Single-channel currents recorded from membrane of denervated frog muscle fibres
TL;DR: A more sensitive method of conductance measurement is reported, which, in appropriate conditions, reveals discrete changes in conductance that show many of the features that have been postulated for single ionic channels.
Abstract: THE ionic channel associated with the acetylcholine (ACh) receptor at the neuromuscular junction of skeletal muscle fibres is probably the best described channel in biological membranes. Nevertheless, the properties of individual channels are still unknown, as previous studies were concerned with average population properties. Macroscopic conductance fluctuations occurring in the presence of ACh were analysed to provide estimates for single channel conductance and mean open times1–3. The values obtained, however, depended on assumptions about the shape of the elementary conductance contribution—for example, that the elementary contribution is a square pulse-like event2. Clearly, it would be of great interest to refine techniques of conductance measurement in order to resolve discrete changes in conductance which are expected to occur when single channels open or close. This has not been possible so far because of excessive extraneous background noise. We report on a more sensitive method of conductance measurement, which, in appropriate conditions, reveals discrete changes in conductance that show many of the features that have been postulated for single ionic channels.
Citations
More filters
TL;DR: 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.
Abstract: 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.
17,136 citations
Cites methods from "Single-channel currents recorded fr..."
...The extracellular patch clamp technique has allowed, for the first time, the currents in single ionic channels to be observed (Neher and Sakmann 1976)....
[...]
TL;DR: It is shown that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane, which could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.
Abstract: We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.
3,251 citations
Book•
05 Jun 1975
TL;DR: Introduction to synaptic circuits, Gordon M.Shepherd and Christof Koch membrane properties and neurotransmitter actions, David A.Brown and Anthony M.Brown.
Abstract: Introduction to synaptic circuits, Gordon M.Shepherd and Christof Koch membrane properties and neurotransmitter actions, David A.McCormick peripheral ganglia, Paul R.Adams and Christof Koch spinal cord - ventral horn, Robert E.Burke olfactory bulb, Gordon M.Shepherd, and Charles A.Greer retina, Peter Sterling cerebellum, Rodolfo R.Llinas and Kerry D.Walton thalamus, S.Murray Sherman and Christof Koch basal ganglia, Charles J.Wilson olfactory cortex, Lewis B.Haberly hippocampus, Thomas H.Brown and Anthony M.Zador neocortex, Rodney J.Douglas and Kevan A.C.Martin Gordon M.Shepherd. Appendix: Dendretic electrotonus and synaptic integration.
3,241 citations
01 Jan 1996
TL;DR: The action potential is triggered when the membrane potential, which was at the resting level, depolarizes and reaches the threshold of excitation, which triggers the action potential.
Abstract: Excitability. Excitability of cell membranes is crucial for signaling in many types of cell. Excitation in the physiological sense means that the cell membrane potential undergoes characteristic changes which, in most cases, go in the depolarizing direction. Single depolarization from the resting potential to potentials near 0 mV has generally been called an action potential. A schematic representation of a neuronal action potential is given in Fig. 12.1 A. The action potential is triggered when the membrane potential, which was at the resting level, depolarizes and reaches the threshold of excitation. This depolarization, which triggers the action potential, is generated by depolarizing synaptic currents, or depolarizing current coming from a membrane region that is already excited (propagation of an action potential), or by pacemaker currents mediated by pacemaker channels, or by current injected externally by an electrode. The duration of different types of action potential varies from seconds to less than 1 ms.
3,016 citations
TL;DR: A review of the basic neuroscience processes of pain (the bio part of biopsychosocial, as well as the psychosocial factors, is presented) and on the development of new technologies, such as brain imaging, that provide new insights into brain-pain mechanisms.
Abstract: The prevalence and cost of chronic pain is a major physical and mental health care problem in the United States today. As a result, there has been a recent explosion of research on chronic pain, with significant advances in better understanding its etiology, assessment, and treatment. The purpose of the present article is to provide a review of the most noteworthy developments in the field. The biopsychosocial model is now widely accepted as the most heuristic approach to chronic pain. With this model in mind, a review of the basic neuroscience processes of pain (the bio part of biopsychosocial), as well as the psychosocial factors, is presented. This spans research on how psychological and social factors can interact with brain processes to influence health and illness as well as on the development of new technologies, such as brain imaging, that provide new insights into brain-pain mechanisms.
2,566 citations
Cites methods from "Single-channel currents recorded fr..."
...First described by Neher and Sakmann (1976), patch-clamp recording in vitro is now a powerful method for studying electrophysiological properties and chemosensitivity of neurons involved in the transduction and transmission of nociceptive stimuli....
[...]
References
More filters
TL;DR: Acetylcholine produced end‐plate current (e.p.c.) noise is shown to be the results of statistical fluctuations in the ionic conductance of voltage clamped end‐plates of Rana pipiens.
Abstract: 1. Acetylcholine produced end-plate current (e.p.c.) noise is shown to be the results of statistical fluctuations in the ionic conductance of voltage clamped end-plates of Rana pipiens.
2. These e.p.c. fluctuations are characterized by their e.p.c. spectra which conform to a relation predicted from a simple model of end-plate channel gating behaviour.
3. The rate constant of channel closing α is determined from e.p.c. spectra and is found to depend on membrane potential V according to the relation α = BeAV (B = 0·17 msec−1±0·04 S.E., A = 0·0058 mV−1±0·0009 S.E. at 8° C) and to vary with temperature T with a Q10 = 2·77, at −70 mV. A and B in this expression both vary with T and therefore produce a membrane potential dependent Q10 for α.
4. Nerve-evoked e.p.c.s and spontaneous miniature e.p.c.s decay exponentially in time with a rate constant which depends exponentially on V. The magnitude and voltage dependence of this decay constant is exactly that found from e.p.c. spectra for the channel closing rate α.
5. The conductance γ of a single open end-plate channel has been estimated from e.p.c. spectra and is found not to be detectibly dependent on membrane potential, temperature and mean end-plate current. γ = 0·32±0·0045 ( S.E.) × 10−10 mhos. Some variation in values for γ occurs from muscle to muscle.
6. It is concluded that the relaxation kinetics of open ACh sensitive ionic channels is the rate limiting step in the decay of synaptic current and that this channel closing has a single time constant. The relaxation rate is independent of how it is estimated (ACh produced e.p.c. fluctuations, e.p.c., m.e.p.c.), and is consistent with the hypothesis that individual ionic channels open rapidly to a specific conductance which remains constant for an exponentially distributed duration.
7. The voltage and temperature dependence of the channel closing rate constant agree with the predictions of a simple dipole-conformation change model.
919 citations
TL;DR: 1. When a steady dose of acetylcholine is applied to an end‐plate, the resulting depolarization is accompanied by a significant increase in voltage noise, and this noise can be significant in both the positive and the negative directions.
Abstract: 1. When a steady dose of acetylcholine (ACh) is applied to an end-plate, the resulting depolarization is accompanied by a significant increase in voltage noise.2. The characteristic properties of this ACh noise (amplitude and time course) are examined under various experimental conditions. The voltage noise is analysed on the assumption that it arises from statistical fluctuations in reaction rate, and in the frequency of the elementary current pulses (;shot effects') produced by the action of ACh molecules.3. The elementary ACh current pulse (amplitude approximately 10(-11) A), arises from a conductance change of the order of 10(-10) Omega(-1) which lasts for approximately 1 ms (at 20 degrees C), and produces a minute depolarization, of the order of 0.3 muV. It is associated with a net charge transfer of nearly 10(-14) C, equivalent to approximately 5 x 10(4) univalent ions.4. At low temperature, and during chronic denervation, the duration of the elementary current pulse increases, and the elementary voltage change becomes correspondingly larger.5. Curare has little or no effect on the characteristics of the elementary event.6. A comparative study of ACh and carbachol actions shows that carbachol produces considerably briefer, and therefore less effective, current pulses than ACh.
895 citations
TL;DR: NET ion movements across biological or synthetic lipid membranes may take place by various mechanisms, underlying all of which there is a rather ill-defined and small ion leakage or background conductance.
Abstract: NET ion movements across biological or synthetic lipid membranes may take place by various mechanisms, underlying all of which there is a rather ill-defined and small ion leakage or background conductance. Most ions permeate by means of pathways involving either permanent or transient modifications of the basic structure of the membrane. If permanent pathways are involved, a given membrane conductance may be accounted for by routes which are either numerous and of low conductance or few and of high conductance. For transient pathways, duration must also be considered. Thus, if a carrier is invoked, the duration will be the time the carrier, complexed with an ion, spends shuttling across the membrane. For a pore, the duration is the time for which it remains open to ions. At present little is known concerning the number, conductance and duration of the ionic pathways in any membrane of the types mentioned. Limited information is available for the nerve membrane, although this is rather imprecise and indirect1.
387 citations
TL;DR: The purpose of the present experiment was to determine directly the time course of the active phase of the e.
Abstract: IT HAS BEEN CONSIDERED that the end-plate potential (e.p.p.) is generated by the brief ionic flux across the end-plate membrane and the later slowly declining phase of the e.p.p. is due to the dissipation of the charge along and across the muscle membrane. This consideration was supported by some authors. Kuffler (21) observed with a single nerve-muscle preparation that the later slowly decaying part of the e.p.p. was destroyed by a propagated muscle impulse and obtained a duration of transmitter action (3-4 msec. at 20°C.) by observing the size of the e.p.p. that was built after the invasion of a propagated muscle impulse. Katz (18) demonstrated that the neuromuscular transmitter produced a brief phase of impedance loss at the end-plate region. Recently Fatt and Katz (10) observed by measuring the displacement of the total charge along and across the muscle membrane during the e.p.p. that the active depolarization process at the end-plate had ceased within 2 msec. On the other hand the time course of the actively depolarizing phase of the e.p.p. was estimated by an analysis of the time course of the e.p.p., it being assumed that the exponentially decaying phase was attributable to the passive repolarization of the muscle membrane (7,19). The purpose of the present experiment was to determine directly the time course of the active phase of the e.p.p. by using the voltage clamp method which was originally described by Hodgkin et al. (14) and was also applied to the squid giant synapse by Tasaki and Hagiwara (29). When the membrane potential is clamped at the resting membrane potential with negative feed-back during the neuromuscular transmission, the electrotonic spread of the charge along the muscle fibre membrane can be eliminated. The feed-back current which flows through the muscle membrane to hold the membrane potential at the resting value is due to the brief electric change at the end-plate, i.e., it will show the active phase of the e.p.p. To simplify the expression, the feed-back current during neuromuscular transmission will be called provisionally the “end-plate current.” A preliminary report of the present experiment appeared in 1958 (27).
327 citations
TL;DR: Frog cutaneous pectoris nerve‐muscle preparations were incubated with collagenase and protease and examined with electrophysiological and electron microscopic techniques.
Abstract: 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.
202 citations