Ionic selectivity of the sodium channel of frog skeletal muscle

The ionic selectivity of the Na channel to a variety of metal and organic cations is studied in frog semitendinosus muscle. Na channel currents are measured under voltage clamp conditions in fibers bathed in solutions with all Na+ replaced by a test ion. Permeability ratios are calculated from measured reversal potentials using the Goldman-Hodgkin-Katz equation. The permeability sequence was Na+ approximately Li+ approximately hydroxylammonium greater than hydrazinium greater than ammonium greater than guanidinium greater than K+ greater than aminoguanidinium in the ratios 1:0.96:0.94:0.31:0.11:0.093:0.048:0.031. No inward currents were observed for Ca++, methylammonium, methylguanidinium, tetraethylammonium, and tetramethylammonium. The results are consistent with the Hille model of the Na channel selectivity filter of the node of Ranvier and suggest that the selectivity filter of the two channels is the same.     

Opentime heterogeneity during bursting of sodium channels in frog skeletal muscle.

Single voltage-activated Na+ channel currents were obtained from membrane patches on internally dialyzed skeletal muscle segments of adult frogs. The high channel density in these membranes permitted frequent observation of the "bursting mode" of individual Na+ channels during 400-ms records. We examined the opentimes within and between bursts on individual membrane patches. We used a new nonparametric statistical procedure to test for heterogeneity in the opentime distributions. We found that although 80% of all bursts consisted of opentimes drawn from a single distribution, the opentime distribution varied significantly from burst to burst. Significant heterogeneity was also detected within the remaining 20% of individual bursts. Our results indicate that the gating kinetics of individual Na+ channels are heterogeneous, and that they may occasionally change in a single channel.     

Chloride channels in toad skeletal muscle fibers

Chloride currents were measured in short lumbricalis fibers of toads (Bufo arenarum) with voltage and patch clamp techniques. For the availability of chloride currents we applied a double-pulse technique in voltage-clamped fibers. When the test pulse was preceded by a positive prepulse, the initial current was larger than with a negative prepulse and exhibited a different rate of decline to its steady-state value. At the single-channel level we found that in most of the experiments with symmetrical 110 mM NaCl solutions, two levels of conductance, 20 (small channel) and 360 pS (maxi channel), occurred with the highest probabilities. The openings of the maxi channels were more frequent at potentials close to 0 mV, whereas for the small channels the openings were at negative potentials. In contrast with the results with the macroscopic currents, a change of 2 orders of magnitude in the pH, from 7.3 to 5, had only minor effects on the channels' conductance. As with some other anion channels, the selectivity of the channels described here is low, the p(Cl)/p(Na) ratio being 1.9 and 3.7 for the small and maxi Cl(-) channels, respectively. The behavior of these Cl(-) channels with a relative high Na(+) permeability could contribute to the relatively low resting membrane potential of the lumbricalis fibers measured in the standard 110 mM NaCl solution.     

Furosemide-sensitive cation transport in frog skeletal muscle fibers

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.     

Blockade of sodium channels by Bistramide A in voltage-clamped frog skeletal muscle fibres.

The effect of Bistramide A, a toxin isolated from Bistratum lissoclinum Sluiter (Urochordata), on the peak sodium current (INa) of frog skeletal muscle fibres was studied with the double sucrose gap voltage clamp technique. External or internal application of Bistramide A inhibited INa without alteration of the kinetic parameters of the current nor of the apparent reversal potential for Na. The steady-state activation curve of INa was unchanged while the steady-state inactivation curve of INa was shifted towards more negative membrane potentials. Dose-response curves indicated an apparent dissociation constant for Bistramide A of 3.3 microM and a Hill coefficient of 1.2 which suggested a one to one relation between the toxin and Na channel. The inhibition of INa occurred at rest, and was more important at more positive holding potentials. Bistramide A exhibited only a weak frequency-dependent effect. The toxin did not interact with the use-dependent effect of lidocaine. It mainly blocked Na channels at more depolarized holding potentials. The toxin blocked Na channels when it was internally applyed and when the inactivation gating system has been previously destroyed by internal diffusion of iodate. The data suggest that Bistramide A inhibited the Na channel both at rest and in the inactivated state and occupied a site which was not located on the inactivation gate.     

Determination of ionic calcium in frog skeletal muscle fibers

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).     

Localization of acid organelles in frog skeletal muscle fibers

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.     

Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a shift in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

Stiffness and force in activated frog skeletal muscle fibers.

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)     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

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.     

A-band shortening in single fibers of frog skeletal muscle.

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.     

Strophanthidin-sensitive sodium fluxes in metabolically poisoned frog skeletal muscle

Strophanthidin-sensitive and insensitive unidirectional fluxes of Na were measured in fog sartorius muscles whose internal Na levels were elevated by overnight storage in the cold. ATP levels were lowered, and ADP levels raised, by metabolic poisoning with either 2,4-dinitrofluorobenzene or iodoacetamide. Strophanthidin-sensitive Na efflux and influx both increased after poisoning, while strophanthidin-insensitives fluxes did not. The increase in efflux did not require the presence of external K but was greatly attenuated when Li replaced Na as the major external cation. Membrane potential was not markedly altered by 2,4-dinitrofluorobenzene. These observations indicate that the sodium pump of frog skeletal muscle resembles that of squid giant axon and human erythrocyte in its ability to catalyze Na-Na exchange to an extent determined by intracellular ATP/ADP levels.     

Kinetic evidence for two Na channels in frog skeletal muscle

The kinetics of voltage-clamped sodium currents were studied in frog skeletal muscle. Sodium currents in frog skeletal muscle activate and inactivate following an initial delay in response to a depolarizing voltage pulse. Inactivation occurs via a double exponential decay exhibiting fast and slow components for virtually all depolarizing pulses used.The deactivation of Na currents exhibits two exponential components, one decaying rapidly, while the other decays slowly in time; the relative amplitude of the two components changes with the duration of the activating pulse. The two deactivation phases remain after pharmacological elimination of inactivation.In individual fibers, the percent amplitude of the slow inactivation component correlates with the percent amplitude of the slow deactivation component.Tetrodotoxin differentially blocks the slow deactivation component.These observations are interpreted as the activation, inactivation and deactivation of two subtypes (fast and slow) of Na channels.Studies of the slow deactivation phase magnitude vs the duration of the eliciting pulse provide a way to determine the kinetics of the slow Na channel in muscle.Ammonium substitution for Na in the Ringer produces a voltage dependent activation and inactivation of current which exhibits only one decay phase, and eliminates the slow decay phase of current, suggesting that adjustments of the ionic environment of the channels can mask the presence of one of the channel subtypes.     

Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.     

Modulation by caffeine of calcium-release microdomains in frog skeletal muscle fibers

The effects of caffeine on the process of excitation-contraction coupling in amphibian skeletal muscle fibers were investigated using the confocal spot detection technique. This method permits to carefully discriminate between caffeine effects on the primary sources of Ca2+ release at the Z-lines where the triads are located and secondary actions on other potential Ca Release sources. Our results demonstrate that 0.5 mM caffeine potentiates and prolongs localized action-potential evoked Ca2+ transients recorded at the level of the Z-lines, but that 1mM only prolongs them. The effects at both doses are reversible. At the level of the M-line, localized Ca2+ transients displayed more variability in the presence of 1 mM caffeine than in control conditions. At this dose of caffeine, extra-junctional sources of Ca2+ release also were observed occasionally.     

Two open states or two different Na channels in skeletal muscle fibers: Markov models of the decay of Na currents in frog skeletal muscle

The rate of sodium current decay at –140 mV was studied as a function of the duration and amplitude of the activating voltage pulse. These sodium current decays or tails of current showed a biexponential decline in amplitude which depended upon the duration of the activating pulse. At 12°C, the two exponential components of the Na tail currents exhibited time constants of 72 and 534 s. As the duration of an activating pulse was lengthened, the relative amplitude of the slow component of the decay increased compared to the fast component, without any changes in the fast and slow time constants. This slowing of the decay of current as a function of the duration of the activating pulse is found only in fibers with inactivation intact.A number of Markov models were tested for their ability to predict the biexponential decays found in muscle fibers with inactivation intact and removed. A homogeneous population of channels having only a single open state fails to predict the behavior. A homogeneous population of channels having two open states predicts the behavior. The behavior can also be predicted by two different types of single open-state sodium channels, with one ensemble of channels carrying a minority of the current and exhibiting a much slower closing rate. If a homogeneous population of channels is present, the simulations show that the observed changes in decay rates are driven by inactivation.     

Potential-dependent changes in ionic selectivity of batrachotoxin-modified sodium channels of frog nerve fiber

Ionic currents through sodium channels modified by batrachotoxin were measured by the voltage clamp method on a myelinated frog nerve fiber membrane. The reversal potential (E_rev) of steady-state currents was shown to be on the average 5 mV less positive than E_rev corresponding to the initial (peak) values of the currents. The results of control experiments using procaine and tetrodotoxin showed that the change in E_rev observed during a depolarizing pulse is not connected with the presence of unmodified sodium channels or unblocked potassium channels, with nonlinearity of leakage, or with a change in transmembrane gradients of current-carrving cations. In experiments with measurement of "instant" currents it was shown that E_rev becomes less positive as the amplitude and duration of preliminary depolarization increase. The results support the view that sodium-potassium selectivity of batrachotoxin-modified sodium channels depends on potential.     

Calcium sparks in intact skeletal muscle fibers of the frog.

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.