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E Neher

Publications and source records attributed to E Neher.

At least 127 records · Page 7Linked to original sources
PubMed1987

Effects of hydrostatic pressure on membrane processes. Sodium channels, calcium channels, and exocytosis.

A patch-clamp study under high hydrostatic pressure was performed by transferring cells or membrane patches into a pressure vessel (Heinemann, S. H., W. Stühmer, and F. Conti, 1987, Proceedings of the National Academy of Sciences, 84:3229-3233). Whole-cell Na currents as well as Ca currents were measured at pressures up to 40 MPa (approximately 400 atm; 1 MPa = 9.87 atm) in bovine adrenal chromaffin cells. Ca currents were found to be independent of pressure within experimental resolution. The mean amplitude and the gating kinetics of Na currents were affected by less than 20% at 10 MPa. This lack of a pronounced effect is surprising since the high-pressure nervous syndrome (HPNS), a disorder at high pressures known to result from impaired nervous transmission, manifests itself at pressures as low as 5 MPa. The results show that ion channels involved in transmission cannot be implicated in HPNS. However, when exocytosis was studied at high pressure by monitoring the cell capacitance (Neher, E., and A. Marty, 1982, Proceedings of the National Academy of Sciences, 79:6712-6716), more drastic effects were seen. The degranulation evoked by dialyzing the cell with 1 microM free Ca2+ could be slowed by a factor of 2 by application of 10 MPa. The same effect was observed for the degranulation of rat peritoneal mast cells stimulated with 40 microM of the GTP analogue GTP-gamma-S. According to these results, the process of exocytosis is the most likely site at which hydrostatic pressure can act to produce nervous disorders. Furthermore, we demonstrate that pressure can be a useful tool in the investigation of other cellular responses, since we were able to separate different steps occurring during exocytosis owing to their different activation volumes.

Adrenal Medulla↗
PubMed1987

Gradual and stepwise changes in the membrane capacitance of rat peritoneal mast cells.

1. The membrane capacitance of mast cells was monitored under voltage clamp, using sinusoidal excitation and a lock-in amplifier. 2. Degranulation was accompanied by stepwise capacitance increases that presumably represent the fusion of single secretory granules with the cell membrane. Besides capacitance steps, we also observed gradual changes in capacitance that occurred even in the absence of degranulation, were independent of the presence of nucleotides in the pipette, and were steeply dependent on cytoplasmic [Ca2+]. 3. Cytoplasmic Ca2+ at concentrations of 0.3-3 microM stimulated a decline in capacitance, with a dose-response curve suggesting control by the binding of Ca2+ to high-affinity intracellular sites. When maximally activated, this mechanism could lead to a loss of about 6% of the cell membrane capacitance, at an average rate of 0.1-0.2% s-1. 4. At even higher cytoplasmic [Ca2+] (greater than 3 microM), the reverse effect was observed. The capacitance increased gradually by up to 40%, at an average rate of 0.4% s-1. Evidently gradual changes in membrane capacitance can occur by two mechanisms, and both are influenced by cytoplasmic [Ca2+]. 5. Ca2+ frequently stimulated an inward current accompanied by an increase in membrane conductance. 6. The effects described above were observed also when only trace amounts of Ca2+ and chelator were added to the cytosol, and when increases in cytosolic [Ca2+] could have occurred only by endogenous mechanisms. It is suggested that these effects occur also in intact cells during the large [Ca2+] increases known to occur before and during degranulation.

Animals↗
PubMed1986

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

The calcium concentration, [Ca]i, in single rat peritoneal mast cells was measured by means of the new Ca indicator dye fura-2. Upon stimulation with antigen or compound 48/80, [Ca]i rose for seconds to values greater than 5 microM. These Ca transients did not depend on the presence of extracellular Ca, and they sometimes occurred spontaneously, especially in the presence of exogenous phosphatidylserine. Calcium transients did not necessarily lead to degranulation. Degranulation usually occurred during periods of somewhat elevated [Ca] (0.5-1 microM) following transients but was sometimes observed at [Ca]i less than or equal to 250 nM. We found no evidence that an antigen-induced Ca influx is required for degranulation.

Animals↗
PubMed1985

The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading.

The Ca concentration ([Ca2+]i) in single rat peritoneal mast cells was measured by means of the new fluorescent Ca-indicator dye fura-2. Dye-loaded cells were made to degranulate with either antigen or compound 48/80. In cells loaded with extracellularly applied, membrane-permeant fura-2 ester, degranulation was accompanied by a permanent loss of 40-60% of the fluorescence, but comparison of fluorescence at different wavelengths indicated no or only small changes in [Ca2+]i. When cells were loaded by microinjection of the impermeant potassium salt of the dye, degranulation resulted in no permanent loss of fluorescence, but instead was preceded by transient fluorescence changes that indicate a rapid, large and transient increase in [Ca2+]i. We suggest that ester-loaded fura-2 accumulates to a significant degree in the secretory granules and is lost from the cell during exocytosis.

Animals↗
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↗
PubMed1984

Substance P reduces acetylcholine-induced currents in isolated bovine chromaffin cells.

Patch-clamp techniques were used to examine the effect of substance P on acetylcholine-induced current in bovine chromaffin cells. Cells had been enzymatically isolated and kept in short-term culture. Experiments were performed at 22 degrees C. Under whole-cell voltage-clamp conditions substance P alone (2-10 microM) did not induce ionic currents. Acetylcholine (ACh, 20 microM) at -60 mV induced an inward current that desensitized in the continued presence of ACh. The time course of desensitization was somewhat variable from cell to cell. In most cases it could be fitted by a single exponential with time constant of 8-10 s. Substance P (2-50 microM) applied simultaneously with ACh induced what appeared to be an acceleration of the desensitization process. The time course in the presence of 10 microM-substance P (20 microM-ACh) was best fitted by the sum of two exponentials with time constants of 0.6 s and 5 s respectively. The effect was reversible. The recovery of ACh-induced current from desensitization was not affected by substance P. The time constant for recovery was approximately 7 s in the presence or absence of substance P. Single-channel records showed that the conductance of individual channels was not changed by substance P. The mean open time of single channels was shortened by substance P both at high (20 microM) and at low (0.5 microM) concentrations of ACh. The inverse mean open time varied linearly with substance P concentration. Single-channel responses appeared in bursts and clusters after almost complete desensitization at 20 microM-ACh, as was previously observed in frog skeletal muscle. Substance P dramatically reduced ACh current by increasing interburst intervals while decreasing burst duration and the number of openings per burst. We conclude that substance P inhibits ACh-induced depolarization of chromaffin cells either by increasing the rate of desensitization or by inducing channel blockade, which indirectly enhances desensitization. Possible models of desensitization in the absence and presence of substance P are discussed.

Acetylcholine↗
PubMed1984

Trifluoperazine reduces inward ionic currents and secretion by separate mechanisms in bovine chromaffin cells.

Using patch-clamp techniques, excitation and secretion in chromaffin cells were studied by measurement of unitary inward currents and of stimulus-evoked increments in membrane capacitance. The effect of the calmodulin inhibitor trifluoperazine (TFP) on Na, Ca and acetylcholine-induced (ACh) currents as well as on capacitance increments was investigated. TFP in concentrations up to 10 microM had no effect on Na channel currents. TFP was a potent anticholinergic agent. TFP in concentrations of 100 nM-1 microM decreased net ACh-induced currents by a slow block or allosteric modification of the channel. The effect was only partially reversible. Recovery from desensitization was retarded in direct relation to [TFP]. At the single channel level, TFP was found to slightly shorten open times in 0.5 and 20 microM-ACh. As reported previously, desensitization can be modelled by at least two desensitized states, as reflected by the bursting and clustering behaviour of single channels. TFP shortened clusters mainly by reducing the number of bursts per cluster. Whole-cell Ca currents (ICa) were reduced in 10 microM-TFP from an average of 29 microA cm-2-13 microA cm-2. Changes in capacitance of 1-200 fF were elicited in controls by maximal activation of the Ca current. We interpreted these steps to be the summed result of many exocytotic vesicular fusion events. Capacitance steps depended on ICa and were absent when extracellular Ca was removed. Application of 10 microM-TFP inhibited capacitance steps. The block of capacitance steps by TFP was shown to be independent of the reduction of ACh and Ca inward ionic currents. We conclude that the prevention of exocytosis by TFP is not completely described by its inhibition of electrical excitability but also results from intracellular actions.

Acetylcholine↗
PubMed1983

The charge carried by single-channel currents of rat cultured muscle cells in the presence of local anaesthetics.

Acetylcholine-induced single-channel currents were measured in the presence of the lignocaine derivative QX222. Unit responses appeared as bursts of short current pulses as a result of the fast blocking action of the drug (QX222). The amplitude of the individual current pulses was not changed by the presence of the drug up to a concentration of 250 microM. The time integral of current during a burst, which for a sequential blocking model should be independent of drug concentration, decreased at concentrations of QX222 higher than 40 microM. The distribution of gap times within a burst could not be fitted by a single exponential for high concentrations of QX222. It is concluded that the simple sequential model of channel blockade does not apply for concentrations of QX222 higher than 40 microM.

Acetylcholine↗
PubMed1982

Dielectric relaxation studies of ionic processes in lysolecithin-packaged gramicidin channels.

Dielectric permitivities have been determined for suspensions of lysolecithin packaged malonyl gramicidin channels over the frequency range of 5 kHz to 900 MHz and under conditions of approximately equimolar concentrations (approximately 10mM) of channels and salts. The salts were lithium chloride, sodium chloride and thallium acetate. A relaxation process unique to the thallium acetate-channel system was observed which on analysis gave rise to a relaxation time at 25 degrees of 120 msec. The permitivity data, as well as a comparison of binding constants, indicate that the relaxation process results from TI+ being bound within the channel and more specifically from an intrachannel ion translocation with a rate constant of approximately 4 x 10(6) sec-1 and with an energy of activation of less than 6.7 kcal/mole. These data compare favorably with data from conductance studies on planar bilayers and with ion and carbon-13 nuclear magnetic studies on the lysolecithin packaged malonyl gramicidin channels which combine to indicate that the relaxation process is due to the jump of the thallium ion across a central barrier.

Circular Dichroism↗
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↗