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1.
Effects of membrane potential on the capacitance of skeletal muscle fibers 总被引:4,自引:4,他引:0
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A method for measuring muscle fiber capacitance using small test pulses applied with the three-microelectrode voltage clamp is presented. Using this method, three membrane potential-dependent changes in capacitance were observed: (a) Capacitance of polarized fibers increased by 5--15% with depolarization from V less then -100 mV to voltages slightly below the contraction threshold. (b) Capacitance of fibers depolarized to -30 mV by 100 mM Rb solution decreased by roughly 8% with further depolarization to about +50 mV and increased with repolarization, exhibiting a maximum increase of about 10% at -80 to -90 mV. (c) Capacitance of fibers depolarized to -15 mV by 100 mM K solution increased by about 19% with further depolarization to +43 mV and decreased by about 23% with repolarization to -62 mV. Effects a and b are attributed to changes in specific membrane capacitance due to voltage-dependent redistribution of mobile charged groups within surface of T-tubule membranes. Effect c is caused by changes in the T-system space constant lambdaT due to the voltage dependence of K conductance (inward rectification). Analysis of c showed that in 100 mM K solution lambdaT congruent to 30 mum when inward rectification was fully activated by hyperpolarization and that the density of inward rectifier channels is about the same in surface and tubular membranes. Fiber internal resistance was found to be independent of voltage, a necessary condition for the interpretation of the capacitance measurements. 相似文献
2.
A quantitative description of the voltage-dependent capacitance in frog skeletal muscle in terms of equilibrium statistical mechanics 总被引:4,自引:0,他引:4
S Duane C L Huang 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1982,215(1198):75-94
We present an analysis of experimental steady-state properties of charge movements in voltage-clamped frog skeletal muscle; by adopting an approach based on equilibrium statistical mechanics, detailed assumptions about the dynamics of the charge movement were avoided. Different components of the charge movements, as characterized by their sensitivities to local anaesthetics, were taken to correspond to different types of independent subsystem (integral membrane proteins). Quantitative agreement with the data for the q beta and q gamma components was obtained for the simplest subsystems, having two energy levels; however, q alpha required three (or more) energy levels. Each of the subsystems can be interpreted as having a charged group, minimum valency z, able to occupy two or more positions within the membrane. The tetracaine-sensitive q gamma charge can be described in terms of an ensemble of subsystems having z = 4.5, each occupying one of two levels whose free energies at zero applied voltage differ by 1.7 x 10(-1) eV. Likewise, the lidocaine-sensitive q beta has z = 1.0, free energy difference 1.9 x 10(-2) eV. However, the simplest model for the lidocaine-resistant q alpha involves a charge of valency 1.7 moving between three levels having relative free energies at zero applied voltage -9.7 x 10(-2), 0, and 2.1 x 10(-2) eV respectively, where the intermediate level 'sees' about 50% of the applied voltage. 相似文献
3.
Effects of sulfhydryl inhibitors on nonlinear membrane currents in frog skeletal muscle fibers 总被引:1,自引:1,他引:0
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《The Journal of general physiology》1993,101(3):425-451
The effect of sulhydryl reagents on nonlinear membrane currents of frog skeletal muscle fibers has been studied using the triple Vaseline gap voltage-clamp technique. These compounds, which are known to interfere with depolarization contraction coupling, also appear to diminish intramembranous charge movement recorded with fibers polarized to -100 mV (charge 1). This effect, however, is accompanied by changes in the fiber membrane conductance and in most cases by the appearance of an inwardly directed current in the potential range between -60 and +20 mV. This current is reduced by both cadmium and nifedipine and does not occur in Ca-free solution, suggesting that it is carried by calcium ions flowing through regular calcium channels that are more easily activated in the presence of SH reagent. These changes in the membrane electrical active and passive properties decrease the quality and reliability of the P/n pulse subtracting procedure normally used for charge movement measurements. These effects can be substantially reduced by cadmium ions (0.1 mM), which has no effect on charge movement. When SH reagents are applied in the presence of cadmium, no effects are observed, indicating that this cation may protect the membrane from the reagent effects. The effects of -SH reagents can be observed by applying them in the absence of cadmium, followed by addition of the cation. Under these conditions the conductance changes are reversed and the effects of the SH reagents on charge movement can be measured with a higher degree of confidence. Maximum charge is reduced by 32% in the presence of 1.5 mM PCMB and by 31% in the presence of 2 mM PHMPS. These effects do not occur in the presence of DTT and in some cases they may be reversed by this agent. Charge 2, recorded in depolarized muscle fibers, is also reduced by these agents. 相似文献
4.
Furosemide-inhibitable components in unidirectional cation fluxes have been identified in frog skeletal muscle. In sodium loaded muscles, placed in sodium-free rubidium lithium media, furosemide (1 mM) inhibits partially rubidium and lithium influxes as well as potassium and sodium outfluxes. The furosemide-inhibitable components were found to depend on the presence of ouabain. They were greatly diminished in sodium-free magnesium media and were present in chloride-free nitrate containing media. The dependence of furosemide-inhibitable sodium efflux on internal sodium content was also described. 相似文献
5.
After a contracture response, skeletal muscle fibers enter into a state of contractile refractoriness or inactivation. Contractile inactivation starts soon after membrane depolarization, and causes spontaneous relaxation from the contracture response. Here we demonstrate that contractile inactivation continues to develop for tens of seconds if the membrane remains in a depolarized state. We have studied this phenomenon using short (1.5 mm) frog muscle fibers dissected from the Lumbricalis brevis muscles of the frog, with a two-microelectrode voltage-clamp technique. After a contracture caused by membrane depolarization to 0 mV, from a holding potential of -100 mV, a second contracture can be developed only if the membrane is repolarized beyond a determined potential value for a certain period of time. We have used a repriming protocol of 1 or 2 s at -100 mV. After this repriming period a fiber, if depolarized again to 0 mV, may develop a second contracture, whose magnitude and time course will depend on the duration of the period during which the fiber was maintained at 0 mV before the repriming process. With this procedure it is possible to demonstrate that the inactivation process builds up with a very slow time course, with a half time of approximately 35 s and completion in greater than 100 s. After prolonged depolarizations (greater than 100 s), the repriming time course is slower and the inactivation curve (obtained by plotting the extent of repriming against the repriming membrane potential) is shifted toward more negative potentials by greater than 30 mV when compared with similar curves obtained after shorter depolarizing periods (10-30 s). These results indicate that important changes occur in the physical state of the molecular moiety that is responsible for the inactivation phenomenon. The shift of the inactivation curve can be partially reversed by a low concentration (50 microM) of lanthanum ions. In the presence of 0.5 mM caffeine, larger responses can be obtained even after prolonged depolarization periods, indicating that the fibers maintain their capacity to liberate calcium. 相似文献
6.
Ionic calcium concentrations were measured in frog skeletal muscle fibers using Ca-selective microelectrodes. In fibers with resting membrane potentials more negative than -85 mV, the mean pCa value was 6.94 (0.12 microM). In fibers depolarized to -73 mV with 10-mM K the mean pCa was 6.43 (0.37 microM). This increase in the intracellular [Ca2+] could be related to the higher oxygen consumption and heat production (Solandt effect) reported to occur under these conditions. Caffeine, 3 mM, also produced an increase in the free ionic calcium to a pCa of 6.52 (0.31 microM) without changes in the membrane potential. Lower caffeine concentrations, 1 and 2 mM, did not change the fiber pCa. Lower Ca concentrations in the external medium effectively reduced the internal ionic calcium to an estimated pCa of 7.43 (0.03 microM). 相似文献
7.
By means of fluorescent and phase-contrast microscopy the distribution of acid membrane organelles in normal and vacuolated frog skeletal muscle fibers has been studied. The vacuolation of the T-system was produced by loading and subsequent removal of glycerol (80-110 mM), or it appeared as a result of Zenker's necrosis. Acridine orange (AO) was used as a marker for acid intracellular compartments. AO accumulated in granules localized near the nuclear poles (more seldom around the nucleus)' and in the intermyofibrillar spaces. Typically the AO granules make up short longitudinal chains or regular pairs, where the distances between neighboring granules are short-dated to sarcomere lengths. Almost all granules emit in red, but about one third of them simultaneously emit in green, which is characteristic of AO monomers. In the vicinity of necrotic boundary or under the influence of brefeldin A, a green component of fluorescence appears in most granules. Treatment with monensin leads to granule disappearance. Vacuoles accompanying the glycerol treatment or developing of necrosis do not accumulate AO and exert no effect on the localization of AO-granules. The nature of cellular organelles accumulating AO in skeletal muscle fibers is discussed. 相似文献
8.
Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers. 总被引:5,自引:0,他引:5
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In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule. 相似文献
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11.
The question of whether A-bands shorten during contraction was investigated using two methods: high-resolution polarization microscopy and electron microscopy. During shortening from extended sarcomere lengths in the passive state, sarcomere-length changes were essentially accounted for by I-band shortening. During active shortening under otherwise identical conditions, the sarcomere length change was taken up approximately equally by A- and I-bands. Several potential artifacts that could give rise to apparent A-band shortening were considered and judged unlikely. Results obtained with polarization microscopy were similar to those obtained with electron microscopy. Thus, modest but significant thick filament shortening appears to occur during active sarcomere shortening under physiological conditions. 相似文献
12.
《The Journal of cell biology》1984,99(4):1391-1397
Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments. 相似文献
13.
Wahr Philip A.; Johnson J. David; Rall Jack. A. 《American journal of physiology. Cell physiology》1998,274(6):C1608
The influences of sarcomere uniformity andCa2+ concentration on the kineticsof relaxation were examined in skinned frog skeletal muscle fibersinduced to relax by rapid sequestration ofCa2+ by the photolysis of theCa2+ chelator, diazo-2, at10°C. Compared with an intact fiber, diazo-2-induced relaxationexhibited a faster and shorter initial slow phase and a fast phase witha longer tail. Stabilization of the sarcomeres by repeated releases andrestretches during force development increased the duration of the slowphase and slowed its kinetics. When force of contraction was decreasedby lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, withthe slow phase being the most sensitive toCa2+ concentration. Twitchlikecontractions were induced by photorelease ofCa2+ from a cagedCa2+ (DM-Nitrophen), withsubsequent Ca2+ sequestration byintact sarcoplasmic reticulum orCa2+ rebinding to cagedCa2+. These twitchlike responsesexhibited relaxation kinetics that were about twofold slower than thoseobserved in intact fibers. Results suggest that the slow phase ofrelaxation is influenced by the degree of sarcomere homogeneity andrate of Ca2+ dissociation fromthin filaments. The fast phase of relaxation is in part determined bythe level of Ca2+ activation. 相似文献
14.
Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
15.
The capacitance of skeletal muscle fibers in solutions of low ionic strength 总被引:2,自引:2,他引:0
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The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 µF/cm2 in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 µF/cm2. It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 µF/cm2 which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential—like mechanism in the tubular membrane. 相似文献
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18.
Pharmacological dissection of charge movement in frog skeletal muscle fibers. 总被引:4,自引:0,他引:4
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C S Hui 《Biophysical journal》1982,39(1):119-122
When charge movement is measured from muscle fibers bathed in a moderately hypertonic solution, a secondary hump appears in the decay phase of the signal during the "on" of the test pulse. The hump can be suppressed by the application of dantrolene sodium or tetracaine. The amount of charge associated with the hump is approximately 20-25% of the total charge. All the observed properties of the hump charge are consistent with the possibility that it is more closely associated with calcium release from the sarcoplasmic reticulum, and thus more relevant to excitation-contraction coupling, than the rest of the charge. 相似文献
19.
X-ray patterns from frog skeletal muscles at rest show a series of relatively weak meridional reflections which may be indexed as the 5, 7, 9, 11 and 13 orders of the repeat period of about 2 x 385 A. According to the model of the thin filaments structure, suggested by V. V. Lednev and G. M. Frank (1977), this period is specific for the activated or "switched on" state of the actin--containing filaments. At the same time, according to the generally accepted model (suggested in 1972), the axial repeat period of the thin filament structure is approximately equal to 385 A and does not depend on the functional state of the muscle. The existence of the repeat period of about 2 x 385 A in the thin filaments of a resting muscle suggests that even at rest the thin filaments of vertebrate skeletal muscle are not completely inhibited. It may be suggested that partial activation of the thin filaments in a resting muscle is the result of formation of long life rigorlike crossbridges, the existence of which was postulated by D. K. Hill in 1968 on the basis of his studies on resting tension in the frog skeletal muscle. 相似文献
20.
S Hollingworth J Peet W K Chandler S M Baylor 《The Journal of general physiology》2001,118(6):653-678
Calcium sparks were studied in frog intact skeletal muscle fibers using a home-built confocal scanner whose point-spread function was estimated to be approximately 0.21 microm in x and y and approximately 0.51 microm in z. Observations were made at 17-20 degrees C on fibers from Rana pipiens and Rana temporaria. Fibers were studied in two external solutions: normal Ringer's ([K(+)] = 2.5 mM; estimated membrane potential, -80 to -90 mV) and elevated [K(+)] Ringer's (most frequently, [K(+)] = 13 mM; estimated membrane potential, -60 to -65 mV). The frequency of sparks was 0.04-0.05 sarcomere(-1) s(-1) in normal Ringer's; the frequency increased approximately tenfold in 13 mM [K(+)] Ringer's. Spark properties in each solution were similar for the two species; they were also similar when scanned in the x and the y directions. From fits of standard functional forms to the temporal and spatial profiles of the sparks, the following mean values were estimated for the morphological parameters: rise time, approximately 4 ms; peak amplitude, approximately 1 DeltaF/F (change in fluorescence divided by resting fluorescence); decay time constant, approximately 5 ms; full duration at half maximum (FDHM), approximately 6 ms; late offset, approximately 0.01 DeltaF/F; full width at half maximum (FWHM), approximately 1.0 microm; mass (calculated as amplitude x 1.206 x FWHM(3)), 1.3-1.9 microm(3). Although the rise time is similar to that measured previously in frog cut fibers (5-6 ms; 17-23 degrees C), cut fiber sparks have a longer duration (FDHM, 9-15 ms), a wider extent (FWHM, 1.3-2.3 microm), and a strikingly larger mass (by 3-10-fold). Possible explanations for the increase in mass in cut fibers are a reduction in the Ca(2+) buffering power of myoplasm in cut fibers and an increase in the flux of Ca(2+) during release. 相似文献