The voltage sensor of excitation-contraction coupling in skeletal muscle. Ion dependence and selectivity

Manifestations of excitation-contraction (EC) coupling of skeletal muscle were studied in the presence of metal ions of the alkaline and alkaline-earth groups in the extracellular medium. Single cut fibers of frog skeletal muscle were voltage clamped in a double Vaseline gap apparatus, and intramembrane charge movement and myoplasmic Ca2+ transients were simultaneously measured. In metal-free extracellular media both charge movement of the charge 1 type and Ca transients were suppressed. Under metal-free conditions the nonlinear charge distribution was the same in depolarized (holding potential of 0 mV) and normally polarized fibers (holding potentials between -80 and -90 mV). The manifestations of EC coupling recovered when ions of groups Ia and IIa of the periodic table were included in the extracellular solution; the extent of recovery depended on the ion species. These results are consistent with the idea that the voltage sensor of EC coupling has a binding site for metal cations--the "priming" site--that is essential for function. A state model of the voltage sensor in which metal ligands bind preferentially to the priming site when the sensor is in noninactivated states accounts for the results. This theory was used to derive the relative affinities of the various ions for the priming site from the magnitude of the EC coupling response. The selectivity sequence thus constructed is: Ca greater than Sr greater than Mg greater than Ba for group IIa cations and Li greater than Na greater than K greater than Rb greater than Cs for group Ia. Ca2+, the most effective of all ions tested, was 1,500-fold more effective than Na+. This selectivity sequence is qualitatively and quantitatively similar to that of the intrapore binding sites of the L-type cardiac Ca channel. This provides further evidence of molecular similarity between the voltage sensor and Ca channels.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium in excitation--contraction coupling of frog skeletal muscle

A principal step in the process leading to muscle contraction is the intracellular release of Ca2+. We have detected and compared some physical and chemical events that reflect Ca2+ release in contracting frog skeletal muscle cells, described the effects of some agents that are believed to alter intracellular Ca2+ release during contraction, and speculated about the role of Ca2+ release in influencing some of the mechanical properties of frog muscle. The specific physical features recorded were changes in striation spacing, myofibrillar orientation, and force development. The chemical feature was the relative change in intracellular [Ca2+] recorded as light emission from cells microinjected with the Ca2+-sensitive protein aequorin. The presence or absence of a correlation among these variables has been used (i) to evaluate the action of some agents thought to change intracellular Ca2+ release in excitation--contraction (E--C) coupling, (ii) to further substantiate the effects of cell length on Ca2+ release, and (iii) to examine some details of models for E--C coupling. The results showed that potentiating agents enhance and prolong intracellular Ca2+ release without changing the rate of Ca2+ removal during E--C coupling. This extra Ca2+ does not produce the same effect on contractions at all lengths. Contractility is inversely related to cell length, and Ca2+-induced activation is normally less than maximum not only at short lengths but also at optimal striation spacings.     

Dissipative processes in human passive skeletal muscle

The measurement of damping of low-amplitude limb oscillations permits to evaluate the energy losses in passive human skeletal muscle during small length changes. The attenuation curve for the limb oscillations is quite different from the classical attenuation curve in the presence of viscous damping. Energy losses per oscillation cycle are practically frequency independent. Thus the damping properties of passive muscles at joint angular velocities up to 100% are due mostly to the velocity-independent resistance of "dry friction" type. The value of this "friction" is about 0.07 N per sm2 of muscle cross-section. The passive muscle also has marked thixotropy, as its resistance to small amplitude low velocity stretches strongly depends on time between stretches.     

Inositol trisphosphate and excitation-contraction coupling in skeletal muscle

The role of inositol trisphosphate as a chemical messenger in excitation-contraction coupling is discussed, both in terms of positive and negative results. The evidence presented includes experiments on the effect of inositol trisphosphate in intact and skinned fibers, in calcium release from isolated sarcoplasmic reticulum vesicles, in activation of single calcium release channels incorporated in planar bilayers, and biochemical experiments that have established the presence of all the intermediate steps involved in the metabolism of phosphoinositides, both in intact muscle and in isolated membranes. From these results, it is clear that a role for inositol triphosphate in skeletal muscle function is highly likely; whether this molecule is the physiological messenger in excitation-contraction coupling remains to be established.     

Perchlorate enhances transmission in skeletal muscle excitation- contraction coupling

The effects of the anion perchlorate (present extracellularly at 8 mM) were studied on functional skeletal muscle fibers from Rana pipiens, voltage-clamped in a Vaseline gap chamber. Established methods were used to monitor intramembranous charge movement and flux of Ca release from the sarcoplasmic reticulum (SR) during pulse depolarization. Saponin permeabilization of the end portions of the fiber segment (Irving, M., J. Maylie, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:1-41) substantially reduced the amount of charge moving during conventional control pulses, thus minimizing a technical error that plagued our previous studies. Perchlorate prolonged the ON time course of charge movement, especially at low and intermediate voltages. The OFFs were also made slower, the time constant increasing twofold. The hump kinetic component was exaggerated by ClO4- or was made to appear in fibers that did not have it in reference conditions. ClO4- had essentially no kinetic ON effects at high voltages (> or = 10 mV). ClO4- changed the voltage distribution of mobile charge. In single Boltzmann fits, the midpoint potential V was shifted -20 mV and the steepness parameter K was reduced by 4.7 mV (or 1.78-fold), but the maximum charge was unchanged (n = 9). Total Ca content in the SR, estimated using the method of Schneider et al. (Schneider, M. F., B. J. Simon, and G. Szucs. 1987. Journal of Physiology. 392:167-192) for correcting for depletion, stayed constant over tens of minutes in reference conditions but decayed in ClO4- at an average rate of 0.3 mumol/liter myoplasmic water per s. ClO4- changed the kinetics of release flux, reducing the fractional inactivation of release after the peak. ClO4- shifted the voltage dependence of Ca release flux. In particular, the threshold voltage for Ca release was shifted by about -20 mV, and the activation of the steady component of release flux was shifted by > 20 mV in the negative direction. The shift of release activation was greater than that of mobile charge. Thus the threshold charge, defined as the minimum charge moved for eliciting a detectable Ca transient, was reduced from 6 nC/microF (0.55, n = 7) to 3.4 (0.53). The average of the paired differences was 2.8 (0.33, P < 0.01). The effects of ClO4- were then studied in fibers in modified functional situations. Depletion of Ca in the SR, achieved by high frequency pulsing in the presence of intracellular BAPTA and EGTA, simplified but did not eliminate the effects of ClO4-.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling

Much recent progress has been made in our understanding of the mechanism of sarcoplasmic reticulum Ca²⁺ release in skeletal muscle. Vertebrate skeletal muscle excitation-contraction (E-C) coupling is thought to occur by a mechanical coupling mechanism involving protein-protein interactions that lead to activation of the sarcoplasmic reticulum (SR) ryanodine receptor (RyR)/Ca²⁺ release channel by the voltage-sensing transverse (T–) tubule dihydropyridine receptor (DHPR)/Ca²⁺ channel. In a subsequent step, the released Ca²⁺ amplify SR Ca²⁺ release by activating release channels that are not linked to the DHPR. Experiments with mutant muscle cells have indicated that skeletal muscle specific DHPR and RyR isoforms are required for skeletal muscle E-C coupling. A direct functional and structural interaction between a DHPR-derived peptide and the RyR has been described. The interaction between the DHPR and RyR may be stabilized by other proteins such as triadin (a SR junctional protein) and modulated by phosphorylation of the DHPR.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Caffeine and length dependence of staircase potentiation in skeletal muscle

Skeletal muscle sensitivity to Ca2+ is greater at long lengths, and this results in an optimal length for twitch contractions that is longer than optimal length for tetanic contractions. Caffeine abolishes this length dependence of Ca2+ sensitivity. Muscle length (ML) also affects the degree of staircase potentiation. Since staircase potentiation is apparently caused by an increased Ca2+ sensitivity of the myofilaments, we tested the hypothesis that caffeine depresses the length dependence of staircase potentiation. In situ isometric twitch contractions of rat gastrocnemius muscle before and after 10 s of 10-Hz stimulation were analyzed at seven different lengths to evaluate the length dependence of staircase potentiation. In the absence of caffeine, length dependence of Ca2+ sensitivity was observed, and the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased length (DT = 1.47 - 0.05 ML, r2 = 0.95, where DT is developed tension). Length dependence of Ca2+ sensitivity was decreased by caffeine when caffeine was administered in amounts estimated to result in 0.5 and 0.75 mM concentrations. Furthermore, the negative slope of the relationship between staircase potentiation and muscle length was diminished at the lower caffeine dose, and the slope was not different from zero after the higher dose (DT = 1.53 - 0.009 ML, r2 = 0.43). Our study shows that length dependence of Ca2+ sensitivity in intact skeletal muscle is diminished by caffeine. Caffeine also suppressed the length dependence of staircase potentiation, suggesting that the mechanism of this length dependence may be closely related to the mechanism for length dependence of Ca2+ sensitivity.     

Deuterium oxide effects on excitation-contraction coupling of skeletal muscle

²H₂O (99.8%) Ringer's solution greatly reduces the twitch and tetanus of frog sartorius muscle and, as specially shown here, slows the onset features of the mechanical output of the twitch by: (a) increasing the time (L_R) from stimulus to start of latency relaxation; (b) slowing the developmet of the latency relaxation, and (c) greatly decreasing the rate of onset of tension development. These changes reflect effects of ²H₂O on excitation-contraction coupling and they represent the critical direct effects of ²H₂O on muscle since it does not depress either the action potential or the intrinsic myofibrillar contractility. The increase in L_R is attributed to slowed inward electrical propagation in the T-tubule. But the critical effect of ²H₂O on frog muscle is to greatly depress mobilization of activator Ca²⁺. The depression of the Ca²⁺ mobilization and of its effects on the activation of contraction evidently result from (a) a lowered rate of release of Ca²⁺ from the sarcoplasmic reticulum, as indicated by the slowed development of the latency relaxation, (b) a decreased amount of Ca²⁺ released in a twitch, and (c) a reduced speed of diffusion of the Ca²⁺ to the contractile filaments. The depressed mobilization of Ca²⁺ is apparently the essential cause of ²H₂O's general depression of twitch and tetanus output.     

Hematopoietic potential cells in skeletal muscle

During mouse embryogenesis, the formation of primitive hematopoiesis begins in the yolk sac on embryonic day 7.5 （E7.5）. Thereafter, definitive hematopoietic stem cell （HSC） activity is first detectable in the aorta-gonadmesonephros （AGM） region on E10, followed by fetal liver and yolk sac. Subsequently, the fetal liver by E12 becomes the main tissue for definitive hematopoiesis. At a later time, HSC population in the fetal liver migrates to the bone marrow, which becomes the major site of hematopoiesis throughout normal adult life.[第一段]     

Role of inositol 1,4,5-trisphosphate in excitation-contraction coupling in skeletal muscle

The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membranous network that controls the myoplasmic Ca2+ concentration and the contraction-relaxation cycle. Ca2+ release from the terminal cisternae (TC) region of the SR evokes contraction. How electrical depolarization of the transverse tubule is linked to Ca2+ release from the junctionally associated TC is still largely unknown. Independent evidence has been recently obtained indicating that either inositol trisphosphate (IP3) or (and) Ca2+ is (are) the chemical transmitter(s) of excitation-contraction coupling. Here we outline the experimental data in support of each transmitter and discuss possible interactive roles of Ca2+ and IP3.     

The role of membrane processes in controlling skeletal muscle function

The role of membrane processes in the activation of skeletal muscle fibers has been investigated in details recently. It seems very probable that the intramembrane charge movement is the potential sensitive step of the excitation-contraction coupling. Considerable efforts were made to monitor the subsequent steps (SR function, Ca release) using optical methods. Experimental data were presented about the relations existing between charge movement process, Ca release and contraction threshold.     

Electrical models of excitation-contraction coupling and charge movement in skeletal muscle

The consequences of ionic current flow from the T system to the sarcoplasmic reticulum (SR) of skeletal muscle are examined. The Appendix analyzes a simple model in which the conductance gx, linking T system and SR, is in series with a parallel resistor and capacitor having fixed values. The conductance gx is supposed to increase rapidly with depolarization and to decrease slowly with repolarization. Nonlinear transient currents computed from this model have some of the properties of gating currents produced by intramembrane charge movement. In particular, the integral of the transient current upon depolarization approximates that upon repolarization. Thus, equality of nonlinear charge movement can occur without intramembrane charge movement. A more complicated model is used in the text to fit the structure of skeletal muscle and other properties of its charge movement. Rectification is introduced into gx and the membrane conductance of the terminal cisternae to give asymmetry in the time- course of the transient currents and saturation in the curve relating charge movement to depolarization, respectively. The more complex model fits experimental data quite well if the longitudinal tubules of the sarcoplasmic reticulum are isolated from the terminal cisternae by a substantial resistance and if calcium release from the terminal cisternae is, for the most part, electrically silent. Specific experimental tests of the model are proposed, and the implications for excitation-contraction coupling are discussed.