K. Peper

Junctional and extrajunctional acetylcholine receptors in normal and denervated frog muscle fibres. Noise analysis experiments with different agonists.

SummaryIonic channel properties of acetylcholine receptors located in, in the vicinity of, or far away from a frog neuromuscular junction were investigated by noise analysis of drug induced current fluctuations. For drugs applied to the junction, in certain cases two Lorentzian curves were necessary to describe the data. It is postulated that the reason for this observation is that a contribution from perijunctional receptors was being observed. The conductance of a single channel in the junction was independent of the nature of the agonist and had an average value of 17.9 pS (temperature range 8–25°C, solution buffered with Tris). After denervation for 21 days the conductance γ was 7.5 pS at extrajunctional locations. In the close neighbourhood of the junction (perijunctional receptors) values were found between 4 and 19 pS.The mean value of the open channel life-time τ in the endplate exposed to acetylcholine was 2.4 ms at 8–11°C. This value was 0.90 ms with carbachol, 0.50 ms with succinylcholine, 0.28 ms with decamethonium and 0.45 ms with nicotine. The receptors outside the endplate exhibited τ-values which at a given temperature were 2–3 times larger than those at the endplate. Raising the temperature to 23°C reduced all τ-values by factors of 2–3. It is concluded that at least two types of ACh-receptors with different properties exist in the muscle membrane, possibly produced by ACh-receptive units in different states of aggregation.

Iontophoretic application of acetylcholine: Advantages of high resistance micropipettes in connection with an electronic current pump

SummaryDesensitization of ACh-receptors occurs when ACh-micropipettes with resistances smaller than 100 MΩ are used, even if an applied “braking” current is set to an optimum. High sensitivity with almost no desensitization can be obtained with pipettes of higher resistances, but the current passing capability is normally reduced and a high time constant makes ACh-pulses of 1 msec duration a problem. These drawbacks can be reduced by using a potent electronic constant current source which simultaneously neutralizes the pipettes capacity. The resistance range from 120 to 200 MΩ seems to be optimal for microapplication of ACh, especially in exploring regions of low sensitivity. Rather uniform pipettes in this range can be obtained by applying a cooling air jet during the pull of the pipettes.

The M. omohyoideus of the mouse as a convenient mammalian muscle preparation

SummaryMuscles from cats, rats, guinea pigs and mice have been investigated as preparations for visualizing mammalian neuromuscular junctions with the aid of Nomarski interference optics. The M. omohyoideus of the mouse was found to be most convenient. Electrophysiological investigations showed that an endplate is normally surrounded by a population of perijunctional receptors. For junctional receptors in the endplate, a Hill coefficient ofnH=2.6 for acetylcholine was determined at 38°C, decreasing to a value of 2.3 at room temperature. For both perijunctional and extrajunctional receptors (the latter occurring after denervation), the coefficientnH was 1.9. Noise analysis revealed a channel conductance γ which changed abruptly from 22.4±1.0 pS (10–23°C) to 45.6±0.9 pS (34–39°C) in a very small temperature range around 25.5°C. The mean channel lifetime τ was 0.3 ms at 39°C and 1.0 ms at 23°C.

The effects of alcohols and diols at the nicotinic acetylcholine receptor of the neuromuscular junction

A series of straight chain aliphatic alcohols from ethanol to octanol were tested at voltage-clamped frog endplates. In the presence of high concentrations of ethanol (greater than 1 M) the individual current responses to ionophoretic pulses of ethanol were reduced in amplitude and the dose-response curve for acetylcholine was shifted to the right. All the alcohols tested had this effect and their potency increased with the length of the carbon chain. The results were interpreted to indicate that as the molecular weight of the alcohol increased, its potency as a channel blocker also increased. The diol derivative of ethanol, which is ethylene glycol (ethanediol), was totally inactive up to 400 mM. However, 1,3-propanediol was a more potent blocker than propanol. After dose-response curves were carried out in high doses of ethanol and propanediol, the number of receptors was found to be permanently reduced. This effect could be due to irreversible denaturation of the receptor and therefore reversible denaturation could account for some of the reversible blocking effects caused by such drugs. An additional effect on the receptor was observed in that low concentrations of ethanol and propanol reduced the apparent dissociation constant for acetylcholine, thus increasing the amplitude of individual responses and shifting the dose-response curve to the left.

Acetylcholine-induced potassium current fluctuations in the rabbit sino-atrial node

Abstract1.Small sinoatrial-node preparations were voltage clamped by two micropipettes. Both the amplitude (mean current) and the fluctuation of acetylcholine-induced potassium current were recorded. The fluctuations were analyzed by both calculating their variance and establishing their power density spectrum.2.It is assumed that acetylcholine receptors, when occupied, open momentarily ionic channels. Each channel is described by its conductance γ and the average open time τ.3.The variance did not increase linearly with the mean current, but reached a saturation and even decayed with large mean currents, indicating that more than 50% of the total number of channels are open.4.The average maximum mean current (all channels open) for a preparation approximately 300 μm ×200 μm×50 μm was 89±22 nA (n=7).5.The average single channel conductance, calculated from the variance, was 3,71±0.48 pS (n=7).6.The power density spectra of the fluctuations were fitted by single Lorentzian curves. The single channel conductance was thus determined to be γ= 3.79±1.25 pS and the corner frequencyfc=0.96±0.25 Hz (n=21). This corresponds to an average open time τ=166 ms.7.TheQ10 of the average open time (determined by the corner frequency (fc) was 2.83±0.38 (n=4).8.The density of channels was approximately 1 per 1 μm2 of cell surface or roughly 2000 channels per cell.9.The muscarinic acetylcholine receptor of S-A node cells may thus be characterized by a relatively long open time (160 ms), a low density of channels and a small elementary conductance of about 4 pS.

Acetylcholine receptor: modification of synaptic gating mechanism after treatment with a disulfide bond reducing agent.

SummaryReduction of a ‘reactive’ disulfide bond in the postsynaptic membrane of the frog neuromuscular junction by dithiothreitol (DTT) decreases both the sensitivity of the membrane to applied acetylcholine (ACh) and the amplitude of the single ‘shot effect’. Analysis of ACh induced conductance fluctuations in voltage clamped frog endplates indicates that DTT reduces both the amplitude γ and duration τ of the elementary conductance events. The mean control value of γ was 18.5·10−12 Ω−1 with no significant dependence on temperature. The mean control values of τ were 2.3 msec at 7–9°C and 0.94 msec at 20–22°C. At 7–9°C 1 mM DTT (20–50 min after application) reduced γ to 61% of the control value and at 20–22°C to 39%, while τ was reduced to 70% at both temperature ranges. The dose-response curve for iontophoretically applied ACh indicates that neither the total number of ionic channels nor the cooperativity within the receptors are changed. However, the affinity of ACh for the receptor sites was reduced. All effects of DTT were fully reversed by the oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB).

A monolayer preparation of innervated skeletal muscle fibres of the m. cutaneus pectoris of the frog.

SummaryThe preparation of a monolayer of innervated skeletal muscle fibres of theM. cutaneus pectoris of the frog is described. The advantage of this preparation is demonstrated by examples of living end-plates as viewed under Nomarskiinterference optics, and of nerve terminal regions investigated by scanning microscopy and freeze-cleavage techniques.

Postjunctional characteristics of the endplates in mammalian fast and slow muscles

We have studied the postjunctional characteristics of motor endplates in the extensor digitorum longus (EDL) and soleus muscles of the rat. At voltage clamped endplates, equilibrium interactions between acetylcholine (ACh) and the ACh receptor were determined from the dose-response curves obtained by quantitative ionophoresis of ACh. These results showed that the maximum ACh induced conductance change per unit endplate surface,gmax, was 21.8±0.9 nS/μm2 in EDL and 8.2±0.9 nS/μm2 in soleus, the apparent dissociation constant,K, was 65.9±4.3 μM in EDL and 43.5±3.3 μM in soleus, and the Hill-coefficient,nH, was 2.3±0.1 in EDL and 2.2±0.1 in soleus.Single channel characteristics were derived from analysis of the ACh-induced endplate current noise. The results showed that at room temperature the mean conductance of the single channel, γ, was 24.6±1.2 pS in EDL and 23.9±1.2 pS in soleus, and the mean life time of the channel, τ, was 0.80±0.05 ms in EDL and 0.71±0.03 ms in soleus.Of all the properties studied, the maximum conductance per unit endplate surface,gmax, was significantly smaller at the soleus endplate than at the EDL endplate. The calculated density of functional ACh receptors was 62% less, and the total number of the functional ACh receptors was 60% less at the soleus endplates than at the EDL endplates. These results suggest that the soleus has a lower margin of safety for neuromuscular transmission than the EDL.

Current-voltage relation and reversal potential at junctional and extrajunctional ACh-receptors of the frog neuromuscular junction

SummaryThe relationship between synaptic current and membrane potential has been examined at junctional and extrajunctional end-plate receptors of the frog. At junctional receptors, the synaptic currentmembrane potential relationship is linear for close range iontophoretic ACh application and non-linear when it is delivered from some distance. At extrajunctional receptors the current-voltage relationship is always non-linear. These non-linearities can be related to the fact that in both cases [ACh] on membrane outlasts the mean life-time of the synaptic channels. When their mean life-time is varied, the currentvoltage relationship obtained at junctional receptors is not longer linear and the peak synaptic conductance increases or decreases with hyperpolarization as the channel life time is shortened or lengthened, respectively.

The acetylcholine sensitivity in the vicinity of the neuromuscular junction of the frog

SummaryFor distancesr larger than 15 μm the ACh-response can be described by the diffusion law: the sensitivity depends onr−3; time to peak,T, and latency,L, depend onr2 (r=distance junction-ACh-pipette). The diffusion coefficient for ACh has been determined to be 11.9 (S.E.±0.4)·10−6 cm2 sec−1 at 23°C. For a depolarization of 2 mV, the range of junctional ACh-sensitivity was normally 150–500 mV/nC with extreme values of 100 and 700 mV/nC. The time to peak was shorter than 6 msec. The range of maximal extrajunctional sensitivity was 0.1–45 mV/nC or, related to the respective junction, 0.1–12%, with low values at winter- and low to high values at summertime. The extension of ACh-sensitivity beyond the endplate (1% of junctional value) was 0 to 400 μm, being high at summertime. In some fibres a low ACh-sensitivity could be demonstrated over the whole length of the muscle. The extrajunctional sensitivity was highest at the entrance of the nerve fibre with 2 to 4 fold decrease within the endplate region. Extrajunctional ACh-receptors can be conveniently investigated on the surface of the muscle fibre antipodal to the neuromuscular junction. No extrajunctional “hot spots” or areas of high sensitivity neighbouring those of low sensitivity could be demonstrated.