANEPPS). Pairs of fluorescence images from the dye-stained membrane of neuroblastoma N1E-115 cells excited at two wavelengths were initially obtained to calculate ratio images corresponding to the resting transmembrane potential. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from -100 to +60 mV; at each voltage, a pair of dual-wavelength images were acquired to develop a calibration of the fluorescence ratio. Using this method, the resting potentials could accurately be measured showing that the differentiated cells were 17 mV more polarized than undifferentiated cells. The combination of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permit a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels.

Animals↗

PubMed1999

Voltage-gated sodium and potassium channels in radial glial cells of trout optic tectum studied by patch clamp analysis and single cell RT-PCR.

Radial glial cells in the visual center of trout were analyzed immunocytochemically and with the whole cell mode of the patch-clamp technique in combination with RT-PCR. By immunostaining with anti-GFAP antibodies radially oriented cell processes spanning the entire width of the tectum were brightly labeled, while with anti-S-100 antiserum the cell bodies residing in a discrete layer close to the ventricular border became most clearly visible. Virtually all radial glial cells examined in brain slices exhibited voltage-gated sodium inward currents that were activated above -40 mV, blocked by micromolar concentrations of TTX and totally eliminated if sodium was substituted for Tris in the bath solution. In contrast with adjacent nerve cells of the same slices radial glial cells did not exhibit spontaneous electrical activity and could not be stimulated to generate action potentials by depolarizing current injections. Two types of voltage-gated potassium outward currents were elicited by depolarizing voltage steps: a sustained current with delayed rectifier properties and a superimposed transient "A"-type current, both being activated at a threshold potential of -40 mV. In cultured radial glial cells subtle differences were noticed regarding current density, inactivation kinetics, and TEA-sensitivity of the potassium currents. Inwardly rectifying potassium currents activating at hyperpolarized voltages were not observed. By single cell RT-PCR the transcripts of two shaker-related potassium channel genes (termed tsha1-a fish homologue to Kv1.2- and tsha3) were amplified, while transcripts for tsha 2 and tsha 4 were not detected.

Animals↗

PubMed1987

The receptor-regulated calcium influx in mouse submandibular acinar cells is sodium dependent: a patch-clamp study.

1. Salivary acini were isolated enzymatically from submandibular glands of adult male mice. The patch-clamp technique was employed to record K+ currents in cell-attached patches of basolateral membrane. Application of acetylcholine (10(-5) M) to the medium bathing the cells results in a pronounced and sustained activation of the K+ channels in the cell-attached patches, an effect mediated by an intracellular second messenger. In the present study we investigate the mechanism by which acetylcholine achieves activation of K+ channels. 2. The effects of acetylcholine on single-channel activity were shown to be dependent on extracellular Ca2+, i.e. due to Ca2+ influx. In cells bathed in Ca2+-free medium acetylcholine activation resulted in no increase in single-channel open probability. This blockade could be reversed by reintroduction of Ca2+ to the extracellular fluid in the continued presence of the agonist. The effects of acetylcholine in control medium (1.2 mM-Ca2+) were mimicked by the Ca2+ ionophore, A23187 (10(-8) M). 3. In K+-depolarized cells (bathed in a Na+-free, 145 mM-KCl solution) there was no evidence of any voltage-activated Ca2+ influx pathway. In the K+-depolarized cells acetylcholine application was no longer associated with any increase in the open probability of the K+ channels. K+ channels could be activated by adding A23187 (10(-8) M) to the high-K+ solution. 4. Cells bathed in another Na+-free (N-methyl-D-glucamine substituted for Na+) but non-depolarizing solution were also refractory to acetylcholine. K+ currents could, however, be activated in patches attached to these cells by application of A23187 (10(-8) M) or by the introduction of 20 mM-Na+ to the extracellular fluid in the presence of acetylcholine. The increased activity associated with the reintroduction of Na+ was totally reversed by atropine, i.e. it was receptor regulated. 5. The data presented above indicate that the cholinergic regulation of K+ channels is secondary to the receptor-regulated activation of a Ca2+ influx pathway. There is no evidence of voltage-activated Ca2+ influx in these cells. The cholinergic activation of Ca2+ influx is abolished in Na+-depleted cells. We conclude that the Na+ dependency indicates either that Na+ is involved in the gating of some voltage-independent Ca2+ channel or that Ca2+ entry is via a coupled Na+-Ca2+ co- or countertransport pathway.

Acetylcholine↗

PubMed1997

Improved preparation of Xenopus oocytes for patch-clamp recording.

We report a new technique for improving the efficiency of single-channel recording in the Xenopus oocyte expression system. Conventional methods of oocyte preparation (adequate for two-electrode voltage-clamp) often leave residual adhesions between the vitelline and plasma membranes. As a result, removal of the vitelline membrane for patch-clamp recording produces microscopic damage to the oocyte plasma membrane that interferes with the formation of high-resistance electrical seals. This problem has been alleviated by a three-phase method of oocyte preparation: (1) a strong initial enzymatic digestion using both collagenase and hyaluronidase, immediately followed by (2) exposure to hypertonic media and mechanical defolliculation, (3) pre-shrinkage and selection of only those oocytes displaying clear separation between vitelline and plasma membranes. Following this selection process, oocytes were returned to solutions of normal osmolarity, incubated overnight at 19 degrees C, and then injected with RNA for expression studies. Comparison of oocytes prepared by different methods indicated that use of these three steps significantly improved the success of high-resistance seal formation, without compromising oocyte survivability or the efficiency of channel expression.

Animals↗

PubMed1997

Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: whole-cell patch-clamp analysis.

1. The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. 3. Concentration-response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4. The polyamine spermine (1 microM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. 5. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 7. Mg2+ (10, 100, 500 microM), (5, 20 microM) or ketamine (2, 10 microM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 8. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor-ionophore complex.

Animals↗

PubMed1986

Spread of the light response along the rod outer segment: an estimate from patch-clamp recordings.

Longitudinal spread of the light response was studied using cell-attached patch-clamp recordings from the outer segments of isolated rod photoreceptors from toad retina. The effective photon-collecting area over which photoisomerizations influenced the membrane current through the recorded patch of membrane was determined by measuring the probability of a detectable response as a function of flash photon density (i.e. a "frequency-of-seeing" experiment). The effective collecting area averaged 0.40 micron2, which corresponds to a total sampling length of approximately 1 micron for the patch electrode.

Animals↗

PubMed1998

Dentate gyrus basket cell GABAA receptors are blocked by Zn2+ via changes of their desensitization kinetics: an in situ patch-clamp and single-cell PCR study.

Although GABA type A receptors (GABAARs) in principal cells have been studied in detail, there is only limited information about GABAARs in interneurons. We have used the patch-clamp technique in acute rat hippocampal slices in combination with single-cell PCR to determine kinetic, pharmacological, and structural properties of dentate gyrus basket cell GABAARs. Application of 1 mM GABA (100 msec) to nucleated patches via a piezo-driven fast application device resulted in a current with a fast rise and a marked biexponential decay (time constants 2.4 and 61.8 msec). This decay could be attributed to strong receptor desensitization. Dose-response curves for the peak and the slow component yielded EC50 values of 139 and 24 microM, respectively. Zn2+ caused a marked blocking effect on both the peak and the slow component via a noncompetitive mechanism (IC50 values of 8 and 16 microM). This led to an acceleration of the slow component as well as a prolongation of recovery from desensitization. Zn2+ sensitivity was suggested to depend on the absence of gamma-subunits in GABAARs. To test this hypothesis we performed single-cell reverse transcription PCR that revealed primarily the presence of alpha2-, beta2-, beta3-, gamma1-, and gamma2-subunit mRNAs. In addition, flunitrazepam increased the receptor affinity for its agonist, indicating the presence of functional benzodiazepine binding sites, i.e., gamma-subunits. Thus, additional factors seem to co-determine the Zn2+ sensitivity of native GABAARs. The modulatory effects of Zn2+ on GABAAR desensitization suggest direct influences on synaptic integration via changes in inhibition and shunting at GABAergic synapses.

Animals↗

PubMed2026

Automated patch clamp data improve variant classification and penetrance stratification for SCN5A-Brugada syndrome.

BACKGROUND AND AIMS: Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. METHODS: All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. RESULTS: Most variants (146/252) were functionally abnormal (Z ≤ -2), with 100 having severe loss-of-function (Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. CONCLUSIONS: This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

Humans↗

PubMed2005

Effects of N-methyl-D-aspartate glutamate receptor antagonists on oscillatory signal propagation in the guinea-pig accessory olfactory bulb slice: characterization by optical, field potential and patch clamp recordings.

To characterize the role of N-methyl-d-aspartate glutamate receptors in oscillations induced by a single electrical stimulation of the vomeronasal nerve layer, optical, field potential and patch clamp recordings were carried out in guinea-pig accessory olfactory bulb slice preparations. Bath application of the N-methyl-D-aspartate receptor antagonists, 2-amino-5-phosphonovaleric acid or MK-801, produced an increase in frequency of oscillating waves (oscillation) in external plexiform and mitral cell layers. The removal of Mg2+ from perfusate abolished oscillations, while subsequent application of 2-amino-5-phosphonovaleric acid or MK-801 restored oscillations. Vomeronasal nerve layer-evoked postsynaptic currents were analyzed by whole-cell clamp recordings from mitral and granule cells. A long-lasting excitatory postsynaptic current and periodic inhibitory postsynaptic currents, which were superimposed on the long excitatory postsynaptic current, were observed in mitral cells. The frequency of the periodic inhibitory postsynaptic currents correlated with the frequency of oscillations observed in the optical and field potential recordings. Furthermore, periodic inhibitory postsynaptic currents were blocked by puff application of bicuculline to the external plexiform layer/mitral cell layer, where mitral cells make dendrodendritic synapses with granule cells. In addition, puff application of the non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, to the external plexiform layer/mitral cell layer suppressed an early phase of periodic inhibitory postsynaptic currents (membrane oscillation), whereas 2-amino-5-phosphonovaleric acid suppressed the late phase of periodic inhibitory postsynaptic currents. These data indicate that periodic excitatory postsynaptic currents of granule cells induce relevantly periodic inhibitory postsynaptic currents in mitral cells via dendrodendritic synapses and suggest that feedback inhibition regulates generation of oscillation via activation of non-N-methyl-d-aspartate glutamate receptors and gradual attenuation of oscillation via activation of N-methyl-D-aspartate receptors on granule cells.

Animals↗

PubMed1998

Coupling of proton source and sink via H+-migration along the membrane surface as revealed by double patch-clamp experiments.

Long-range proton transfer along the surface of black lipid bilayers was observed between two integral membrane channels (gramicidins), one operating as a proton source, the other as a sink, by patch-clamp technique. In contrast, potassium ions were shown to equilibrate with the aqueous bulk phase before being consumed. Both channels opened and closed simultaneously only if the charge between them was carried by protons. In this case an anomalous high conductance between two patched membrane fragments was measured, each of them containing one single gramicidin channel. The coupled state disappeared when the distance between these two channels was increased above the critical value. The latter was shown to increase with the channel lifetime. Our results support the idea of the 'localized' proton coupling, in which protons that have been pumped across membranes migrate along the membrane surface to reach another membrane protein that utilizes the established pH gradient.

Electric Conductivity↗

PubMed1997

Synaptic excitation in the dorsal nucleus of the lateral lemniscus: whole-cell patch-clamp recordings from rat brain slice.

The synaptic events underlying the excitation of neurons in the rat's dorsal nucleus of the lateral lemniscus were studied by whole-cell patch-clamp recordings in a brain slice preparation of the auditory midbrain. Both current-clamp and voltage-clamp data were obtained with the brain slice submerged in artificial cerebrospinal fluid. The rats were between 21 and 35 days of age at the time the recordings were made. Synaptic responses were evoked by a bipolar stimulating electrode placed on the lateral lemniscus just ventral to the dorsal nucleus. To eliminate glycinergic inhibitory responses, all physiological data were gathered with 0.5 microM strychnine added to the saline bath. Under current-clamp conditions, excitatory postsynaptic potentials could be subdivided into early and late components. The early component produced a single, highly reliable, short-latency spike and the later component produced a more variable, long-latency spike or train of spikes. The non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, completely blocked the early excitatory postsynaptic potential and its associated action potential. The N-methyl-D-aspartate antagonist, D,L-2-amino-5-phosphonovaleric acid, blocked the later excitatory postsynaptic potential and its action potentials. Typically, both early and late excitatory postsynaptic potentials could be recorded from the same cell, but the early excitatory postsynaptic potential was evoked at lower stimulus levels and had a larger amplitude than the later excitatory postsynaptic potential. Under voltage-clamp conditions, dorsal nucleus of the lateral lemniscus neurons responded to stimulation of the lateral lemniscus with excitatory postsynaptic currents. Outward excitatory postsynaptic currents were recorded with holding potentials that depolarized the cell membrane and inward currents were seen when the cell was hyperpolarized. The current-voltage (I-V) relation of the early peak portion of the excitatory postsynaptic current was nearly linear, whereas the I-V relation of the later excitatory postsynaptic current (12 ms after the peak) was non-linear over the range between -50 and - 100 mV. The outward excitatory postsynaptic current consisted of an early current that was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and a later current that was blocked by D,L-2-amino-5-phosphonovaleric acid. In artificial cerebrospinal fluid with normal concentrations of Mg2+, the inward excitatory postsynaptic current was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, but was not affected by D,L-2-amino-5-phosphonovaleric acid. In Mg2+-free artificial cerebrospinal fluid. however, the early component of the inward excitatory postsynaptic current was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and a later component was blocked by D,L-2-amino-5-phosphonovaleric acid. The results indicate that both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated synaptic responses are present in dorsal nucleus of the lateral lemniscus neurons of rats at 21-35 days of age. The N-methyl-D-aspartate component had a longer time-course and a higher threshold than the non-N-methyl-D-aspartate component, and was subject to a voltage-dependent Mg2+ block when the cell's membrane was hyperpolarized. The long-duration N-methyl-D-aspartate component is probably responsible for the prolonged inhibitory effect of dorsal nucleus of the lateral lemniscus neurons on physiological responses in the rat's inferior colliculus.

Action Potentials↗

PubMed1991

Two types of chloride channels in hen colon epithelial cells identified by patch-clamp experiments.

Freshly isolated epithelial cells from hen colon were investigated using the patch-clamp technique. The aim of this investigation was to characterise the cellular conducting site for Cl- secretion. In cell-attached mode two types of Cl(-)-channels were found. Both showed distinct outward rectification. The channel types differed in single channel conductances and the marked voltage dependence of the open probabilities. A low conductance Cl(-)-channel was observed with a mean conductance at negative holding potentials of g- = 9 pS, and of g+ = 34 pS at positive potentials. This channel was predominantly open at negative potentials, corresponding to cell hyperpolarization. The second channel type observed had conductances of g- = 35 pS and g+ = 77 pS, and showed increasing open probabilities with increasing holding potentials (cell depolarisation). Both channel types were blockable by the Cl(-)-channel blocker NPPB. These data in combination with previously published transepithelial transport data on hen colon indicate that these channels are the Cl- secretory sites in colon epithelium.

Animals↗

PubMed1990

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells.

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.

Aging↗

PubMed1994

JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements.

The JPCalc program has been designed to graphically illustrate how junction potential contributions arise in various electrophysiological situations, to enable the magnitude and direction of those values to be readily calculated and to show clearly how the resultant appropriate corrections need to be applied to experimental measurements. In particular, the program covers the analysis of junction potential contributions and corrections in all the various different patch-clamp configurations (intact patches, inside-out patches, outside-out patches, whole-cell recording and perforated patches), intracellular recording, epithelial and bilayer measurements. It can also be used to correct experimental measurements designed to try to directly measure junction potentials and to compare both the magnitude and direction of such corrected measurements with their theoretical values. Although it is recommended that all of the above measurements be done with salt-filled electrodes or salt bridges, the program will allow the use of Ag/AgCl reference electrodes in direct contact with bathing solution in most situations. JPCalc also makes use of a library file of mobility and valency data for about 30 ions which can be extended by the user. In addition to its value in correcting electrical measurements, the program with its graphical displays may also be of value in focusing discussion about the contribution of junction potentials to such measurements and in helping to design experiments to either minimize their contribution or to measure their values. The JPCalc program, designed for IBM-compatible computers with color or monochrome monitors, can also be run on Macintosh computers with SoftPC emulation software.

Animals↗

PubMed2006

Controlled hidden markov models for dynamically adapting patch clamp experiment to estimate Nernst potential of single-ion channels.

This paper presents novel kernel-based stochastic learning algorithms for controlling the kinetics of single-ion channels in a patch clamp experiment. The algorithms yield efficient estimates of the equilibrium (Nernst) potential of an ion channel. The equilibrium potential of an ion channel is the applied external potential difference required to maintain electrochemical equilibrium across the ion channel. The algorithm adaptively controls the exploration of the learning algorithm to achieve an optimal balance between exploration and exploitation. An important feature of the resulting algorithm is that it is guaranteed to minimize the experimental effort. We illustrate the efficiency of the algorithms for the experimentally determined current voltage curve of a bi-ionic single potassium ion channel.

Adaptation, Physiological↗

PubMed1996

ADP-induced rapid inward currents through Ca(2+)-permeable cation channels in mouse, rat and guinea-pig megakaryocytes: a patch-clamp study.

1. The rapid inward currents in mouse megakaryocytes evoked by adenosine diphosphate (ADP), a ubiquitous platelet-activating substance, were studied. Time and current resolution were improved by using patch-clamp recording and an extracellular fast perfusion ("Y tube') technique. 2. Application of ADP (40 microM) to megakaryocytes immersed in physiological saline evoked rapid inward currents (80-340 pA at -42 mV). The cellular responses to a second ADP application were markedly reduced, but in the absence of external Ca2+, responses to repeated ADP application were maintained and did not deteriorate. 3. The ADP-induced current recorded in Ca(2+)-free external media showed short latency (less than 20 ms) and approximately exponential decay (time constant, 300-500 ms), which was independent of the holding potential and seemed to be caused mainly by receptor desensitization; it took over 5.5 min for complete recovery. 4. The ADP concentration response relationship of the megakaryocytes revealed that the half-maximal concentration and the Hill coefficient were 12.6 microM and 1.4, respectively. 5. An ion replacement experiment showed that the ADP-induced currents could be carried by Na+, Cs+ and K+, but not Cl-, and the cation channels were permeable to Ca2+, Ba2+ and Mg2+. 6. Neither Ca2+ chelators (10 mM EGTA and 10 mM BAPTA) nor hydrolysis-resistant guanine nucleotides (2 mM GDP-beta-S and 0.4 mM 5'-guanylylimidodiphosphate) in the internal saline affected the rapid responses to ADP, and ADP-induced currents were recorded in excised membrane patches, suggesting that the ADP receptor site and the molecular structure forming the cation channel are tightly coupled and/or parts of the same molecule. 7. In rat and guinea-pig megakaryocytes, ADP-induced rapid inward currents showed the same properties as in mouse megakaryocytes.

Adenosine Diphosphate↗