Potassium Exchange and Afterpotentials in Frog Sartorius Muscles Treated with Glycerol

The potassium exchange properties of glycerol-treated sartorius muscles of the frog were determined. Potassium (⁴²K) uptake, efflux, and net flux were measured in the presence of glycerol and at various times after exposure to glycerol and return to isotonic Ringer solution. Potassium uptake was not altered by the presence of glycerol but was reduced on the average 53% after glycerol treatment. Efflux transiently increased in the presence of glycerol and was reduced 37% after glycerol removal. Consequently, there was a net loss of intracellular potassium as well as a gain of sodium. In contrast to the irreversible alterations of potassium exchange induced by glycerol treatment, action potentials with normal negative afterpotentials (N.A.P.) were elicited 4–5 hr after glycerol removal. The reappearance of the N.A.P. was associated with a return of the membrane potential to normal values (90 ± 2 mv). However, the response of these muscles to reduced extracellular potassium was anomalous. In K⁺-free Ringer solution the average resting membrane potential was 74 ± 3 mv and a positive afterpotential of 11 ± 3 mv was associated with the action potential.     

Swelling of the Transverse Tubular System in Frog Sartorius

Electron microscopy shows that the transverse tubular system of frog sartorius swells in Ringer fluid in which NaCl is partially replaced by sucrose (sucrose isotonic solutions). At constant tonicity, the degree of swelling is roughly proportional to the decrease in ionic strength and to the sucrose concentration of the bathing solution. Swelling is time-dependent and reversible within 2 hr. The late after potential which follows a train of impulses is prolonged with swelling, but not to the extent expected from the model of Adrian and Freygang. This discrepancy remains unexplained, as does the mechanism of swelling of the transverse tubular system, although some suggestions are offered. One is that the transverse tubular system contains fixed charges and swells like a fixed charge gel.     

The Roles of Calcium in Excitation-Contraction Coupling in Various Muscles of the Frog, Mouse, and Barnacle

In rectus abdominis muscles of the frog the active shorteningprovoked by 15–40 mM K was supported by Ca, Sr, and Ba,but not by Ni, Co, Mn, Cd, or Zn ions. Addition of the lattercations to a solution containing Ca decreased the responsesin a manner suggesting competitive inhibition. The shorteningof the rectus muscle found in divalent cation-free, low K solutionsis abolished by Ni, Co, Mn, and Mg. In rat muscles a transientincrease in the contractural responses to elevated potassiumwas observed when Ni was applied following partial washout ofCa. In single muscle fibers of the barnacle, development oftension was supported by Ca and Sr, and the other divalent cationswere without effect. Retention of Ca⁴⁵ in barnacle resting musclefibers soaked in solutions containing 10 mM Ca for 2 min andsubsequently washed for 10 min was 60 ± 3.1 mµMCa/g, whereas retention of Ca⁴⁵ in contracting muscles similarlyexposed to Ca⁴⁵ was 156 + 17 mµM Ca/g of fresh muscle.The results are compatible with the idea that activation ofcontraction in some types of muscles is due to entry of extracellularCa.     

Evidence for Anion-Permselective Membrane in Crayfish Muscle Fibers and Its Possible Role in Excitation-Contraction Coupling

Under certain conditions only, isolated crayfish skeletal muscle fibers change in appearance, becoming grainy, darkening, and seemingly losing their striations. These changes result from development of large vesicles on both sides of the Z-line. The longitudinal sarcoplasmic reticulum remains unaffected. The vesicles are due to swelling of a transverse tubular system (TTS) which is presumably homologous with the T-system tubules of other muscle fibers. The vesiculations occur during efflux of water or on reducing external K or Cl, but only when KCl can leave the fiber. They never result from osmotic, ionic, or electrical changes when KCl cannot leave. Inward currents, applied through a KCl-filled intracellular cathode, also cause the vesiculations. These are not produced when the cathode is filled with K-propionate, nor by outward or longitudinal currents. Thus the transverse tubules swell only when Cl leaves the cell. Accordingly, their membrane is largely or exclusively anion-permselective. These findings also indicate that the TTS forms part of a current loop, connecting with the exterior of the fiber probably through radial tubules (RT) possessing membrane of low conductivity. Thus, part of the current flowing inward across the sarcolemma during activity can return to the exterior through the membrane of the TTS. The structure and properties of the latter offer the possibility for an efficient electrical mechanism to initiate excitation-contraction coupling.     

Contraction and Action Potentials of Frog Heart Muscles Soaked in Sucrose Solution

Isolated auricles or ventricles from the frog continue to contract, either spontaneously or when stimulated, for from 2 to 4 hours after they are placed in isotonic sucrose solution. After the muscles stop contracting in sucrose solution, contractility is partially restored when the muscles are placed in chloride Ringer's. However, contractility is usually not restored if the muscles are placed in sulfate Ringer's. Ventricles soaked in sucrose solution at 4–7°C continue to contract for 12 to 24 hours and during the first few hours in sucrose solution the contractions often are enhanced. Several types of experiment indicate that the sucrose solution does replace the Ringer's in the extracellular space. Auricles and ventricles also continue to conduct action potentials, with an overshoot, for from 30 to 360 minutes after being placed in sucrose solution. Muscles soaked in sucrose until they are inexcitable rapidly recover in chloride Ringer's but often fail to recover in sulfate Ringer's. The results are discussed in relation to theories about the generation of the action potential in cardiac muscle, and the role of the extracellular fluid in contraction.     

Action Potential in the Transverse Tubules and Its Role in the Activation of Skeletal Muscle

The double sucrose-gap method was applied to single muscle fibers of Xenopus. From the "artificial node" of the fiber, action potentials were recorded under current-clamping condition together with twitches of the node. The action potentials were stored on magnetic tape. The node was then made inexcitable by tetrodotoxin or by a sodium-free solution, and the wave form of the action potential stored on magnetic tape was imposed on the node under voltage-clamp condition (simulated AP). The twitch height caused by the simulated AP's was always smaller than the twitch height produced by the real action potentials, the ratio being about 0.3 at room temperature. The results strongly suggest that the transverse tubular system is excitable and is necessary for the full activation of twitch, and that the action potential of the tubules contributes to about 70 % of the total mechanical output of the normal isotonic twitch at 20°C. Similar results were obtained in the case of tetanic contraction. At a temperature near 10°C, twitches produced by the simulated AP were not very different (85 % of control amplitude) from the twitches caused by real action potentials. This indicates that the excitability of the tubules becomes less necessary for the full activation of twitch as the temperature becomes lower.     

兴奋-收缩偶联中DHPR与RyR的相互作用

兴奋-收缩偶联（E—C coupling）依赖纽胞膜二氢吡啶受体（DHPR）／L型电压门控Ca^2＋通道和肌浆网兰诺定受体（RyR）／Ca^2＋释放通道的相互作用。在骨骼肌细胞中,DHPR与RyRl在结构上二机械偶联,不依赖细胞外Ca^2＋即可激活RyRl;在心肌细胞中,去极化激活DHPR,细胞外Ca^2＋内流,内流的Ca^2＋通过钙诱导钙释放（CICR）机制激活RyR2。最近的研究表明,DHPR与RyR之间的信号转导通常是双向的。DHPR与RyR机械和化学的双向偶联机制调节这两种Ca^2＋通道的效率、精确度和活性。     

The Mode of Transverse Spread of Contraction Initiated by Local Activation in Single Frog Muscle Fibers

Isolated single frog muscle fibers were locally activated by applying negative current pulses to a pipette whose tip was in contact with the fiber surface. In contrast to the graded inward spread of contraction initiated by a moderate depolarization, the contraction in response to a strong negative current was observed to spread transversely around the whole perimeter but not through the center of the fiber. This response was elicited only with pipettes of more than 6 µ diameter. The response was still present if the sodium of the Ringer solution was replaced by choline, or the chloride was replaced by nitrate or propionate. The duration of the response appeared to be independent of the duration of stimulating current in fresh fibers, while the contraction lasted as long as the current went on in deteriorated fibers. The contraction was first initiated at the area of fiber surface covered by the pipette, and spread around the perimeter of the fiber with a velocity of 0.8–6 cm/sec. Possible mechanisms of the response are discussed in connection with the properties of the transverse tubular system, the possibility of some self-propagating process along the walls of the tubules being suggested.     

Organization of Ryanodine Receptors, Transverse Tubules, and Sodium-Calcium Exchanger in Rat Myocytes

Confocal and total internal reflection fluorescence imaging was used to examine the distribution of caveolin-3, sodium-calcium exchange (NCX) and ryanodine receptors (RyRs) in rat ventricular myocytes. Transverse and longitudinal optical sectioning shows that NCX is distributed widely along the transverse and longitudinal tubular system (t-system). The NCX labeling consisted of both punctate and distributed components, which partially colocalize with RyRs (27%). Surface membrane labeling showed a similar pattern but the fraction of RyR clusters containing NCX label was decreased and no nonpunctate labeling was observed. Sixteen percent of RyRs were not colocalized with the t-system and 1.6% of RyRs were found on longitudinal elements of the t-system. The surface distribution of RyR labeling was not generally consistent with circular patches of RyRs. This suggests that previous estimates for the number of RyRs in a junction (based on circular close-packed arrays) need to be revised. The observed distribution of caveolin-3 labeling was consistent with its exclusion from RyR clusters. Distance maps for all colocalization pairs were calculated to give the distance between centroids of punctate labeling and edges for distributed components. The possible roles for punctate NCX labeling are discussed.     

Fractionation of Sodium Efflux in Frog Sartorius Muscles by Strophanthidin and Removal of External Sodium

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q₁₀ of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q₁₀ of about 7.4.     

Visual Stimuli Evoked Action Potentials Trigger Rapidly Propagating Dendritic Calcium Transients in the Frog Optic Tectum Layer 6 Neurons

The superior colliculus in mammals or the optic tectum in amphibians is a major visual information processing center responsible for generation of orientating responses such as saccades in monkeys or prey catching avoidance behavior in frogs. The conserved structure function of the superior colliculus the optic tectum across distant species such as frogs, birds monkeys permits to draw rather general conclusions after studying a single species. We chose the frog optic tectum because we are able to perform whole-cell voltage-clamp recordings fluorescence imaging of tectal neurons while they respond to a visual stimulus. In the optic tectum of amphibians most visual information is processed by pear-shaped neurons possessing long dendritic branches, which receive the majority of synapses originating from the retinal ganglion cells. Since the first step of the retinal input integration is performed on these dendrites, it is important to know whether this integration is enhanced by active dendritic properties. We demonstrate that rapid calcium transients coinciding with the visual stimulus evoked action potentials in the somatic recordings can be readily detected up to the fine branches of these dendrites. These transients were blocked by calcium channel blockers nifedipine CdCl₂ indicating that calcium entered dendrites via voltage-activated L-type calcium channels. The high speed of calcium transient propagation, >300 μm in <10 ms, is consistent with the notion that action potentials, actively propagating along dendrites, open voltage-gated L-type calcium channels causing rapid calcium concentration transients in the dendrites. We conclude that such activation by somatic action potentials of the dendritic voltage gated calcium channels in the close vicinity to the synapses formed by axons of the retinal ganglion cells may facilitate visual information processing in the principal neurons of the frog optic tectum.     

Multimodal Distribution of Frog Miniature Endplate Potentials in Adult, Denervated, and Tadpole Leg Muscle

Amplitude histograms of spontaneous miniature endplate potentials (MEPPs) from adult sartorius muscle cells show a definite bimodality with the mean amplitude of the larger mode five to seven times that of the smaller mode which accounted for 2–5 % of the total MEPPs. Histograms were plotted after high frequency MEPP generation induced by increasing temperature, increasing external calcium or nerve stimulation. These plots showed a reversible left-shift of the major mode as well as a reversible increase in the proportion of small mode MEPPs. Repeated challenges shifted almost all MEPPs into the small mode. An increase in the percentage of small mode MEPPs also occurred spontaneously during the course of denervation before the quiescent period and some of the histogram profiles showed multiple modes whose means were integer multiples of the small mode mean. In the early stages of hind leg development the greatest proportion of MEPPs were of the small mode size; as metamorphosis progressed, the histograms showed a definite multimodality with the mean of each mode being an integer multiple of the small mode mean and with the proportion of MEPPs in each mode about the same. During tail resorption the percentage of larger MEPPs increased until the adult histogram profile was reached. Thus, the changes in MEPP amplitude histograms over the course of metamorphosis are the reverse of those found with denervation.     

Multiple Sequence Variants in STAC3 Affect Interactions with CaV1.1 and Excitation-Contraction Coupling

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Analysis of Combined Action of Antagonists of Excitatory and Inhibitory Amino Acids on Motoneuron Postsynaptic Potentials of the Frog Rana ridibunda

The effect of blockers of excitatory and inhibitory amino acid receptors on postsynaptic potentials (PSP) evoked by activation of three synaptic inputs of the lumbar motoneuron (stimulation of the dorsal root, reticular formation, ventral and lateral columns) was studied on preparation of the isolated spinal cord of the frog Rana ridibunda. It has been shown that sensitivity of PSP to antagonists differs in different motoneurons, in the same motoneuron at activation of different inputs, and in the same input in different PSP components. It has been found that many descendent (DC) PSPs resistant to kynurenate or CNQX [1] were inhibited by blockers of inhibitory receptors. In this case the early component of DC-PSP varied considerably by amplitude and changed its polarity from positive to negative on the background of a low transmembrane depolarizing current. These changes were absent under conditions of replacement of chlorine ion by sulfate in the perfusion solution or treatment of the spinal cord with a blocker of inhibitory amino acids. All this allows suggesting that these DC-PSPs or their components were inhibitory. A part of PSPs resistant to kynurenate and CNQX were also resistant to the blockers of inhibitory amino acids (strychnine, picrotoxin, and bicuculline). In some cases, as a result of treatment with convulsants, the same blockers of excitatory receptors inhibited the initially resistant PSPs.     

Caffeine- and Potassium-Induced Contractures of Frog Striated Muscle Fibers in Hypertonic Solutions

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm² of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.     

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners,STIM1 and STIM2

Our recent work identified store-operated Ca²⁺ entry (SOCE) as the critical Ca²⁺ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370–5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca²⁺ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca²⁺-content during repeated KCl-induced Ca²⁺ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.     

Electrical Coupling, Potentials, and Resistances in Oat Coleoptiles: Effects of Azide and Cyanide

Electrophysiological measurements were made on oat coleoptile(Avena sativa L. cv. Victory) parenchyma cells. Both 1 mM potassiumcyanide and 1 mM sodium azide cause reductions in cell restingpotential and electrical coupling and an increase in the combinedtonoplast and plasmalemma resistance. The reduction in coupling is probably attributable to a decreasein current flow through plasmodesmata, resulting from an increasein plasmodesmatal resistance. Potassium cyanide also induces some callose formation withincell walls and this may contribute to the observed reductionin coupling. However, sodium azide does not induce callose formation.Presumably other processes are involved in the reduction ofcoupling which are not attributable to callose.     

Structure and Function of the Transverse Tubular System in Crustacean Muscle Fibers

The structure and function of the transverse tubular system(TTS) in two types of crustacean muscle fibers are examined.Giant fibers from the barnacle,Balanus nubilus, which are gradedlycontracting, are compared with allor-none twitch fibers fromthe crab, Carcinus maenas. Both fiber types were found to havedeep sarcolemmal invaginations which serve both to increasethe fiber surface area and to kfeep the length of the tubulesshort enough for electrotonic propagation.The ultrastructureof the tubular system in both types of fiber is compared.Thesystem is better developed in Carcinus than in Balanus, butthe slow Balanus fibers do have a relatively well developedTTS and sarcoplasmic reticulum in contrast to slow vertebratefibers. The apparent high, membrane-capacitance values of crustaceanfibers are the result of investigators not taking into considerationthe large increase in surface area due to the sarcolemmal infoldings.Thetubular membranes in Carcinus fibers were found to be permselectiveto chloride ions, and could be made to swell (as confirmed byelectron microscopy) by establishing an outward gradient forchloride across them. The capacitance of the tubular membranerelative to the plasma membrane was found to increase when thetubuleswere swollen. The implication of a fiber having two spatiallyseparated, differentially permeable membranes on excitation-contractioncoupling is discussed.     

Tension in Skinned Frog Muscle Fibers in Solutions of Varying Ionic Strength and Neutral Salt Composition

The maximal calcium-activated isometric tension produced by a skinned frog single muscle fiber falls off as the ionic strength of the solution bathing this fiber is elevated declining to zero near 0.5 M as the ionic strength is varied using KCl. When other neutral salts are used, the tension always declines at high ionic strength, but there is some difference between the various neutral salts used. The anions and cations can be ordered in terms of their ability to inhibit the maximal calcium-activated tension. The order of increasing inhibition of tension (decreasing tension) at high ionic strength for anions is propionate^- SO₄^-- < Cl^- < Br^-. The order of increasing inhibition of calcium-activated tension for cations is K⁺ Na⁺ TMA⁺ < TEA⁺ < TPrA⁺ < TBuA⁺. The decline of maximal calcium-activated isometric tension with elevated salt concentration (ionic strength) can quantitatively explain the decline of isometric tetanic tension of a frog muscle fiber bathed in a hypertonic solution if one assumes that the internal ionic strength of a muscle fiber in normal Ringer's solution is 0.14–0.17 M. There is an increase in the base-line tension of a skinned muscle fiber bathed in a relaxing solution (no added calcium and 3 mM EGTA) of low ionic strength. This tension, which has no correlate in the intact fiber in hypotonic solutions, appears to be a noncalcium-activated tension and correlates more with a declining ionic strength than with small changes in [MgATP], [Mg], pH buffer, or [EGTA]. It is dependent upon the specific neutral salts used with cations being ordered in increasing inhibition of this noncalcium-activated tension (decreasing tension) as TPrA⁺ < TMA⁺ < K⁺ Na⁺. Measurements of potentials inside these skinned muscle fibers bathed in relaxing solutions produced occasional small positive values (<6 mV) which were not significantly different from zero.