Nystatin-, mycoheptin- and levorin-induced conductance in the membrane of frog skeletal muscle fibres.

The effects of the polyene antibiotics nystatin (2 x 10(-5)-10(-4) mol/l), mycoheptin (1.3 x 10(-6)-10(-5) mol/l) and levorin (10(-8)-5 x 10(-5) mol/l) on isolated frog skeletal muscle fibres and whole sartorius muscles of the frog have been investigated. Cation conductance was measured under current clamp conditions using a double sucrose-gap technique. Cation effluxes were studied by means of flame emission photometry. All three antibiotics increased the cation conductance and efflux rates; however, differences between the polyenes were found in the steady state values of induced cation transport at a given concentration. The values of both induced conductance gA infinity and efflux rate constants KA formed the following sequence: levorin > mycoheptin > nystatin, demonstrating a correlation with the order of antifungal activities. The dose-response curves of lg polyene-induced cation transport against lg of antibiotic concentration in our experiments had slope values which were much lower than those in bilayers: 1.7 and 1.3 for nystatin and mycoheptin, respectively, whereas the aromatic heptaene levorin had an even smaller concentration dependence. The decline in the equilibrium conductance caused by nystatin- and mycoheptin removal was very fast (during the first minute tau = 0.74 and 2.39 min, respectively). In contrast, levorin-induced conductance was irreversible. It is proposed that the processes which limit the rate of channel formation are different in biological and model membranes.

[The effect of dihydropyridines on the strontium and calcium content in frog muscle]. / Vliianie digidropiridinov na soderzhanie strontsiia i kal'tsiia v mushtse liagushki.

2,6-Dimethyl-3,5-dimethoxycarbonyl-4-(O-difluromethoxyphenyl)-1,4- dihydropyridine (ryodipine) (2 x 10(-4) M) significantly blocks the resting Sr++ uptake in sartorius and iliofibularis muscles of Rana temporaria without changes in Ca++ and Mg++ content. Dihydropyridine CGP (2.10(-4) M) increased both [Sp++] and [Ca++] (mumol/g dr. w.) by about 20 and 13%, respectively, as compared with paired control muscles, whereas [Mg++] remained unaffected. The [Sr++] changes are interpreted as specific dihydropyridine-effect on the Ca(++)-channels in the muscle membrane.

Effects of channel-forming antibiotics on the membrane of skeletal muscle fibre.

Gramicidin A, amphotericin B and nystatin increased the cation conductance and efflux rates from frog skeletal muscle fibres. Concerning gramicidin A, the characteristics tested are similar to those reported for BLM. Therefore, a channel formation like that in BLM is assumed for the muscle cell membrane. On the other hand, the kinetics of channel formation are different in muscle membrane and BLM. This suggests that the processes which limit the rate of channel formation are different.

[Effect of dihydropyridines on cation transport across the skeletal muscle membrane in the frog]. / Vliianie digidropiridinov na transport kationov cherez membranu skeletnoi myshtsy liagushki.

2,6-Dimethyl-3,5-dimethoxycarbonyl-4-(o-difluromethoxyphenyl)- 1,4-dihydropyridine (ryodipine) blocks Ca-channels in phasic muscle fibres from ileofibularis and semitendinosus muscle of Rana esculenta. Ryodipine and some other newly synthesized dihydropyridines (10(-7)-10(-4) M) exerted a slight, if any, effect on the steady-state of potassium chord conductance in isotonic K+-sulfate solution. The effluxes of potassium and rubidium from the sartorius muscle of Rana temporaria also remained unchanged after addition of 2 x 10(-4) M ryodipine. Thus, the nonspecific dihydropyridine effect on ion transport seems to be poorly expressed.

[The cation specificity of the potassium and gramicidin channels of muscle fiber membrane]. / Kationnaia spetsifichnost' kalievykh i gramitsidinovykh kanalov membrany myshechnogo volokna.

Conductance ratios (Gi/Gk) and permeability ratios (Pi/Pk) for monovalent cations in frog muscle fibres have been defined under constant current conditions using a double sucrose gap method. Selectivity determined from potassium channel conductance is: K+ greater than Rb+ greater than Cs+ greater than greater than NH4+ greater than Na+ greater than Li+. In gramicidin channels both the permeability and conductance sequences are identical: NH4+ greater than Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+. In isotonic K+-sulfate solution with one-sided addition of external [Tl+] (2.5 x 10(-3)-20 x 10(-3) M), differences in the conductance and permeability ratios for gramicidin channel were observed.

Selectivity for cations in potassium and gramicidin channels of the muscle fibre membrane.

The sequences of conductance ratios (GX/GK) and permeability ratios (PX/PK) for monovalent cations (X = Rb+, Cs+, Na+, Li+, NH4+) have been studied in frog skeletal muscle before and after addition of the channel-forming antibiotic gramicidin A. The experiments were carried out under current clamp conditions using a double sucrose gap technique. For inwardly rectifying potassium channels the selectivity measured by membrane conductance ratios was K+ greater than Rb+ greater than Cs+ greater than NH4+ greater than Na+ greater than Li+. In gramicidin channels of the same muscle fibre both the permeability and the conductance ratios had the identical sequence NH4+ greater than Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+. However, after external addition of Tl+ (2.5 x 10(-3) M to 20 x 10(-3) M) conductance and permeability ratios were different. The effect may result from specific binding of Tl+ in gramicidin channels.

Effect of temperature on the inward rectifier and gramicidin A-induced channels in the membrane of frog skeletal muscle fibres.

The conductance of frog skeletal muscle fibres in isotonic K2SO4 solution has been measured. Experiments were carried out under current-clamp conditions using a double sucrose-gap technique. The potassium conductances of the inward rectifier and the gramicidin channel in the same muscle fibre were compared. Potassium conductance of the inward rectifier increased with the temperature, with a value of Q10 1.55 +/- 0.09 (n = 8) under hyperpolarization, and Q10 2.38 +/- 0.23 (n = 6) for the depolarizing stimulus, the difference between Q10 of potassium and gramicidin channels being statistically insignificant.

Effect of gramicidin A and thallium ions on cation effluxes in frog sartorius muscle.

The effluxes of potassium, rubidium, sodium and lithium from the sartorius muscle of Rana temporaria in magnesium-Ringer solution free of sodium and potassium have been studied with the flame-emission technique. The channel-forming antibiotic gramicidin A (2.5 X X10(-7)-1 X 10(-6) mol/l) enhanced the efflux of potassium and rubidium and increased the rate constants of these effluxes. Gramicidin had small if any effect on sodium and lithium effluxes and rate constants. After 60-100 min in a gramicidin-containing medium, the potassium efflux and the corresponding rate constant reached a steady-state level. This steady-state value depended on gramicidin concentration. Effect of gramicidin on both the potassium efflux and the rate constant was partially reversible. Thallium ions (2.5 X 10(-3) and 5 X 10(-3) mol/l) in sodium- and potassium- free magnesium Ringer solution caused a large increase in effluxes of all the cations examined (K+, Rb+ and Na+) both in presence and absence of gramicidin. Possible mechanisms of gramicidin and thallium effects are discussed.

[Effect of temperature on the interaction of gramicidin A and its dimer analog with the muscle fiber membrane]. / Vliianie temperatury na vzaimodeistvie gramitsitina A i ego dimernogo analoga s membranoi myshechnogo volokna.

Potassium conductance of single muscle fibres from Rana esculenta was studied in isotonic K2SO4 solution under constant current conditions using double sucrose gap method. At room temperature the channel formation by gramicidin was much faster than that of the synthetic head to head covalently linked gramicidin dimer. The increase of temperature by 8-10 degrees C resulted in a considerable rise of both dimer- and gramicidin-induced conductances. The effect was much greater than in the case of bilayers indicating a remarkable entropy change in the muscle fibre membrane. The temperature dependence of adsorption was more pronounced than that of desorption: there was no effect on desorption of dimer and only 20% of the temperature-activated desorption of gramicidin irreversibly bound at room temperature.

[Effect of glycerin removal on the electrical resistance of isolated muscle fiber in the frog]. / Vliianie otmyvki glitserina na élektricheskoe soprotivlenie izolirovannogo myshechnogo volokna liagushki.

Fibres isolated from iliofibularis muscles of the frog Rana esculenta were studied under current-clamp conditions with a double sucrose-gap technique. An increase of membrane resistance in muscle fibres (Rm) was demonstrated during the first 10-15 min of glycerol removal in K2SO4 solution. After a 30 min treatment in glycerol-containing K2SO4 Rm was 1.40 +/- 0.12 k omega X X cm2. The transfer of muscles from the glycerol-K2SO4 solution to an isotonic K2SO4 solution resulted in a progressive increase in the Rm which after a 15 minutes removal of glycerol reached the mean value of 2.02 +/- 0.22 k omega X cm2. It is suggested that this increase may reflect detubulation of the fibres caused by glycerol removal in muscles.

[Effect of thallium ions on gramicidin-induced conductance of muscle fiber membranes]. / Deistvie ionov talliia na indutsirovannuiu gramitsidinom provodimost' membrany myshechnogo volokna.

Conductance of single fibres from m. ileofibularis of Rana esculenta was studied in isotonic K2SO4 solution under constant current conditions using the double sucrose gap method. It was found that Tl+ (at concentrations 5, 10, and 20 mM) blocked K+ currents in the gramicidin channel. The decrease of K+ conductance caused by Tl+ was associated with the changes of the membrane potential. Both the decrease of K+ conductance and value of permeability ratio (PTl/PK) found from the membrane potential changes depended on Tl+ concentration in the bathing solution. No effect of Tl+ on the potassium channels was registered in the absence of gramicidin channels. The Tl+ block described here proves the existence of Tl+ ion binding within gramicidin channels of the muscle membrane and interactions among ions in the channels.

[Gramicidin A-induced membrane conductance of isolated muscle fiber]. / Indutsirovannaia gramitsidinom A provodimost' membrany myshechnogo volokna.

The conductance of single fibres from m. ileofibularis of Rana esculenta was studied in isotonic K2SO4 solution under constant--current conditions using the double sucrose gap method. The antibiotic gramicidin A effects a drastic increase of membrane conductance. Variation of the gramicidin-induced conductance with gramicidin concentration in the bathing solution (10(-8)-5,5 . 10(-7) M) was investigated. Gramicidin-induced conductance was proportional to the square of gramicidin concentration in the solution, as expected in a channel made of gramicidin dimer.

The effect of Rb+, Cs+, and T1+ on the gramicidin A-induced conductance changes of the skeletal muscle cell membrane.

The channel-forming antibiotic gramicidin A increases the K+ conductance of frog skeletal muscle fibres in isotonic K2SO4 solution. The conductance of the gramicidin channel is not affected by Rb+ or Cs+, but is reduced by T1+. In contrast, the conductance of the normal K+ channel is decreased by Rb+ and Cs+ but is nearly unaffected by 5 and 10 mM T1+. The results suggest differences in the cation permeation through the gramicidin and the K+ channel of the muscle cell membrane.

[Vacuolization of muscle fiber exposed to colchicine, cytochalasin B and agents altering cell metabolism]. / Vakuolizatsiia myshechnogo volokna pri deistvii kolkhitsina, tsitokhalazina B i agentov, izmeniaiushchikh metabolizm kletki.

The vacuolation of transverse tubules during the washing out of glycerol was examined under the light microscope in muscle fibers from the m. iliofibularis of Rana temporaria. The appearance of vacuoles was found to be unaffected by colchicine (2 x 10(-5)--1 x 10(-3) M), cytochalasin B (10 mkg/ml), ATP (5 x 10(-3) M), monoiodoacetate (1 x 10(-3) M), and 2.4-DNP (1 x 10(-4)--1 x 10(-3) M). At low temperatures (3.8 +/- 0.2 degrees C), the time of vacuole-formation increased up to 20 minutes, however after a 1 hour incubation the vacuolation was perfectly developed in both the control (18.5 +/- 0.4 degrees C) and the "low-temperature" fibers. It is suggested that the initial stage of vacuole-formation does not require either metabolic energy or intact system of cytoplasmic microtubules and microfilaments. The effect of low temperature might be due to the loss of the membrane rather than to the lack of metabolic energy.

Effect of glycerol treatment on sodium and potassium in isolated muscle fibres of the frog.

The sodium concentration in single frog muscle fibres vacuolated by glycerol treatment was significantly higher than in devacuolated fibres. Intracellular potassium concentration did not show any significant change. It is concluded that the transverse tubular system forms vacuoles with a high NaCl concentration upon glycerol removal.

[Effect of vacuolization on the sodium concentration in an isolated muscle fiber]. / Vliianie vakuolizatsii na kontsentratsiiu natriia v izolirovannom myshechnom volokne.

Using the Perkin Elmer flame photometer sodium and potassium concentrations have been measured in muscle fibers from the m. ileofibularis of Rana temporaria. After 30 minutes preincubation in the Ringer solution, made hypertonic by the addition of 0.22M glycerol, the muscle fibers were incubated in the normal Ringer solution for 30 min. These fibers showed a vacuolation and an increase in total fiber sodium up to 37.2 mmol/l +/- 5.9 S. E., or 45.8 mmol/kg H2O +/- 7.3 S. E. No significant changes in potassium concentration were observed. Then, the fibers were exposed again to the Ringer solution containing 0.22 M glycerol. This procedure caused the disappearance of vacuoles and decrease in fiber sodium concentration down to 17.7 mmol/l +/- 1.6 S. E., or 21.8 mmol/kg H2O +/- 2.0 S. E. The effect of vacuolation was not blocked by ouabain (1.10(-4) M). It is suggested that the vacuoles have a high NaCl concentration. A model for NaCl and water accumulation in T-tubules is presented.

[Effect of gramicidin on the potassium conductance of an isolated frog muscle fiber]. / Vliianie gramitsidina na kalievuiu provodimost' izolirovannogo myshechnogo volokna liagushki.

The antibiotic gramicidin A (1.10(-6) M) increases the K+ conductance of normal and detubulated frog skeletal muscle fibres in isotonic K2SO4 solution to a steady-state level, which is reached in 6--9 min, and corresponds to 8058 +/- 1669 and 5767 +/- 902 Om-1. 10(-6)/cm2, resp. There is no correlation between the initial K+ conductance and the value of the steady state of gramicidin A-induced conductance (r = 0.24). According to the dimer hypothesis, the dissociation rate constant of the garmicidin channels was found to be 0.006 +/- 0.0001 sec-1. This result supports the suggestion of a higher stability of gramicidin channels in muscle compared to the bimolecular lipid membranes.

[Modeling changes in the muscle fiber T-system during direct current flow]. / Modelirovanie izmenenii ob'ema T-sistemy myshechnogo volokna v usloviiakh propuskaniia postoiannogo toka

Quantitative calculations of the dynamics of changes in K+, Na+, and Cl- concentrations in the T-system (TS) with changeable volume have been made using the computer "Dniepr-24". It was assumed that the current is transferred throught the TS membrane by K+ and in the lumen of the TS by K+, Na+ and Cl- ions in accordance with their mobilities and concentrations. The model predicts the accumulation of salt and water in the TS at the site of outgoing current. The swelling of TS due to DC effect according to the model was found to be about 9 and 18% for the current densities of 0.39 X 10(-8) a- cm-2 and 2.34 X 10(-8) a-cm-2, resp. The model shows that changes in the ion concentration and in the TS volume with current densities used experimentally reach a steady-state level. The increase of the current by almost 4 times, according to the model, suggests some disturbance in the steady-state and in the TS swelling comparable with experimental observations of vacuole formation.