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A Marty

Publications and source records attributed to A Marty.

At least 91 records · Page 5Linked to original sources
PubMed1986

Calcium-dependent chloride currents in isolated cells from rat lacrimal glands.

Isolated cells from rat lacrimal glands were studied with the tight-seal whole-cell recording technique. Cells were dialysed with K-free solutions containing a high concentration of Ca2+ buffer in order to record Ca-dependent Cl- currents at a Ca2+ level fixed between 0.1 and 10 microM. HEDTA was preferable to EGTA as a Ca2+ buffer, even under conditions of equivalent equilibrium buffering power. After replacement of all internal K+ with Na+, the cells displayed a small conductance component which could be abolished by removal of external K+ or by external application of 2 mM-tetraethylammonium. It is suggested that this conductance is due to Ca-dependent K+ channels. The main part of the cell current was Cl selective. The Cl- conductance was negligible at 0.5 microM-Ca2+, and fully activated at 2 microM-Ca2+. The dose-response curve relating Cl- currents to the internal Ca2+ concentration, [Ca]i, was steeper than predicted by a simple binding isotherm reaction. Relaxations observed in response to voltage jumps could, in most cases, be fitted with single exponentials. At [Ca]i 0.5 microM, the curve relating the relaxation time constant, tau, to the membrane potential, displayed a maximum near +20 mV and 250 ms. At hyperpolarized potentials, tau varied by an e-fold factor in 130 mV. At [Ca]i 1 microM, tau decreased from 100 ms at -120 mV to 60 ms at +60 mV. Relaxation analysis gave an estimate of the variation of the channel open state probability, Po, with potential. At [Ca]i 0.5 microM, Po varied by an e-fold factor in 50-70 mV at hyperpolarized potentials, and saturated above +60 mV. At [Ca]i 1 microM, Po varied e-fold in 100 to 110 mV at hyperpolarized potentials, and saturated near +20 mV. External Cl- was substituted with various anions. From reversal potential measurements, the following permeability sequence was obtained: I- greater than NO3- greater than Br- greater than Cl- greater than F- greater than isethionate, methanesulphonate greater than glutamate. The corresponding normalized permeability coefficients were 2.7, 2.4, 1.6, 1, 0.2, 0.1, 0.1, 0.05. Replacement of external Cl- with Br-, isethionate, methanesulphonate or glutamate did not alter current kinetics as obtained during or after a depolarizing voltage jump.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Muscarinic response in rat lacrimal glands.

A large variety of responses has been uncovered by recent investigations of conductance changes elicited by muscarinic agonists. In exocrine glands, the permeability to K+, Cl- and Na+ ions is increased, and internal Ca2+ serves as a second messenger. Patch-clamp analysis of the secreting cells has revealed three types of Ca2+-dependent channels, which are respectively selective for K+, for Cl-, and for monovalent cations. The channels differ in their sensitivity to the internal Ca2+ concentration, Cai. K+-selective channels are partially activated at rest, with Cai approx. 10 nmol l-1; Cl(-)-selective channels are activated between 100 nmol l-1 and 1 mumol l-1; activation of cationic channels requires micromolar Cai levels. Cell-attached recordings, performed either on isolated cells or on cell clusters, show an activation of all three channel types upon application of acetylcholine. In whole-cell recordings, mostly K+- and Cl(-)-selective channels are activated. The cell currents display slow oscillations linked to variations of Cai. Whole-cell currents rise after a delay of approx. 1 s, and decay with a time constant of approx. 0.7 s upon removal of acetylcholine. They do not depend on extracellular Ca2+. The recent demonstration that Ca2+-dependent currents can also be obtained when dialysing the cells with inositoltrisphosphate or with GTP gamma S, a non-hydrolysable analogue of guanosine triphosphate, opens promising leads to an analysis of intracellular events regulated by acetylcholine.

Acetylcholine↗
PubMed1985

Potassium channels in cultured bovine adrenal chromaffin cells.

K channels of bovine adrenal chromaffin cells were studied using patch-clamp techniques. Whole-cell K currents measured near +10 mV were much larger in 1 mM-external Ca than in Ca-free saline. Noise analysis suggested that this Ca-dependent current was carried by a large unitary conductance channel, called BK channel, which was previously described in inside-out patches (Marty, 1981). The Ca-dependent K current near +10 mV declined with time due to 'run-down' of Ca channels. At the same time, a fraction of the outward current observed above +50 mV was also eliminated. This outward current component probably represents K efflux through Ca channels. Whole-cell Ca-dependent K currents were studied using various Ca buffers. EGTA buffers were surprisingly inefficient: in order to block the current entirely, it was necessary to use an isotonic EGTA solution and to increase internal pH. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was at least five times more efficient than EGTA. In isolated patches three types of single-channel K currents were observed. Under normal ionic conditions (140 mM-K inside, 140 mM-Na outside), the unitary conductances measured between -20 and +40 mV were 96 pS, 18 pS and 8 pS. The 96 pS channels are the Ca-dependent BK channels. 18 pS and 8 pS channels were both activated and then inactivated by membrane depolarization. Both displayed complex kinetics; single-channel currents were grouped in bursts. Activation and inactivation kinetics were faster for the 18 pS channel (therefore termed FK channel, for fast K channel) than for the 8 pS channel (SK channel, for slow or small amplitude channel). The voltage dependence of opening probability was steeper for the FK channel as compared to the SK channel.

Action Potentials↗
PubMed1985

[Ionic currents and secretion in the exocrine glands].

Exocrine glands extrude both proteins and salt. Fluid secretion is related to a modification of the membrane permeability of secreting cells. This permeability change may be measured as an increase of labelled ion fluxes or as a rise of membrane conductance. It involves Na+, K+, Cl- and Ca2+ ions. Intracellular Ca2+ acts as "second messenger" in the development of the electrical response. Recent recordings using the "patch-clamp" technique have revealed three types of ion channel activated by secretory agents. These channels are sensitive to internal Ca2+ ions. They are respectively selective to K+, Cl- and positively charged monovalent ions. Two models suggesting possible roles for these channels in the secretion process are presented. However, evaluation of such models is presently restricted by numerous uncertainties on the function of secreting cells in vivo. Information is notably lacking concerning the exact composition of the secreted fluid, and the exchanges between exocrine glands and blood circulation.

Animals↗
PubMed1984

Incorporation of chromaffin granule membranes into large-size vesicles suitable for patch-clamp recording.

Incubation of chromaffin granules with excess liposomes at pH 6.0 resulted in the formation of cell-size structures, which were purified by centrifugation on sucrose gradients. Experiments with fluorescein-labeled granules indicated incorporation of granule membrane to these structures. The preparation contained various vesicular structures with a diameter up to 15 micron. The largest elements were studied by the 'patch-clamp' technique. 'Cell-attached' and 'whole-cell' recordings indicated the presence of currents corresponding to unitary conductances ranging from 100 to 500 pS.

Animals↗
PubMed1984

Activation of Ca-dependent K channels by carbamoylcholine in rat lacrimal glands.

Electrical properties of the membranes of lacrimal gland cells were investigated using patch-clamp techniques [Hamill, O.P., Marty A., Neher, E., Sakmann, B. & Sigworth, F.J. (1981) Pflügers Arch. 391, 85-100]. The membranes were found to contain a specific kind of voltage- and Ca2+ -activated K+ channel ("BK channels"). These channels account for the strong rectification of the cell current-voltage curve as obtained in tight-seal whole-cell recordings. Application of low concentrations of carbamoylcholine (CbmCho, 0.5 microM) activated the BK channels. No effect was obtained in the presence of atropine (2 microM) or when dialyzing the cell with a strong CaEGTA buffer. The latter result, together with other findings, suggests that CbmCho exerts its action on BK channels by increasing the intracellular Ca2+ concentration. This Ca2+ concentration increase presumably occurred via liberation from a cytoplasmic Ca2+ store, because the response remained unaffected in the absence of extracellular Ca2+. At higher CbmCho concentration (2 microM), an inward current was observed, which was assumed to result from activation of another type of Ca2+ -regulated channel.

Acetylcholine↗
PubMed1984

Three types of calcium-dependent channel in rat lacrimal glands.

Isolated cells from rat lacrimal glands were studied with patch-clamp techniques. Whole-cell and cell-attached recordings were obtained while the cells were stimulated by application of carbamylcholine or of the Ca ionophore, A23187. The results were compared with recordings of Ca-dependent channels obtained in isolated patches. Whole-cell recordings revealed two types of carbamylcholine-induced current. At low levels of stimulation, a specific class of Ca-dependent K channels was selectively activated ('BK channels'). With more intense stimulation an inward current, Ii, was obtained at the cell resting potential. Ii rose rather abruptly after a long delay. In several cells, Ii currents presented spontaneous oscillations. Both K and Ii current responses to carbamylcholine were due to activation of muscarinic receptors. Both responses were elicited by a rise of the intracellular Ca concentration. The immediate source of Ca was intracellular. Replacement of intracellular K with either Na or Cs blocked BK channels entirely, thus allowing the study of Ii currents free from K currents. Ii responses to carbamylcholine were, however, less frequently obtained in Na- or Cs-dialysed cells than in K-dialysed cells. In symmetrical NaCl solutions, Ii inverted at 0 mV. When replacing part of the intracellular or extracellular Cl with glutamate the reversal potential, Ei, was found to vary in the same direction as the equilibrium potential for Cl ions, ECl. In some experiments, Ei was close to ECl but in others Ei deviated strongly from ECl. These experiments suggested that Ii was mainly due to a Cl-selective conductance, and that another conductance type was contributing to Ii in variable proportions. It was found that, in K-free solutions, Ii had a reversal potential very close to ECl. Noise analysis showed that the Cl channels involved in Ii current had a unit conductance of about 1-2 pS in symmetrical NaCl solutions. At -60 mV, the mean channel open time derived from noise power spectra was about 200 ms. The activation of the Ca-dependent Cl channels was increased by depolarization. Voltage jumps elicited slow exponential relaxations. At -60 mV, the time constants of the relaxations were in the range 100-250 ms. Cell-attached recordings suggested that internal Ca activated three types of channel, depending on the Ca concentration: BK channels, 2-4 pS channels and 25 pS channels. Inside-out and outside-out patch conditions allowed a rough estimate to be made of the Ca concentration needed to activate each class of channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Blocking of large unitary calcium-dependent potassium currents by internal sodium ions.

The effects of internal Na on the outflow of K ions through a certain type of Ca-dependent K channels of chromaffin cells (Marty, 1981) were studied in isolated patches. Application of 20 mM Na ions to inside-out patches (Hamill, Marty, Neher, Sakamann and Sigworth, 1981) on the cytoplasmic (called hereafter internal) side of the membrane induced short interruptions of the single channel currents. This effect was voltage dependent. In addition, internal Na ions reduced the opening probability of the channels. Outside-out patch recordings were obtained with 20 mM internal Na and varying external K concentrations. The blocking effects of internal Na ions were relieved by increasing the external K concentration.

Animals↗
PubMed1982

Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.

The capacitance of the surface membrane of small adrenal chromaffin cells was measured with patch-clamp pipettes. Continuous and discrete changes of capacitance were observed. They were interpreted as changes of surface area connected to exocytotic or endocytotic processes. Most of the measurements were performed in the "whole-cell" recording configuration [Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100], which allows the intracellular Ca2+ concentration to be controlled. With an internal solution highly buffered to low values of Ca2+ concentration (10 nM), the surface capacitance usually decreased and could not be markedly changed by electrical stimulation. At low buffering capacity and medium Ca2+ concentrations (0.1-1 microM), the capacitance measurement showed large fluctuations and discrete steps, reflecting both capacitance decrease and increase. A large transient increase of capacitance could be induced by electrical stimulation under these conditions. It was linked to Ca2+ currents through the membrane. Relatively large (2-6 x 10(-14) F) steps of capacitance decrease were common after extensive stimulation. The size distribution of step-like capacitance changes is well compatible with the idea that steps of capacitance increase reflect individual events of exocytosis of chromaffin granules, whereas steps of the opposite polarity reflect the formation of vesicles or vacuoles by endocytosis.

Animals↗
PubMed1982

A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine.

1. Bovine chromaffin cells were enzymatically isolated and kept in short term tissue culture. Their electrical properties were studied using recent advances of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). 2. When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached configuration') current wave forms due to intracellular action potentials could be observed. The frequency of the wave forms was altered by changing the pipette potential. When acetylcholine was present in the pipette solution, acetylcholine-induced single channel currents were evident in the patch recording. Action potential wave forms were then often seen to follow acetycholine-induced single channel currents. 3. In the cell-attached configuration, large single channel current events did not resemble square pulses but showed exponential relaxations with time constants of the order of 50 ms. 4. After rupture of the patch of membrane, the pipette--cell seal remained stable ('whole-cell recording', Hamill et al. 1981). Chromaffin cells were found to have a resting potential of -50 to -80 mV, and an input resistance around 5 G omega. The high cell resistance accounts for the relaxing currents evident in the cell-attached configuration. 5. In the best cases, the effective time constant of the voltage clamp in the whole-cell recording mode was 15 microseconds. Exchange of small ions such as Na+ ions between pipette and cell interior solutions was then complete within 15 s. 6. Acetylcholine-induced currents were obtained at various acetylcholine concentrations. Single acetylcholine-induced channels had a slope conductance of 44 pS between -100 and -55 mV, and a mean duration of 27 ms at -80 mV (at room temperature).

Acetylcholine↗
PubMed1982

Sodium and calcium channels in bovine chromaffin cells.

1. Inward currents in chromaffin cells were studied with the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The intracellular solution contained 120 mM-Cs(+) and 20 mM-tetraethylammonium (TEA(+)). Na(+) currents were studied after blockade of Ca(2+) channels with 1 mM-Co(2+) applied externally. Ca(2+) currents were recorded after eliminating Na(+) currents with tetrodotoxin (TTX). The current recordings were obtained in cell-attached, outside-out and whole-cell recording configurations (Hamill et al. 1981).2. Single channel measurements gave an elementary current amplitude of 1 pA at -10 mV for Na(+) channels. This amplitude increased with hyperpolarization between -10 and -40 mV, but did not vary significantly between -40 and -70 mV.3. The mean Na(+) channel open time was 1 ms at -30 mV. This open time decreased both with depolarization and hyperpolarization. Its value was close to the time constant of inactivation, tau(h), above -20 mV.4. Ensemble fluctuation analysis of Na(+) currents gave results consistent with those of single channel measurements. Noise power spectra obtained between -35 mV and 0 mV could be fitted with a single Lorentzian. A range of Na(+) channel densities of 1.5-10 channels per mum(2) was calculated.5. Cell-attached single Ca(2+) channel recordings were obtained in isotonic BaCl(2) solution. The single channel amplitude was 0.9 pA at -5 mV, and it became smaller for positive potential values.6. At -5 mV, single Ba(2+) currents appeared as bursts of 1.9 ms mean duration containing on the average 0.6 short gaps. The burst duration was larger at positive potentials.7. Ensemble fluctuation analysis of Ca(2+) channels was performed on whole-cell recordings in external solutions containing isotonic BaCl(2) or external Ca(2+) (Ca(o)) concentrations of 1 and 5 mM. The unit amplitude calculated in the former case was similar to that obtained in single channel measurements.8. Noise power spectra of Ca(2+) or Ba(2+) currents could be fitted by the sum of two, but not one, Lorentzian components.9. Tail currents could be fitted by the sum of two exponential components. The corresponding time constants had values close to those obtained with noise analysis.10. The rising phase of Ca(2+) and Ba(2+) currents was sigmoid. It could be fitted by the sum of three exponentials. The time constant of the largest amplitude component, tau(1), was similar to the time constants of the slow component observed in noise and tail experiments. This time constant also corresponded to the burst duration obtained in single channel measurements.11. The value of tau(1) was larger in 5 mM-Ca(o) and in isotonic Ba(2+) than in 5 mM-Ba(o). Thus, the kinetic properties of Ca(2+) channels depend on the nature and concentration of the permeating ion.12. A simple kinetic scheme is proposed to model the activation pathway of Ca(2+) channels.13. Currents in 1 mM-Ca(o) and 5 mM-Ca(o) showed clear reversals around +53 mV and +64 mV respectively. The outward currents observed above these potentials are most probably due to Cs(+) ions flowing through Ca(2+) channels.14. The instantaneous current-voltage relation was obtained from tail current data in the range -70 to +100 mV in 5 mM-Ca(o). The resulting curve displayed an inflexion point around the reversal potential.15. Very little inactivation of Ca(2+) currents was observed. However, a slow current decline was observed in some cells above +10 mV.16. Conditioning prepulses to positive potentials had potentiating or depressing effects on Ca(2+) currents depending on whether the test pulse lay below or above the maximal current potential. The potentiating effect may be linked to the slowest component of the current rise observed below +10 mV. The depressing effect may be related to the slow decline obtained above +10 mV.17. Analysis of ensemble variance and of tail current amplitudes suggested that the opening probability of Ca(2+) channels was at least 0.9 above +40 mV.18. A slow rundown of Ca(2+) currents was observed in whole-cell recordings. The speed of the rundown was dependent on intracellular Ca(2+) concentration. The rundown was apparently due to a progressive elimination of the channels available for activation.19. The density of Ca(2+) channels (before rundown) was estimated at 5-15/mum(2).20. In cell-attached experiments, inward current channels were often seen to follow action potentials. These events did not appear to be the usual Na(+) and Ca(2+) currents. They were probably due to cation influx of either Na(+) or Ba(2+), depending on the pipette solution, through Ca(2+)-dependent channels. Voltage-independent single channel activity observed in whole-cell and outside-out recordings may be due to the same channels.

Adrenal Medulla↗
PubMed1981

Single channel activity associated with the calcium dependent outward current in Helix pomatia.

A recently improved version of the extracellular patch clamp technique (9, 13) was used to record currents from microscopic membrane areas of Helix neurons with predominant Ca2+ dependent outward currents. Current fluctuations in the patches consisted mainly of frequently interrupted, one-sided steps indicating discrete open-closed state changes of single channels with an ohmic conductance of approximately 19 pS. Frequency of occurrence of the elementary events compares with amplitudes of macroscopic currents during depolarizing voltage steps of varied amplitude. Average delays in appearance of the events vary in line with delayed time courses of the cell's outward current.

Animals↗
PubMed1981

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

Acetylcholine↗
PubMed1980

Action of glucosamine on acetylcholine-sensitive channels.

The action of glucosamine was studied on voltage clamped neurones of Aplysia, presenting an excitatory response to acetylcholine. Noise and relaxation experiments show that glucosamine increases the mean channel open time and reduces the amplitude of the elementary current associated with the acetylcholine response. Both effects are enhanced by hyperpolarization of the cell membrane. The results are interpreted by a model assuming glucosamine binding to open channels. This binding impedes the flow of permeant ions and decreases the closing rate of the channels.

Acetylcholine↗