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Biomedical subjects

B Sakmann

Publications and source records attributed to B Sakmann.

At least 145 records · Page 8Linked to original sources
PubMed1987

Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones.

1. The ion-selective and ion transport properties of glycine receptor (GlyR) and gamma-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations of transmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl- intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl- activity indicating activation of Cl(-)-selective channels. The permeability ratio of K+ to Cl- (PK/PCl) was smaller than 0.05 for both channels. 4. The permeability sequence for large polyatomic anions was formate greater than bicarbonate greater than acetate greater than phosphate greater than propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5.2 and 5.6 A, respectively. The sequence of relative permeabilities for small anions was SCN- greater than I- greater than Br- greater than Cl- greater than F- for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cell-attached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels. The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel. 7. Single-channel conductances measured in equal 140 mM concentrations of small anions on both membrane faces revealed a conductance sequence of Cl- greater than Br- greater than I- greater than SCN- greater than F- for both channels. This is nearly the inverse sequence of that found for the permeability of these ions indicating the presence of binding sites for ions in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.

Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane of Xenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Whole-cell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR of Torpedo electric organ, expressed functional AChR channels at a much higher density. Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (greater than or equal to 5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane. The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20 +/- 1 degree C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials. The density of s.a.-channels is estimated to be 0.5-2 channels per micron 2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3-5 micron 2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches. AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding for Torpedo AChR. In normal frog Ringer's solution (20 +/- 2 degrees C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implanted Torpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate.

The fine structure of ion-channel activations by junctional nicotinic receptors in adult frog muscle fibres has been investigated. The agonists used were acetylcholine (ACh), carbachol (CCh), suberyldicholine (SubCh) and decan-1,10-dicarboxylic acid dicholine ester (DecCh). Individual activations (bursts) were interrupted by short closed periods; the distribution of their durations showed a major fast component ('short gaps') and a minor slower component ('intermediate gaps'). The mean duration of both short and intermediate gaps was dependent on the nature of the agonist. For short gaps the mean durations (microseconds) were: ACh, 20; SubCh, 43; DecCh, 71; CCh, 13. The mean number of short gaps per burst were: ACh, 1.9; SubCh, 4.1; DecCh, 2.0. The mean number of short gaps per burst, and the mean number per unit open time, were dependent on the nature of the agonist, but showed little dependence on agonist concentration or membrane potential for ACh, SubCh and DecCh. The short gaps in CCh increased in frequency with agonist concentration and were mainly produced by channel blockages by CCh itself. Partially open channels (subconductance states) were clearly resolved rarely (0.4% of gaps within bursts) but regularly. Conductances of 18% (most commonly) and 71% of the main value were found. However, most short gaps were probably full closures. The distribution of burst lengths had two components. The faster component represented mainly isolated short openings that were much more common at low agonist concentrations. The slower component represented bursts of longer openings. Except at very low concentrations more than 85% of activations were of this type, which corresponds to the 'channel lifetime' found by noise analysis. The frequency of channel openings increased slightly with hyperpolarization. The short gaps during activations were little affected when (a) the [H+]o or [Ca2+]o were reduced to 1/10th of normal, (b) when extracellular Ca2+ was replaced by Mg2+, (c) when the [Cl-]i was raised or (d) when, in one experiment on an isolated inside-out patch, the normal intracellular constituents were replaced by KCl. Reduction of [Ca2+]O to 1/10 of normal increased the single-channel conductance by 50%, and considerably increased the number of intermediate gaps. No temporal asymmetry was detectable in the bursts of openings. Positive correlations were found between the lengths of successive apparent open times at low SubCh concentrations, but no correlations between burst lengths were detectable. The component of brief openings behaves, at low concentrations, as though it originates from openings of singly occupied channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1984

Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart.

Single ventricular cells were enzymatically isolated from adult guinea-pig hearts (Isenberg & Klöckner, 1982). The patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was used to examine the conductance properties of an inward-rectifying K+ channel present in their sarcolemmal membrane. When the K+ concentration on the extracellular side of the patch was between 10.8 and 300 mM, inward current steps were observed at potentials more negative than the K+ equilibrium potential (EK). At more positive potentials no current steps were detectable, demonstrating the strong rectification of the channel. The zero-current potential extrapolated from the voltage dependence of the inward currents depends on the external K4 concentration [K+]o in a fashion expected for a predominantly K+-selective ion channel. It is shifted by 49 mV for a tenfold change in [K+]o. The conductance of the channel depends on the square root of [K+]o. In approximately symmetrical transmembrane K+ concentrations (145 mM-external K+), the single-channel conductance is 27 pS (at 19-23 degrees C). In normal Tyrode solution (5.4 mM-external K+) we calculate a single-channel conductance of 3.6 pS. The size of inward current steps at a fixed negative membrane potential V increases with [K+]o. The relation between step size and [K+]o shows saturation. Assuming a Michaelis-Menten scheme for binding of permeating K+ to the channel, an apparent binding constant of 210 mM is calculated for a membrane potential of -100 mV. For this potential the current at saturating [K+]o is estimated as 6.5 pA. The rectification of the single-channel conductance at membrane potentials positive to EK occurs within 1.5 ms of stepping the membrane potential from a potential of high conductance to one of low conductance. In addition to the main conductance state, the channel can adopt several substates of conductance. The main state could be the result of the simultaneous opening of four conducting subunits, each of which has a conductance of about 7 pS in 145 mM-external K+. The density of the inward-rectifying K+ channels in the ventricular sarcolemma is 0-10 channel/10 micron2 of surface membrane; the average of twenty-eight patches was 1 channel/1.8 micron2. It is concluded that the inward-rectifying K+ channels mediate the resting K+ conductance of ventricular heart muscle and the current termed IK1 in conventional voltage-clamp experiments.

Action Potentials↗
PubMed1984

Voltage-dependent inactivation of inward-rectifying single-channel currents in the guinea-pig heart cell membrane.

Inward currents through single K+ channels in isolated ventricular heart cells of the guinea-pig were recorded using the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The voltage-dependent gating properties of the channels were examined in the potential range between 0 and -120 mV with 145 mM-KCl on the extracellular side of the membrane patch, i.e. with approximately symmetrical transmembrane K+ concentrations. When voltage pulses from 0 mV to negative test potentials were applied to patches containing several channels, more channels were open at the beginning of the pulses than in the steady state. Averages of many current responses showed inactivation of the mean current in response to the hyperpolarizing voltage pulses. The inactivation was stronger and faster at larger hyperpolarization. The lifetimes of the open and closed states of the channel and the probability of the open state p were estimated from records of the elementary currents at various constant potentials. As indicated by the inactivation of the averaged currents, the value of p was smaller at more negative potentials, approximately 0.15 at -50 mV and 0.02 at -110 mV. This caused a negative slope in the current-voltage relation of the time-averaged current at potentials more negative than -50 mV. The channel openings were grouped in complex bursts. At least three exponentials were needed to fit the frequency histogram of the lifetimes of all closed states (time constants at -50 mV: 1.1 ms, 16 ms and 3.2 s). The lifetimes of the individual openings were exponentially distributed (time constant: 70 ms). The kinetics of the channel were interpreted by two different models involving three states of a channel (closed-closed-open or closed-open-closed). The rate constants and their voltage dependence were estimated for both models. Both models describe the data equally well; the reason for this ambiguity is discussed. The channels are blocked by Cs+ or Ba2+. Cs+ (0.1 mM) caused frequent and short interruptions of the individual channel openings. Ba2+ (0.5 mM) also shortened the openings and in addition decreased the number of openings per burst. The results suggest that the inward-rectifying current IK1 in heart ventricular cells is partially inactivated by hyperpolarization. The inactivation could account for part of the time-dependent decrease in the whole-cell current previously ascribed to depletion of K+.

Action Potentials↗
PubMed1983

Neurotrophic control of channel properties at neuromuscular synapses of rat muscle.

1. Ectopic neuromuscular synapses formed when the fibular nerve was implanted into the proximal part of rat soleus muscle and the soleus nerve was cut. The gating properties of acetylcholine (ACh) receptor channels in the newly formed ectopic and in the denervated original end-plates were examined at various stages of ectopic synapse formation.2. At ectopic end-plates the apparent mean open time of ACh receptor channels changes during synaptic development. Channels in immature ectopic end-plates, examined 1 week after cutting the soleus nerve, have apparent mean open times of approximately 4 ms (at -70 mV, 22 degrees C), similar to those of the extrasynaptic ACh receptor channels of completely denervated fibres. The channel gating of mature ectopic end-plates, examined 3-7 weeks after nerve section, is characterized by a mean open time of approximately 1 ms and resembles that found in normal end-plates of adult fibres.3. The conversion of end-plate channel gating occurs during the second and third week of synapse formation. During this period two discrete classes of channels with different gating behaviour are present in the ectopic end-plate.4. Examination of ectopic end-plates in the electron microscope reveals that junctional folds begin to appear in the subsynaptic membrane during the period of channel conversion.5. At the denervated original end-plates of ectopically innervated fibres the apparent mean open time of ACh receptor channels remains similar to that of normally innervated end-plates. Original end-plates retain the normal synaptic class of channel for at least 42 days after denervation. At this time, most of the ACh receptors present originally in the membrane have been replaced by newly inserted receptors.6. At former end-plates of completely denervated fibres ACh activates two classes of channels, even when most of the ACh receptors originally present in the end-plate have been replaced by new receptors.7. The results show that during synapse formation a neurally controlled conversion of ACh receptor channels occurs about 2-3 weeks after establishment of the nerve muscle contact. Thereafter end-plate channel properties are independent of neural influences. These observations are consistent with a mechanism of channel conversion whereby the nerve modifies not the ACh receptor channel itself, but another constituent of the end-plate membrane which determines the gating properties of end-plate channels.

Acetylcholine↗
PubMed1983

Patch-clamp measurements of elementary chloride currents activated by the putative inhibitory transmitter GABA and glycine in mammalian spinal neurons.

Methods for measuring whole-cell and unitary Cl- currents in the soma membrane of cultured spinal cord neurons by the patch-clamp technique are described. To separate single channel Cl- currents activated by the two putative inhibitory transmitters GABA and glycine from other membrane currents, isotonic KCl pipette solutions are used for measurements on "cell-attached" membrane patches. To isolate unitary Cl- currents in "cell-free" membrane patches, symmetrical, isotonic Tris Cl- solutions are used in both the pipette and the bath. Single Cl- channels are opened following binding of two agonist molecules to their respective receptors. The gating behavior of single receptor-channel complexes is described in terms of the activation and desensitization mechanism suggested originally by del Castillo and Katz (1957) and Katz and Thesleff (1957).

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↗
PubMed1981

Agonist-activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayers.

Planar lipid bilayers were formed with the mixed chain phospholipid 1-stearoyl-3-myristolglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Single-channel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonist-activated ionic channel seems, therefore, to be contained within the receptor monomer.

Acetylcholine↗
PubMed1980

Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist.

High resolution measurements of the current through individual ion channels activated by acetylcholine (AChR- channels) in frog muscle have shown that these currents are discrete pulse-like events with durations of a few milliseconds. Fluctuation and relaxation measurements of end-plate currents have led to the conclusion that the rate of channel opening increases with agonist concentrations, and that the channel, once open, closes spontaneously. Katz and Thesleff have shown, however, that in the continued presence of ACh, the initial end-plate current declines to an equilibrium value with a time constant of several seconds. This reversible phenomenon is referred to as receptor desensitization. We report here that in the presence of ACh concentrations sufficient to cause desensitization, single channel current pulses appear in groups. From the temporal sequence of the pulses, we have derived estimates of the rates of activation and desensitization of the AChR-channel.

Acetylcholine↗
PubMed1978

The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes.

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at mu-molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10--100ms. In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1--3 micrometer internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had be to found in which channels open at a relatively low frequency.

Animals↗