Effect of inhibitors of aerobic and anaerobic metabolism on the membrane potential oscillation induced by veratrine.

1. Veratrine-induced membrane potential oscillation is still apparent after the muscle has been treated with 1 mM KCN, or 1 mM NaN3, or 0.2 mM DNP, to inhibit oxidative metabolism. 2. The frequency of the membrane potential oscillation decreases reversibly in response to 1 mM phlorrhizin either in Na- or Li-Ringer solution. 3. Veratrine causes no membrane potential oscillation but only depolarization in muscles pretreated with 0.5 mM iodoacetic acid, eventually a few abortive waves can be observed. Iodoacetic acid also abolishes oscillation which has already developed. 4. On the basis of the present data it is suggested that rhythmic changes of metabolism may be related to the oscillation of membrane potential.     

The physostigmine depolarization potentiating effect of salicylate in frog skeletal muscle.

1) The frog's sartorius muscle was depolarized depending on the degree of concentration 2--4 times more intensely by physostigmine salicylate than by physostigmine sulphate. 2) In normal Ringer's solution, 1 mM physostigmine salicylate decreased the sensitivity of the membrane to potassium depolarization by about 90%. Under similar experimental conditions, physostigmine sulphate and Na salicylate, respectively, decrease the sensitivity of the membrane to potassium depolarization by about 30%. 3) The difference manifested in the depolarizing effect of salicylate and other physostigmine salts (chloride, sulphate, phosphate, formiate, acetate, monochloracetate, benzoate and para-oxy-benzoate) is expressed already at 1 mM concentration (about 10-fold), if the muscle had been equilibrated in chloride-free glucuronate or sulphate milieu. 4) The depolarization develops slowly. It takes 30--60 minutes for the new steady state to develop even in the superficial sartorius fibres. If depolarization has reached its maximum on an average 100 mV, the membrane potential remains unchanged for hours. 5) Depolarization ensues at an unchanged degree in the presence of Na-free (choline) Ringer as well as in the presence of 2X10(-8) g/ml tetrodotoxin; therefore, it is not a Na-dependent process. 6) Under the influence of 1 mM physostigmine salicylate the membrane's resistance to the inward potassium current increased about twofold, while the increase was only 15% to the outward potassium current. It is assumed that the salicylate anion is characteristically capable of potentiating the decreasing effect of physostigmine on potassium permeability, though the role of the metabolic effect of salicylate cannot be excluded.     

Effects of membrane potential on mechanical activation in skeletal muscle

The effect of subthreshold depolarization on mechanical threshold was investigated in tetrodotoxin-poisoned mammalian and amphibian skeletal muscle fibers using a two-microelectrode voltage-clamp technique. Mechanical threshold was determined with a 2-ms test pulse. The immediate effect of depolarization was inhibition of the mechanical system. The consequent increase in the test pulse threshold was linearly related to the size of the depolarization and there was, on the average, a 10% increase in threshold for a 10-mV depolarization in mammalian fibers. The duration of the inhibitory period was also related to the size of the depolarization. Inhibition was interrupted by the onset of activation (seen as a reduction in the test pulse threshold), and in rat soleus fibers this occurred within 100 ms with a 20-mV depolarization, inhibition decayed within 10 ms. The decay of activation after brief conditioning pulses was initially rapid (on the average, the test pulse threshold recovered to 80% of its control value within 1 ms) and then slow (full recovery took 100-500 ms). After long conditioning pulses, activation often decayed into a period of inhibition. When depolarization (of 20 mV or more) was maintained for several seconds, the fibers became inactivated. Rat extensor digitorum longus and sternomastoid fibers were strongly inactivated by depolarization to -40 mV and the test pulse to +40 mV did not cause contraction.     

Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers.

Indo-1 fluorescence signals were measured from one extremity of enzymatically isolated skeletal muscle fibers of mice. An original and simple method was developed to allow the measurements to be made under voltage-clamp control: the major part of a single fiber was embedded in silicone grease, so that only a short portion of one end of the fiber, from which the fluorescence measurements were taken, was in contact with the external solution. Membrane potential was held and varied by using a patch-clamp amplifier in whole-cell configuration with a single microelectrode, the tip of which was inserted across the silicone grease within the insulated portion of the fiber. In response to 100-ms depolarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qualitatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+]i) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release. Although the peak [Ca2+]i elicited by a 100-ms depolarization at 0 or +10 mV varied from fiber to fiber, it could clearly reach a value high enough to saturate Indo-1. The overall results show that this method represents an efficient way of measuring depolarization-induced [Ca2+]i changes in enzymatically dissociated skeletal muscle fibers.     

Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers

The effect of micromolar intracellular levels of ryanodine was tested on the myoplasmic free calcium concentration ([Ca(2+)](i)) measured from a portion of isolated mouse skeletal muscle fibers voltage-clamped at -80 mV. When ryanodine-injected fibers were transiently depolarized to 0 mV, the early decay phase of [Ca(2+)](i) upon membrane repolarization was followed by a steady elevated [Ca(2+)](i) level. This effect could be qualitatively well simulated, assuming that ryanodine binds to release channels that open during depolarization and that ryanodine-bound channels do not close upon repolarization. The amplitude of the postpulse [Ca(2+)](i) elevation depended on the duration of the depolarization, being hardly detectable for pulses shorter than 100 ms, and very prominent for duration pulses of seconds. Within a series of consecutive pulses of the same duration, the effect of ryanodine produced a staircase increase in resting [Ca(2+)](i), the slope of which was approximately twice larger for depolarizations to 0 or +10 mV than to -30 or -20 mV. Overall results are consistent with the "open-locked" state because of ryanodine binding to calcium release channels that open during depolarization. Within the voltage-sensitive range of calcium release, increasing either the amplitude or the duration of the depolarization seems to enhance the fraction of release channels accessible to ryanodine.     

The effect of tetraethylammonium and the decrease in the extracellular chloride concentration on membrane depolarization and contraction of skeletal muscle fiber induced by a hyperpotassium solution

1.--The tetraethylammonium (TEA) effects on K+ contracture and membrane depolarization are compared in both crab and frog skeletal muscle fibres. 2.--The mechanical tension of the contracture is reduced by the TEA in frog skeletal muscle fibre; it is increased in crab skeletal fibre. 3.--When no mechanical phenomenon is observed in frog skeletal muscle, the amplitude and the velocity of membrane depolarization induced by an increase of outward K+ concentration is reduced by the TEA. These effects are in opposition in crab muscle fibre. 4.--In crab muscle fibre, the results obtained tend to show that the C1-ions are not distributed on each side of the membrane according to Donnan equilibrium.     

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.     

Internal citrate ions reduce the membrane potential for contraction threshold in mammalian skeletal muscle fibers.

An effect of internal citrate ions on excitation-contraction coupling in skeletal muscle is described. The threshold for contraction was measured in rat extensor digitorum longus, (EDL), and soleus muscle fibers using a two microelectrode voltage clamp technique with either KCl-filled or K3 citrate-filled current electrodes. Contraction thresholds were stable for many minutes with KCl current electrodes. In contrast, thresholds fell progressively towards the resting membrane potential, by as much as -15 mV over a period of 10 to 20 min of voltage-clamp with citrate current electrodes. In addition, prepulse inhibition was suppressed, subthreshold activation enhanced and steady-state inactivation shifted to more negative potentials. Fibers recovered slowly from these effects when the citrate electrode was withdrawn and replaced with a KCl electrode. The changes in contraction threshold suggest that citrate ions act on the muscle activation system at an intracellular site, since the citrate permeability of the surface membrane is probably very low. An internal citrate concentration of 5 mM was calculated to result from citrate diffusion out of the microelectrode into the recording area for 20 min. 5 mM citrate added to an artificial cell lowered the free calcium concentration from 240 to 31 microM. It is suggested that citrate modifies excitation-contraction coupling either by acting upon an anion-dependent step in activation or by reducing the free calcium and/or free magnesium concentration in the myoplasm.     

Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells

We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30–60 mW. Simultaneous detection of intracellular Ca²⁺ concentration and membrane potential revealed coincident photogeneration of Ca²⁺ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca²⁺ which acts on Ca²⁺ activated K⁺ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses.     

Slow membrane potential changes in skeletal muscle induced in Cl(-)-free medium.

1. Conventional microelectrode techniques were used to measure simultaneous changes in membrane potential (Vm) and conductance (Gm) induced by single electrical stimuli in muscles bathed in Cl(-)-free solution containing 40 mM of tetraethylammonium (TEA+). 2. Stimulation induced slow transient depolarizations (slow response) accompanied by increased calcium conductance, while the potassium conductance was first elevated and later reduced. 3. Stepwise elevation of [K+]0 from 2.5 to 5 or 10 mM during the slow response evoked an abrupt repolarization of 42.3 +/- 8.9 mV (n = 4; p less than 0.001), and 24.8 +/- 3.5 +/- mV (n = 5; p less than 0.001), respectively, while Gm was increased to 1.45 +/- 0.25-fold (n = 5; p less than 0.05). Neither the slow response nor K(+)-induced changes in Vm or Gm were sensitive to tetrodotoxin (3 microM), however, nifedipine (10 microM) abolised the slow response. 4. It was concluded that beyond the increase of calcium conductance, the ionic conductance of the inward rectifier K+ channel was reduced during the slow response, which could be restored by the elevation of [K+]0. The results suggest the possible contribution of these mechanisms to the electrical instability of myotonic muscles. Potential therapeutic consequences are discussed.     

The dependence of calcium-activated potassium currents on membrane potential.

Previous experiments on cholinergic synapses in chick cochlear hair cells have shown that calcium entering through acetylcholine-activated synaptic channels in turn activates calcium-dependent potassium currents, resulting in synaptic inhibition. In voltage-clamp experiments such currents would be expected to increase with depolarization (as the driving force for potassium entry is increased) and then decrease towards zero as the membrane approaches the calcium equilibrium potential (when calcium entry is suppressed). In the hair cells, however, such currents approached zero at about +20 mV, more than 170 mV negative to the calcium equilibrium potential. Another feature of the synapse is its post-junctional morphology: a uniform 20 nm cleft is formed between the postsynaptic membrane and the outermost membrane of an underlying cisterna. Here we present a model in which synaptic activation results in calcium influx into the subsynaptic cleft and thence into the bulk of the cytoplasm. The model suggests that the voltage dependence of the calcium-activated potassium current can be accounted for by only two basic assumptions: (i) entry of calcium through the activated synaptic channels by simple diffusion; and (ii) activation of the potassium channels by the cooperative action of four calcium ions. In addition, the model suggests that during activation the calcium concentration in the restricted subsynaptic space can reach levels adequate to activate the potassium channels, without requiring additional, more complicated, considerations (for example, secondary calcium release from the cisterna).