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Biomedical subjects

E Neher

Publications and source records attributed to E Neher.

29 records · Page 2Linked to original sources
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↗
PubMed1978

Ionic selectivity, saturation, and block in gramicidin A channels. II. Saturation behavior of single channel conductances and evidence for the existence of multiple binding sites in the channel.

A theory, recently developed by Sandblom, Eisenman and Neher (1977) for the conductance of single gramicidin A cha-nelspred icts three limiting behaviors of the relation between conductance and salt concentration. These are: (i) a saturating behavior resembling a simple adsorption isotherm at medium and high concentrations, (ii) a decrease in conductance at the highest obtainable concentrations and (iii) deviations from the isotherm at very low concentrations. Features i and ii have been described before. Experimental evidence for point iii is given here. The new feature points towards interactions among ions in the channel at ionic concentrations as low as 1--10 mM. Particular emphasis is given to the behavior at very low salt concentrations and the experimental problems encountered in this situation. In addition, mutual blocking effects among monovalent ions in symmetrical salt mixtures are characterized and found to be in satisfactory agreement with theoretical expectations, based upon the single salt conductance data presented here and zero-current potentials in salt mixtures to be described in a subsequent paper.

Binding Sites↗
PubMed1978

Interactions in cation permeation through the gramicidin channel. Cs, Rb, K, Na, Li, Tl, H, and effects of anion binding.

As a prototype for binding and interaction in biological Na and K channels, the single channel conductances for Li, Na, K, Rb, Cs, H, and Tl and the membrane potentials for Tl-K mixtures are characterized for gramicidin A over wider concentration rangers than previously and analyzed using an "equilibrium domain" model that assumes a central rate-determining barrier. Peculiarities in the conductance-concentration relationship for TlF, TlNO3, and TlAc suggest that anions bind to Tl-loaded channels, and the theory is extended to allow for this. For concreteness, the selectivity of cation permeation is characterized in terms of individual binding and rate constants of this model, with the conclusions that the strongest site binds Cs greater than Rb greater than K greater than Na greater than Li, while the next strongest binds Na greater than K greater than Li greater than Rb greater than Cs. However, because Schagina, Grinfeldt, and Lev's recent finding of single filing (personal communication) indicates that the channel sites in gramicidin cannot be at equilibrium with the solution, and work in progress with Hägglund and Enos (Biophys. J. 21:26a. [Abstr.]) indicates that the simplest model adequate to account for the observed concentration-dependences of flux-ratio, conductance, I--V characteristic, and permeability has three barriers and four sites, some implications of additional rate-determining barriers at the mouth of the channel are discussed. The results are summarized using phenomenological "experimental" parameters that provide a model-independent way to represent that data concisely and which can be interpreted physically in terms of any desired model.

Anions↗
PubMed1978

Local anaesthetics transiently block currents through single acetylcholine-receptor channels.

1. Single channel currents through acetylcholine receptor channels (ACh channels) were recorded at chronically denervated frog muscle extrajunctional membranes in the absence and presence of the lidocaine derivatives QX-222 and QX-314. 2. The current wave forms due to the opening and closing of single ACh channels (activated by suberyldicholine) normally are square pulses. These single pulses appear to be chopped into bursts of much shorter pulses, when the drug QX-222 is present in addition to the agonist. 3. The mean duration of the bursts is comparable to or longer than the normal channel open time, and increases with increasing drug concentration. 4. The duration of the short pulses within a burst decreases with increasing drug concentration. 5. It is concluded that drug molecules reversibly block open end-plate channels and that the flickering within a burst represents this fast, repeatedly occurring reaction. 6. The voltage dependence of the reaction rates involved, suggested that the site of the blocking reaction is in the centre of the membrane, probably inside the ionic channel.

Animals↗
PubMed1977

Ionic selectivity, saturation and block in gramicidin A channels: I. Theory for the electrical properties of ion selective channels having two pairs of binding sites and multiple conductance states.

A model for the gramicidin A channel is proposed which extends existing models by adding a specific cationic binding site at each entrance to the channel. The binding of ions to these outer channel sites is assumed to shift the energy levels of the inner sites and barriers and thereby alter the channel conductance. The resulting properties are analyzed theoretically for the simplest case of two inner sites and a single energy barrier. This for-site model (two outer and two inner) predicts that the membrane potential at zero current (Uo) should be a Goldman-Hodgkin-Katz equation with concentration-dependent permeability ratios. The coefficients of the concentration-dependent terms are shown to be related to the peak energy shifts of the barrier and to the binding constants of the outer sites. The thory also predicts the channel conductance in symmetrical solutions to exhibit three limiting behaviors, from which the properties of the outer and inner sites can be characterized. In two-cation symmetrical mixtures the conductance as a function of mole fraction is shown to have a minimum, and the related phenomenon of inhibition and block exerted by one ion on the other is explained explicitly by the theory. These various phenomena, having ion interactions in a multiply occupied channel as a common physical basis, are all related (by the theory) through a set of measurable parameters describing the properties of the system.

Electric Conductivity↗
PubMed1977

The influence of phospholipid polar groups on gramicidin channels.

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of N-methylation and comprised phosphocholine, -N,N-dimethylethanolamine, -N-methylethanolamine and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude lambda of the unit conductance step and a decrease in the average channel life-time or duration tau. The series of tau-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension gamma is a relevant parameter for the prediction of tau-values.

Binding Sites↗
PubMed1976

Fluctuation and relaxation analysis of monazomycin-induced conductance in black lipid membranes.

Fluctuation and relaxation analyses were performed on monazomycin-induced conductance of lipid bilayer membranes. With both methods a slow (sec) and a fast (msec) current component are apparent; however, the amplitude of the slow, voltage-dependent process is greater than that of the fast component in the step relaxation experiment and less in the fluctuation experiment. The fluctuation analysis showed principally a rapid voltage-dependent process which appears to be related to the multistate character of the conducting channel. The experimental results are interpreted in terms of a simplified kinetic model which is used to calculate relaxation and noise amplitudes.

Alamethicin↗
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↗