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Biomedical subjects

B Sakmann

Publications and source records attributed to B Sakmann.

At least 163 records · Page 9Linked to original sources
PubMed1978

Decamethonium both opens and blocks endplate channels.

Miniature endplate currents, endplate current fluctuations ("membrane noise"), and voltage-jump current relaxations were studied in voltage-clamped frog muscle fibers during decamethonium action. All three types of experiments revealed two kinetic processes controlling the opening of endplate channels, one that reflects agonist action and another that reflects local anesthetic-like blocking activity. The kinetic constants for these two steps were evaluated from measurements of the fast and slow time constants as a function of decamethonium concentration. At -130 mV membrane potential and 13 degrees, the mean open time of decamethonium-activated channels is 2.8 msec. The forward and backward rate constants for channel blocking are 1.7 X 10(7) M-1 sec-1 and 10(3) sec-1. The voltage dependencies of the channel lifetime and of the blocking equilibrium are similar to those seen with pure agonists and local anesthetics, respectively.

Animals↗
PubMed1978

A comparison of current-voltage relations for full and partial agonists.

1. Local conductance changes produced by various bath-applied agonists at frog end-plate membrane were measured using focal recording of extracellular potential in voltage-clamped muscle fibres. The potential difference between a focal micropipette placed on the nerve terminal and another micro-pipette placed on or near inactive membrane was taken as proportional to the agonist-induced current through a small patch of an end-plate membrane. 2. The current-voltage (I--V) relation of active membrane was obtained directly by increasing the membrane potential in a ramp fashion. The change in membrane potential was slow enough for post-synaptic gating processes to reach equilibrium during the ramp. 3. During application of sufficiently low concentrations of full agonists (carbachol, (ACh) and partial agonists (choline and decamethonium) the I--V relation of end-plate membrane showed strong curvature in the range of -60 to -130 mV. The slope of I--V relations increased exponentially with membrane hyperpolarization, an e-fold change in conductance occurring for about 50 mV potential shift. 4. The curvature of the I--V relation of end-plate-membrane activated by the partial agonists choline and decamethonium became less as the agonist concentration was increased, and with high concentrations (choline 15 mM; decamethonium 250 micrometer) the I--V relation became almost straight. 5. When end-plate currents produced by high concentrations of partial agonists were matched by application of equi-active concentrations of carbachol, the carbachol-activated membrane still showed as much curvature in its I--V relation as when low concentrations of carbachol were used. 6. Choline and decamethonium concentrations for which the I--V relation was straight produced much greater depression of miniature end-plate currents than did carbachol concentrations which produced the same membrane current at the holding potential. 7. I--V relations for full agonists at high concentrations were obtained after alpha-bungarotoxin pre-treatment. During application of carbachol (400--500 micrometer) and ACh (30--40 micrometer; after complete inhibition of acetylcholinesterase activity) the I--V relation of end-plate membrane is much less curved than during application of low concentrations. 8. It is concluded that either the voltage sensitivity of agonist-induced end-plate conductance reflects voltage sensitivity of agonist binding, or the partial agonists used can exert a voltage-dependent 'local anaesthetic' action in addition to their agonist activity.

Acetylcholine↗
PubMed1978

Acetylcholine-induced ionic channels in rat skeletal muscle.

This paper briefly reviews the evidence for ionic channels mediating the conductance increase caused by acetylcholine application to the end-plate of skeletal muscle fibers. "Membrane noise" observed during application of constant low concentrations of acetylcholine to an end-plate is thought to arise from the random superposition of many elementary events corresponding to the opening and closing of discrete ion channels. Statistical analysis of acetylcholine-induced noise reveals an elementary conductance event of of 34 pS (1 S = 1 omega-1) amplitude and 1 msec duration at room temperature in rat muscle fibers. Both size and duration of the elementary event are temperature dependent. Analysis of currents induced by application of acetylcholine to the extrasynaptic membrane of chronically denervated fibers shows that the elementary conductance has a similar size but is of much longer duration. Direct recording of square pulse-like currents by a patch clamp method confirms some of the conclusions drawn from fluctuation analysis.

Acetylcholine↗
PubMed1976

Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres.

1. Voltage clamp currents were recorded during iontophoretic application of steady doses of acetylcholine (ACh), carbachol or suberyldicholine to hyperpersensitive extrasynaptic regions of chronically denervated frog muscle fibers. Autocorrelation functions of drug induced current fluctuations were calculated and estimates of conductance gamma and average open time tau of the extrasynaptic ion channels were derived. 2. The average open time of an extrajunctional channel induced by acetylcholine is tauACh = 11 +/- 1-6 msec (+/- S.E.) at -80 mV and 8 degrees C. Carbachol and suberyldicholine open channels of tauCarb = 3-9 +/- 0-4 msec and tauSubCh = 19 +/- 2-5 msec (+/- S.E.) duration under the same conditions. The average open time of the extrasynaptic channel produced by each drug is three to five times longer than the value found for junctional channels in normal fibres. 3. The average open time of the extrajunctional channel is dependent on temperature and membrane potential. Lowering the temperature or increasing the membrane potential increases the average open time of the channels induced by any one of the drugs. 4. The conductance of a single extrajunctional channel opened by the action of acetylcholine is estimated to be gammaextra = 15 +/- 1-8 pmho (+/- S.E.). This is somewhat lower than the value of gammaep = 23 +/- 2 pmho (+/- S.E.) found for the conductance of a single open channel in the junctional membrane of normal fibres. The extrasynaptic channels opened by the action of carbachol and suberyldicholine have similar conductances to those produced by ACh. 5. The autocorrelation function of drug-induced current fluctuations, recorded at the former end-plate region of chronically denervated fibres often shows both a fast and a slow time constant. They correspond in value to the time constant of the autocorrelation function obtained from end-plate currents in normal fibres and from extrasynaptic currents in denervated fibres respectively. This could indicate that two populations of channels exist at the former end-plate region of denervated muscle fibres.

Acetylcholine↗
PubMed1975

Voltage-dependence of drug-induced conductance in frog neuromuscular junction.

Membrane currents from voltage-clamped frog muscle fibers were recorded during iontophoretic application of steady doses of carbachol, acetylcholine, and suberyldicholine to the endplate region. In the presence of these drugs, an exponentially relaxing current was observed after step changes of membrane potential. The time constant of relaxation was found to be voltage-dependent. It was equal to the time constant obtained from the autocorrelation function of drug-induced conductance fluctuations measured under similar conditions. Analysis of instantaneous current at the on- and offsets of voltageclamp pulses showed that there is no shift in equilibrium potential during the pulses.

Acetylcholine↗
PubMed1975

1-Pyrene-butyrylcholine: a fluorescent probe for the cholinergic system.

The action of 1-pyrene-butyrylcholine, a new cholinergic fluorescent probe, has been studied at the cellular level using electrophysiological and fluorescence techniques. The spectroscopic properties of the probe were found to be similar to those pf pyrene-butyric acid, the excited-state lifetime in air-saturated aqueous solutions being 92 nsec. At micromolar concentrations the probe was found to exert a nondepolarizing, reversible blocking action at the neuromuscular junction of the frog. The same cholinolytic effect was observed in hypersensitive denervated muscles. The synaptic localization of the probe could be observed with fluorescence microscopy using sub- and micromolar concentrations. Treatment of the nerve-muscle preparations with proteolytic enzymes, resulting in the separation of the nerve ending from the muscle end-plate, enabled a distinction to be made between the fluorescence arising from these two parts of the synapse. Intense presynaptic fluorescence was observed, and was not altered by micromolar concentrations of alpha-bungarotoxin, d-tubocurarine, hemicholinium, or cholinesterase inhibitors. Faint reversible staining of the end-plate region was observed in enzymically treated muscles and was inhibited by prior treatment with alpha-bungarotoxin. Fluorescent alpha-toxin revealed similar patterns of fluorescence in the end-plate of enzyme-treated muscles. The postsynaptic localization of the fluorescent probe is therefore tentatively identified as the one producing the cholinolytic effect upon binding to acetylcholine receptor sites.

Animals↗
PubMed1974

The effect of contractile activity on fibrillation and extrajunctional acetylcholine-sensitivity in rat muscle maintained in organ culture.

1. The effect of contractile activity on the initiation of spontaneous action potentials (fibrillation) and on extrajunctional acetylcholine-sensitivity has been studied in single fibres in strips of previously denervated rat diaphragm maintained in organ culture for up to 10 days.2. Following removal of the diaphragm from the animal, fibrillation slowed and usually stopped altogether for about 24-36 hr. Thereafter, spontaneously active fibres were found in all cultured muscle strips.3. At any one time, about (1/4) to (1/3) of fibres impaled with micro-electrodes were active (defined as more than one action potential/10 sec), with a mean discharge frequency of 4.5/sec (range 0.1-24/sec).4. The duration of continuous activity in single fibres was, on average, 21-22 hr; a period of activity was followed by a longer inactive interval. Thus activity in single fibres is cyclical.5. Direct stimulation of fibrillating strips for 24 hr at 10/sec suppressed spontaneous activity for 1-3 days.6. Conversely, blockade of spontaneous activity with tetrodotoxin for 72 hr led to a two- to threefold increase in the number of fibrillating fibres when the drug was washed out; in some strips nearly all fibres became spontaneously active.7. The mean rate of activity of diaphragm fibres during normal breathing, determined by recording single units from the phrenic nerve in lightly anaesthetized animals, is about 18/sec.8. Direct stimulation of cultured diaphragm strips in a pattern similar to breathing for 7-8 days at an average rate of 10-12/sec (or 5/sec in some experiments), resulted in a marked reduction (about 95% in experiments at 10/sec) in extrajunctional sensitivity to ionophoretically applied ACh.9. Direct stimulation for 24 hr at 10/sec (comparable to a period of spontaneous activity) caused only a small reduction in extrajunctional ACh-sensitivity.10. We conclude that spontaneous activity in single fibres under these conditions occurs cyclically because activity, over a period of hours, inhibits the ability of the fibrillating fibre to initiate further action potentials. Repeated self-inhibition of spontaneous activity probably explains why denervated muscle fibres remain highly sensitive to extrajunctionally applied ACh.

Acetylcholine↗
PubMed1974

Membrane properties underlying spontaneous activity of denervated muscle fibres.

We have examined the events underlying the initiation of spontaneous action potentials (fibrillation) in fibres of previously denervated rat diaphragm maintained in organ culture for up to 10 days.1. Based on discharge pattern, two classes of spontaneously active fibres were found: rhythmically discharging fibres, and fibres in which action potentials occur at irregular intervals.2. Sites of action potentials initiation were located by exploration along the fibre length with two independent extracellular recording electrodes. The majority of sites of origin in both regular and irregular fibres were at the former end-plate zone; however, there was no region along the length that could not, at least in some fibres, be a site of origin.3. Intracellular recording at or near sites of origin of action potential discharge showed two types of initiating events. Irregularly discharging fibres were brought to threshold by discrete depolarizations of up to 15 mV in amplitude, while regularly occurring action potentials were associated with oscillations of the membrane potential.4. Discrete depolarizations (called fibrillatory origin potentials or f.o.p.s) at sites of origin in irregularly discharging fibres have the following properties: (a) random occurrence and nearly constant amplitude outside a refractory period during which both amplitude and probability of a second f.o.p. are reduced; (b) associated inward current flow which is localized to about 100 mum or less along the fibre length, and (c) dependence of amplitude and frequency on membrane potential.5. Oscillation of membrane potential found at sites of origin of action potential discharge in regular fibres also occurred locally along the fibre length and was sensitive to changes in membrane potential.6. Both f.o.p.s and oscillations of membrane potential were reversibly abolished by low Na(+)-Ringer fluid or tetrodotoxin.7. Neither type of initiating event was appreciably affected by concentrations of D-tubocurarine which blocked extrajunctional sensitivity to acetylcholine.8. We conclude that spontaneous action potentials under these conditions arise from a localized Na(+)-conductance change in the membrane of the active fibre; this conductance change is distinct from the increased Na(+)-conductance which follows the interaction of acetylcholine with its receptor. Spontaneous activity in single, denervated muscle fibres is cyclical and self-inhibiting (Purves & Sakmann, 1974); thus the Na(+)-conductance change underlying the initiation of spontaneous action potentials is affected by muscle fibre activity.

Action Potentials↗
PubMed1973

Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions.

1. Frog cutaneous pectoris nerve-muscle preparations were incubated with collagenase and protease and examined with electrophysiological and electron microscopic techniques.2. The physiological properties and intracellular ultrastructural appearance of individual muscle and nerve cells were not affected by the enzyme treatment. However, neuromuscular transmission and the morphology of the nerve-muscle junction were altered.3. Collagenase produced an irreversible loss of activity of end-plate cholinesterase and a partial loss of stainable ;synaptic cleft material'.4. Protease produced these changes and, in addition, the entire basement membrane was digested, which led to ;synaptic disjunction' of nerve terminals and muscle end-plates.

Acetylcholinesterase↗