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At least 91 records · Page 5Linked to original sources

PubMed1999

Series resistance compensation for whole-cell patch-clamp studies using a membrane state estimator.

Whole-cell patch-clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the series resistance (R(s)) of the recording pipette limits the bandwidth of the whole-cell configuration, making it difficult to measure rapid ionic currents. To increase bandwidth, it is necessary to compensate for R(s). Most methods of R(s) compensation become unstable at high bandwidth, making them hard to use. We describe a novel method of R(s) compensation that overcomes the stability limitations of standard designs. This method uses a state estimator, implemented with analog computation, to compute the membrane potential, V(m), which is then used in a feedback loop to implement a voltage clamp; we refer to this as state estimator R(s) compensation. To demonstrate the utility of this approach, we built an amplifier incorporating state estimator R(s) compensation. In benchtop tests, our amplifier showed significantly higher bandwidths and improved stability when compared with a commercially available amplifier. We demonstrated that state estimator R(s) compensation works well in practice by recording voltage-gated Na(+) currents under voltage-clamp conditions from dissociated neonatal rat sympathetic neurons. We conclude that state estimator R(s) compensation should make it easier to measure large rapid ionic currents with whole-cell patch-clamp techniques.

Animals↗

PubMed2003

Adapting the Quesant Nomad atomic force microscope for biology and patch-clamp atomic force microscopy.

The Quesant Nomad atomic force microscope (AFM) was modified to produce a reliable patch-clamp AFM for demanding biologic applications. The AFM's laser optics forms the basis of a condenser that allows simultaneous Köhler illumination and AFM imaging on an inverted optical microscope. The original AFM scan head was replaced with plastic and glass to make it biologically inert. A bevel cut in the new scan head permits clearance for patch clamp pipets. Cantilevers are attached to the scan head with a quick setting silicone rubber that is readily removable. Software was developed to (a) automate a gentle approach and set a specific feedback force, (b) provide a mouse-driven control of the X-Y position of the probe tip and recall of saved locations, and (c) measure force-distance curves over user defined paths. Additional modifications were made to minimize mechanical noise. The patch-clamp AFM achieves 600 fA (3 kHz bandwidth) and 1 A RMS noise levels (10 kHz bandwidth). The correlation of electrical and mechanical information allows signal averaging and measures sub-Angstrom, sub-millisecond electromotile responses from cells.

Cell Membrane↗

PubMed1996

[Study of electrophysiological properties of large conductance K(+)-channels in outer hair cells from the guinea pig with patch-clamp technique].

The basic electrophysiological characteristics of large conductance K(+)-channels in the basolateral cell membrane of outer hair cells (OHC) from the guinea pig cochlea were studied with patch-clamp technique. Acutely isolated OHC preparations were obtained by enzymatic digestion and excised patches were made in cell-attached configuration to record single-channel currents led by patch-clamp micropipette and fed to patch-clamp amplifier. Data were sampled and analyzed on-line with a computer. The result showed that the typical properties of single channels of large conductance K(+)-channels were: 1. The channel was activated by depolarization of membrane and it showed a marked voltage-dependence. The channel had a unitary conductance of about 133 pS and the channel current reversed at about -30mV under asymmetrical K+ concentration gradient. 2. Tetraethylammonium (TEA, > 10 mmol/L), a potassium channel blocker, dose-dependently abolished the outward current. 3. The open probability (Po) and mean open time of this channel was markedly increased with elevation of cytoplasmic Ca2+ concentration. These results suggest that a population of voltage-dependent and Ca(2+)-activated K+ channel exists in the basolateral membrane of OHC of the guinea pig; it is characterized by a very high unitary conductance and high sensitivity to Ca2+ and is blocked by K+ channel blocker. This work provided valuable basic materials for further study on the regulation and drug influence of the type of single channels.

Animals↗

PubMed1997

Patch clamp detection in capillary electrophoresis.

We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.

Animals↗

PubMed2003

Analysis of the molecular mechanisms controlling synaptic transmission by patch-clamp recording in brainstem slices.

The first article describing the patch-clamp recording from neurons in the mammalian brain slice appeared in 1989. Since that article, there have been substantial scientific successes in the neuropharmacological and neurophysiological fields using this promising technique, which itself advanced largely owing to the progress in microscopic techniques such as infrared differential interference contrast (IR-DIC) video-enhanced microscopy. This article describes recent advances in the methods for the patch-clamp recording in the brainstem slices, which is now more and more important due to the increased needs in this post-genomic era for identification of the mechanisms underlying cell-to-cell communication in the central nervous system. Here we introduce some of the technical tips developed and being used in our laboratory, which include methods for making the best brainstem slices, pre-recording identification of neuron types using fluorescent tracers, markers, and green fluorescent protein (GFP) signal in transgenic mice. We also describe a method for rapid and secure drug application onto the recorded cell using electromagnetic valves, which we term the "macro Y-tube" method. These techniques may help to accelerate the understanding of the molecular mechanisms underlying dynamic regulation of central nervous function.

Animals↗

PubMed1998

Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2001

Modulation of Ca(2+) signaling by microtubule disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration.

In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30-36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I(Ca,L) density and [Ca(2+)](i) transient amplitude and depressed the response of these parameters to the beta-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 micromol/L colchicine had no effect on shortening or [Ca(2+)](i) transient amplitude or on the amplitude of I(Ca,L) in perforated patch-clamped cells, under basal conditions and after stimulation with 1 micromol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I(Ca,L) was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I(Ca,L) did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca(2+) signaling or the response to beta stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I(Ca,L) and attenuates the response to beta stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.

Amphotericin B↗

PubMed1998

Patch clamp recording from the intact dorsal root ganglion.

A method for patch-clamp recording from intact dorsal root ganglion (DRG) cells in rat is described. The L4 and L5 DRGs with sciatic nerve attached were excised from rats (10-15 days old) and placed in a recording chamber after removing the ganglion sheath and dissolving the connective tissue with dilute collagenase. The somata of individual cells were exposed by gentle surface cleaning through a perfusion micropipette. Somata were classified as Abeta, Adelta or C based on the cell size and the shape of the action potential (AP). Under current clamp, axonal conduction velocity (CV) was calculated from the distance between a stimulating electrode and the center of the ganglion divided by the latency of the AP elicited by stimulation of the sciatic nerve. CVs ranged from 0.2-0.8 m/s for C cells, 0.8-2.4 for Adelta and 3.2-5.0 for A/beta cells. AP threshold occurred at a significantly more positive potential in C cells than in Adelta and Abeta cells. Under voltage clamp, sodium currents were recorded from C cells. Both TTX-resistant (TTX-R) and TTX-sensitive (TTX-S currents) were demonstrated in the present study. The results demonstrate the feasibility of patch-clamp recording from intact, identified DRG cells in vitro.

Animals↗

PubMed2005

[Method for human peripheral blood eosinophil isolation for patch-clamp study].

OBJECTIVE: To establish a rapid and economic method for isolating human peripheral blood eosinophils with high viability for patch-clamp studies and investigate the electrophysiological properties of Ca(2+)-activated K(+) channel of the isolated cells. METHODS: Peripheral blood eosinophils were isolated by modified discontinuous Percoll density gradient centrifugation, and the electric currents in the single Ca(2+)-activated K(+) channels of the cells were recorded using patch-clamp technique with cell-attached configuration. RESULTS: The purity of the eosinophils from healthy donors reached (90.5+/-1.6)%, with a viability rate over 99% and recovery rate of (48.2+/-6.9)%. The isolated cells were morphologically intact, from which Ca(2+)-activated K(+) channel activity could be detected. CONCLUSION: The peripheral blood eosinophils isolated using this rapid, simple and highly efficient method are characterized by high purity and viability without obvious cellular injuries, which are ideal for patch-clamp studies.

Cell Separation↗

PubMed2003

[Analysis of central synaptic transmission with the slice-patch-clamp technique].

More than ten years have passed since the slice-patch-clamp technique was established as a powerful method for the analysis of central synaptic transmission. Although this technique was restricted only to young animal preparations, we can now apply it to several synapses in slices obtained from adult animals, owing to recent advances in optics or slicers. In addition, advanced techniques have been recently available such as paired whole-cell recording from two or more synaptically connected neurons, recording from dendrites or some presynaptic terminals. Further developments are expected in both of the two directions: more microscopic analysis such as investigating glutamatergic sensitivities of single dendritic spines in combination with two-photon photolysis of a caged-glutamate compound and analysis in a more physiological function-oriented manner such as investigation of pain perception mechanisms using in vivo patch-clamp technique.

Animals↗

PubMed2000

Dendrimer-assisted patch-clamp sizing of nuclear pores.

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be approximately equal to 10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5-10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Starburst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions.

Animals↗

PubMed2001

The "patch-clamp" technique and its application in investigations of the properties of human T lymphocyte potassium channels.

The first part of this review presents a historical outline on the development of experimental methods in electrophysiology starting from the first experiments performed in the 1920s and ending with the "patch-clamp" technique. Recording configurations of the "patch-clamp" technique are briefly reviewed in the second section. The areas of application of the configurations are shown. The last section contains a short review on the available data of "patch-clamp" studies on the expression and properties of potassium channels in human T lymphocytes (TL). Problems that require further investigation are briefly presented.

Electrophysiology↗

PubMed1999

Effects of choline and other nicotinic agonists on the tectum of juvenile and adult Xenopus frogs: a patch-clamp study.

We have used anatomical methods and whole-cell patch-clamp recording to assess the distribution of nicotinic receptors in the tectum of Xenopus frogs and to measure effects of nicotinic ligands (carbachol, cytisine and nicotine) on glutamatergic spontaneous miniature excitatory postsynaptic currents. Our results confirm that retinotectal axons account for the majority of nicotinic receptors in the tectum and that nicotinic agonists exert presynaptic effects that increase the rate of transmitter release on to tectal cells. The nicotinic blockers mecamylamine and methyllycaconitine reduced responses to carbachol and cytisine. A small percentage of cells also showed postsynaptic responses. We have assessed whether there are developmental changes in the frequency of occurrence of spontaneous miniature excitatory postsynaptic currents. The first three months post-metamorphosis fall within the critical period for the dramatic plasticity displayed by binocular inputs during development in Xenopus. During this period, visual activity governs the formation of orderly maps relayed from the ipsilateral eye via the cholinergic projection from the nucleus isthmi to the tectum. In this study, we have found that critical-period tecta (two to 12 weeks postmetamorphosis) tend to have higher spontaneous activity than do older tecta (two to 69 weeks postmetamorphosis), and that nicotinic agonists increase that activity in both groups, with the result that the peak rates in response to nicotinic agonists are higher during the critical period than later. We also investigated the possible role of choline as an agonist of nicotinic receptors in the tectum. We have found that choline, as well as carbachol and cytisine, can cause a reversible increase in the rate of miniature excitatory postsynaptic currents. This result may help to explain how the isthmotectal projection, which accounts for the overwhelming majority of cholinergic input to the tectum, can exert effects on retinotectal terminals even though there are no morphologically identifiable synapses between the two populations. We have examined the morphology of cells filled with biocytin during the patch-clamp experiments, and we find that cells with dendrites in the stratum zonale, a layer with particularly dense input from the contralateral nucleus isthmi, have higher spontaneous activity than cells with dendrites that do not extend into that layer. Nicotinic agonists increased the activity recorded in both classes of cells. In addition, four pretectal cells were identified. Nicotinic agonists increased the rate of spontaneous activity recorded in that population. The results indicate that retinotectal transmission in the superior colliculus can be increased presynaptically by activity of the cholinergic projections of the nucleus isthmi. This modulation may be the basis for observations that blocking of cholinergic input disrupts the formation of topographic retinotectal projections. Moreover, the ability of choline to activate these receptors suggests that this metabolite of acetylcholine may permit paracrine activation of presynaptic receptors even though the tectum contains high acetylcholinesterase activity.

Aging↗

PubMed1989

Anion channels in a leaky epithelium. A patch-clamp study of choroid plexus.

We have used the patch-clamp technique to characterize three anion channels in the ventricular membrane of the choroid plexus epithelium from Necturus. The most frequently occurring channel had a nonlinear IV-curve. The conductance in excised patches with 112 mM chloride at both sides was 28 pS at 0 mV, increasing towards positive membrane potentials. The selectivity ratios were PNa:PCl less than or equal to 0.1 and PNO3:PCl:PHCO3 = 1.6:1:0.43. SITS and furosemide (1 mM) on the inside reduces chloride flux to 0.15 and 0.37 times the control value. In attached patches, the most commonly observed channel had a conductance of 7.5 pS. The single-channel current for this channel reversed direction at 15 mV hyperpolarization, indicating accumulation of chloride to a factor of 1.8 above equilibrium. External stimulation of the tissue by theophylline, IBMX and dbcAMP, or by hypotonic shock did not increase the activity of this channel. In very few excised patches, we have observed a chloride channel with a conductance of 7 pS with 112 mM chloride at both sides. The 7 pS channel appears to be identical to a 2 pS channel found in attached patches. The 2 pS channel was not normally active in attached patches but was activated in 28% of the patches by external stimulation. Finally, in few excised patches we have found a 375 pS channel which inactivates within seconds when membrane potential is stepped from 0 mV to a value that differs more than 10-20 mV from zero. The channel did not conduct gluconate but PNO3:PCl = 1.08 and PNa:PCl less than or equal to 0.1. Internal SITS and furosemide (1 mM) reduced chloride flux to 0.3 and 0.5 times the control value. The channel was never seen in attached patches. The current carried through these channels can not account for the transepithelial steady state Cl- -flux measured by microelectrodes. KCl exit from the cell is suggested to be carried by KCl-cotransport or by channels that are too small to be seen in patch-clamp experiments.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia.

The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (Vh -60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 +/- 15.2 (SE) pA (n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 +/- 10.6 pA (n = 18). Although the hypoOUT reversed at -83.7 +/- 3.8 mV (n = 18), the posthypoOUT did not reverse. The K+ATP-blocking sulphonylureas tolbutamide (100 microM) and glibenclamide (30 microM), significantly reduced the peak of the hypoOUT by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 microM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings (Vh -60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 microM), glibenclamide (10-30 microM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 microM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.

Adenosine Triphosphate↗

PubMed1996

Electrophysiological and pharmacological characteristics of ionotropic glutamate receptors in medial vestibular nucleus neurons: a whole cell patch clamp study in acutely dissociated neurons.

A patch clamp study was performed to determine which subtype of ionotropic glutamate receptors is involved in the glutamate-induced excitation of the medial vestibular nucleus (MVN) neurons. Whole cell recording was performed on MVN neurons that were acutely dissociated by enzymatic and mechanical treatments. Application of glutamate at a concentration of 100 microM produced a current with a reversal potential of approximately 0 mV. The glutamate-induced current was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), a non-N-methyl-D-aspartate (NMDA)-receptor antagonist. Application of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and kainic acid (KA), non-NMDA-receptor agonists, at concentrations of 30 and 100 microM produced a concentration-dependent depolarization concomitantly with an increase in firing rates during current clamp recording. During voltage clamp recording, glutamate, AMPA and KA elicited a concentration-dependent current with an equilibrium potential of approximately 0 mV. To clarify whether NMDA receptors are present in MVN neurons, the effects of glycine on the glutamate- and NMDA-induced current were examined. Two types of NMDA receptor-mediated current (types 1 and 2) were obtained in terms of the difference in sensitivity to both magnesium ion and MK-801, which act on the NMDA-receptor channel. In the type 1 neurons, the NMDA-induced current was not apparently blocked by magnesium ion or MK-801, although a larger current was obtained in the absence of magnesium ion. In the type 2 neurons, marked blockade of the NMDA-induced current was seen in the presence of magnesium ion and MK-801, as previously reported in other neurons of the central nervous system. These findings indicate the presence of both non-NMDA and NMDA receptors, which are involved in primary afferent transmission, in the MVN neuron, and two distinct types of NMDA receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMedDate not supplied

A single electrode voltage, current- and patch-clamp amplifier with complete stable series resistance compensation.

An input headstage for single electrode voltage and current clamping is described which permits selecting desired operational modes during an experiment. These include cell attached or whole cell patch clamping, single micro-electrode voltage or current clamping, potential recording, iontophoresis and voltammetry. Input electrode series resistance with the electrode inserted in a cell or during whole cell patch clamping can be measured at any time. The unique circuitry allows complete compensation of the series resistance with high frequency response. The input series electrode resistance can also vary considerably around an initially set 100% compensation with intrinsic stability. An accelerator 'supercharger' or 'booster circuit' is included which can shorten the rise time of membrane potential to the command potential by a hundredfold. This improves the recording of early ion channel currents. Feedback resistors are selected remotely for the specific experimental need. Signal resolution in patch clamping is that expected from the thermal noise of the feedback resistor and electrode to membrane seal resistance. This headstage circuitry design thus allows interchangeable modes of operation and parameters during an experiment to obtain optimal conditions for signal detection and frequency response.

Electrodes↗

PubMed1992

Purification and patch clamp analysis of a 40-pS channel from rat liver mitochondria.

Patch clamp analysis of membranes reconstituted with a fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine earlier indicated the presence of two classes of ion channels, of about 40- and 140-pS conductance in medium including 150 mM KCl. Now a 57-kDa constituent of the quinine-affinity column eluate has been identified as the 40-pS channel. Protein fractions derived from the quinine-affinity column eluate by preparative isoelectric focusing with a Rotofor cell have been reconstituted into phospholipid vesicle membranes by detergent dialysis, and vesicles have been enlarged for patch clamping by dehydration and rehydration. Voltage clamp analysis has been carried out on excised patches bathed symmetrically in buffered medium containing 150 mM KCl and 100 microM CaCl2. Patches of membrane incorporating the 57-kDa protein exhibit 40-pS conductance transitions. The magnitude of conductance transitions is similar when Na+ replaces K+ in the bathing medium, indicating little selectivity of the 40-pS channel for K+ relative to Na+. Another fraction derived from the quinine-affinity column eluate is found to contain the larger channel, now estimated to have an average conductance of about 130 pS. Patches of control membrane prepared in the same way but without protein exhibit no channel activity.

Animals↗