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EXCITATION-CONTRACTION

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COUPLING AND THE MECHANISM OF MUSCLE CONTRACTION Setsuro Ebashi National Institute for Physiological Sciences, Okazaki 444 , Japan KEY WORDS:

Ca2+ antagonist-binding protein as voltage sensor, ryanodine-binding protein as Ca release channel, Ca-induced Ca release, troponin as Ca2+ receptor,

connectin

INTRODUCTION Muscle contraction including excitation-contraction coupling (E-C coupling) was one of the most fascinating subjects of biological sciences in the 1950s and 1960s. Numerous articles were published and attracted much attention, even from those who were not involved in muscle research. Today scientists in many fields seem to feel that the essential part of muscle physiology has been clarified. As will be described later, however, my impression.is rather the opposite. Muscle research has just reached a new starting line. In 1976 I wrote an article for the Annual Review of Physiology titled E-C Coupling and promised the reader that fundamental progress would be made very soon. Although I was a little too hasty, recent remarkable progress based on new means, including a molecular genetic approach, has rekindled my optimism. The subjects to be dealt with here will be confined in principle to the events of skeletal muscle, where we can see the most simplified features of contrac­ tion. Although I have been working on smooth muscle for some fifteen years, I will not refer to this attractive muscle because my opinion about its con­ tractile mechanism is quite at variance with the current concept held by the majority of scientists. 0066-4278/91/0315-0001$02.00

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EBASHI While writing this article, which is concerned primarily with skeletal

muscle physiology, I feel myself something of a stranger in this field. The words of an outsider, such as I am, are usually scoffed at, but they may be

occasionally amusing to the reader because the lack of the depth of his knowledge makes him so bold that he tends to focus on his favorite subjects and to depict them in a caricature-like manner, paying no attention to other eminent achievements that others might think far more worthy. I hope that this article will somehow prove enjoyable to the reader, even if not profitable

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at all.

HISTORICAL OVERVIEW OF E-C COUPLING RESEARCH From Galvan; to Ca2+ The discovery by Galvani (1791) (32) might have led to the research of E-C coupling, but his successors instead focused on electricity, not on muscle contraction. In the studies on biological electricity, movement of muscle induced by electrical stimulation was an unnecessary disturbance. Con­ sequently, the nerve became a more favorable tissue for research, and the new field of nerve physiology opened. Electrophysiology was often employed as a synonym for nerve physiology. It should be noted, however, that illuminating discoveries from the view­ point of general physiology were made using muscle as experimental materi­

al. For instance, Bernstein (1902) (2) found the dependence of membrane

potential on the potassium gradient across the muscle membrane, and Overton

(1902) (71) noticed the requirement of sodium (and lithium) for excitability. It was Hill (1949) (39) who brought a new phase to the relationship between electrical activity and contraction. He examined the time course of

contraction with some mathematical consideration and concluded that a sub­ stance diffusing from the surface membrane could not reach the interior part of a muscle cell within the latent time, i.e. the time from the electrical stimulation to onset of the active state of contraction. Hill's comment, being critical rather than encouraging, became a counterattack to Heilbrunn' s pro­ posal (see below), which was made with undeniable ardor but lacked quantita­ tive analysis. Although his comment was not favorable to the Ca2+ concept, Hill was perhaps the first person who consciously distinguished between the events at the surface membrane and subsequent processes inside the cell. This thought corresponds to today's most popular concept of biological science, transmem­ brane signaling or signal transduction. In the meantime, it became clear that only the electric current across the

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surface membrane, not the current running inside the muscle cell along its longitudinal axis, could induce the contraction (58). In this way, people gradually became aware of a new genre of physiology, excitation-contraction coupling, the term given by Sandow in 1952 (76). Since E-C coupling is, in a sense, a matter how to deal with Ca2+ , it may not be unreasonable at this point to make a brief survey of how Ca2+ became recognized as the intracellular trigger (the historical work of Ringer (74) will not be referred to here because it is not directly connected with the current Ca2+ concept).

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Prior to the proposal of Heilbrunn that Ca2+ must be the physiologic factor inducing muscle contraction (1940) (36), there were a few reports that indicated the importance of Ca2+ in the movement of protozoa (e.g. 6). Even in muscle, we can find notable work of Chambers & Hale (1932) (5) and of Keil & Sichel (1936) (52), the latter especially full of suggestive findings.

None of them, however, stated that Ca2+ must be the physiologic factor. Heilbrunn's proposal was supported by the more persuasive findings of

Kamada & Kinosita (1943) (51) and Heilbrunn & Wiercinski (1947) (37).

In spite of these, Ca2+ was not widely recognized until the work of Weber and that of Ebashi carried out around 1959 (10, 11, 89, cf. 16), which unequivocally showed that Ca2+ exerted its effect directly on the contractile proteins; the latter demonstrated that only a few JLM Ca2+ were enough for nearly full activation, even 0.2 JLM being definitely effective. Thus the first recognition of Ca2+ on the molecular level was made in muscle research, and Ca2+ in this sense was confined to muscle until the discovery of Ca2+ dependence of brain phosphodiesterase in 1970 (SO). The question arises then of why Ca2+ had been disregarded for so long after Heilbrunn's proposal, in spite of his strong assertion and supporting data. One explanation was that Heilbrunn's claim was overshadowed by the discovery of the actomyosin-ATP system by A. Szent-Gyorgyi and his colleagues (cf.

80), the first success in reproducing an important physiologic function in vitro. The fact that this remarkable system apparently did not require Ca2+ furthered the disbelief in Heilbrunn's hypothesis by some people, especially those on biochemical side. Another explanation might be the view of Hill (39) whose proposal was so clear and elegant that many physiologists might have thought that the contraction could not be induced by a simple chemical substance, but rather through a more sophisticated process of a physical nature such as crystallization. Recognition of the T-system (T-tubule, transverse tubule) as the device transferring various factors from the outer medium into the interior of the cell

(19, 44) confirmed the suggestions from anatomical (73) and physiologic (42) viewpoints and eventually answered the question raised by Hill (39) and allowed scientists to make a reasonable compromise with the Ca concept.

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As emphasized in another article ( 16), the development of the Ca2+ concept was indebted to studies on the relaxing factor contained in a simple extract of muscle (4, 53, 59, 61 ). In view of the findings that the factor was the sarcoplasmic reticulum (SR) itself in its fragmel1ted state (18) and that it showed a strong and rapid Ca-binding activity that was able to remove Ca from the actomyosin system (11 ), it was not difficult to depict the scheme for E-C coupling (12) that was not essentially different from that known today (e.g. 14). In this scheme the step that was not backed by experimental evidence was that of Ca release from SR induced by the depolarization of the T-system. Subsequent discovery of troponin (17, cf. 13, 16) as the sole Ca receptor site for contraction further simplified the situation. Search for a Ca Release Mechanism

In 1968 Endo et al (23, 24) and Ford & Podolsky (28, 29) reported that Ca release from the SR was facilitated by Ca2+ itself. It was plausible that this interesting regenerative process, Ca-induced Ca release (ClCR), could be the mechanism involved in E-C coupling. Endo, however, presented various pieces of evidence against its physiologic significance (20, 21). This was correct, but recently affairs concerning CICR have taken a dramatic tum, as will be described in a later section. In the early 1970s Chandler & his colleagues (7, 77) demonstrated the voltage-dependent charge movement at the T-system membrane. They thought that this charge movement was directly involved in the process to open the Ca2+ channel located at the foot of the terminal cisternae (called cistern hereafter) of SR; their idea was well illustrated in a model (see Figure II in 7). The nature of the charge transfer coincided well with various properties of E-C coupling, e.g. inactivation of E-C coupling simultaneously abolished the charge movement. Recent gains reached through biochemical and molecular genetic approaches (see below) are expected to shed new light on this attractive idea. Reference must also be made to the Ca2+ release from SR by depolarization (depolarization was induced by changing the ionic composition in the medium surrounding skinned fibers) noted by Endo's group (22). Since it was later shown that the ionic composition in the cistern is essentially the same as that in the myoplasm except for Ca2+ (79), there seems no ionic basis for electrical disturbances at the SR membrane and, therefore, the depolarization hypothesis does not hold in its original form. As we have seen in the case of CICR (see below), however, we cannot deny the possibility that an un­ revealed mechanism hidden behind this finding may have a significant role in the Ca release process.

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FRONTIERS IN E-C COUPLING RESEARCH Ca2+ Antagonist-Binding Protein as a Voltage Sensor As stated above, E-C coupling is the process by which the depolarization at the T-system induces the release of Ca from the cistern of SR. Thus E-C coupling is the oldest and still up-to-date subject of signal transduction across the membrane. For better understanding it may be convenient to divide this process into two steps. First, the depolarization is transduced into a different

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type of signal; this transduction system resides in the T-system membrane. Second, the transduced signal is transferred to the interior system that is dedicated to Ca release; the system is composed of a foot region

(30) and a

part localized in the cistern membrane. The developments in the studies of Ca2+ antagonists have revealed that an enormous amount of L-type Ca2+ channel or, strictly speaking, Ca2+ an­ tagonist-binding protein [dihydroxypyridine (DHP) derivatives were the most useful tool in this research; the DHP-binding protein, or DHP receptor used hereafter] exists in the T-system membrane

(3, 8, 27). The whole primary 170 kd, which

structure of DHP-binding protein of a molecular mass of about

is responsible for the first step, i.e. the process of transduction of electrical signal, has been deduced from its complementary DNA (cDNA) by Numa &

his colleagues; it has a feature common to that of the Na + channel

(84). The

structure of the cardiac DHP-binding protein has also been determined (see below) and has a high homology with the skeletal one

(65).

The postulation that the DHP-binding protein is the voltage transducer, or the voltage sensor, has been substantiated using a dysgenic mouse that genetically lacks the DHP receptor

(82); cultured myotubes of the mouse

restored E-C coupling by introducing the corresponding cDNA. It is worthy of note that the restoration of E-C coupling was also achieved by introducing cardiac DHP-receptor's cDNA

(83). Interestingly and rather unexpectedly,

the mode of coupling thus induced was very much like that of cardiac muscle, e.g. quick rise of Ca2+ current and abolition of the coupling in the absence of Ca2+. The step of transduction is complicated by its intricate nature, inactivation, i.e. the prolonged depolarization abolishes this transducing step and a fairly long time for recovery is required following repolarization (cf. p.

297 in 14).

The process of inactivation in skeletal muscle is controlled by Ca2+ in a complex manner:

1 . The twitch is not affected even by the complete removal of Ca2 from the outer medium under normal conditions, i.e. Ca2+ in the outer medium has no direct effect on E-C coupling.

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2. The inactivation upon depolarization, however, is markedly facilitated by

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the removal of Ca2+, e.g. the fall of sustained contracture induced by high K + solution is intensely quickened by the removal of Ca2+ from the outer medium. 3. The effect of Ca antagonists is essentially the same as that of the removal of Ca2+. Under physiologic conditions, E-C coupling in skeletal muscle is not liable to inactivation; the L-type Ca2+ channel has the least tendency to inactivate among various types of voltage-dependent Ca2+ channels. This does not mean, however, that inactivation is not significant in muscle research. On the contrary, this phenomenon can be utilized as an important clue, as ex­ emplified by the aforementioned work of Chandler & his colleagues (7). While no attention was paid to T-system proteins other than DHP-binding proteins in this section (cf. 84), it is possible that they have supporting roles, especially in steric arrangement of DHP- and ryanodine-binding proteins. Ryanodine-binding Protein: Ca Release and CICR

Ryanodine has been known to cause irreversible contracture of skeletal muscle and repression of cardiac contractility. Fleischer & his colleagues (26) showed that ryanodine firmly bound to the open state of Ca release channels thus making this drug a useful tool for isolating Ca release channel. The conditions for ryanodine binding were very similar to those for CICR, which suggested that the proteins for both processes were closely related. Indeed, isolated ryanodine-binding protein embedded in the lipid bilayer membrane showed all the properties of both the Ca channel and CICR (46-48, 60). Surprisingly, electron microscopic structure of ryanodine-binding protein is identical with the foot protein (30, 48, 75). Its primary structure was sub­ sequently determined by Numa's group (81) also. It was a single protein of a molecular mass about 565 kd, composed of the foot region and the Ca release channel part. Although Endo's disbelief in the physiologic role of CICR is reasonable (see above), the CICR channel thus appears to be identical with the channel utilized in the physiologic process (see below). Furthermore, CICR now seems to be the mechanism common to the endoplasmic reticulum of almost all kinds of tissues (e.g. 57). Since the events occurring in non-muscle cells are not as fast, it is quite possible that CICR has a physiologic significance in intracellular mobilization of Ca2+ of these tissues. In view of these new facts, it is necessary to expand on the properties of CICR. First a little pharmacology of CICR (cf. 14; 20, 2 1): enhancers, i.e. Ca2+

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(this is CICR itselt), caffeine, ATP, adenine [much weaker activity than ATP with substantial affinity for the site comparable to ATP, sO it can be utilized as an antagonist of ATP and also as a tool to detect CICR under physiologic conditions (49)] and its derivatives, and halothane (see below), facilitate the opening of the ryanodine receptor. Repressors, i.e. Mg2+, procaine, ruthe­ nium red, and so on prevent its opening. CICR seems to have crucial role in the pathogenesis of malignant hyper­ thermia, for which halothane is often the cause (21 , 25). Those who suffer from this disease have been genetically determined to have increased inclina­ tion for CICR. Dantrolene, the remedy for the disease, also depresses CICR at body temperature, but not at lower temperature; its mechanism is complicated (cf. 21). In skeletal muscle, E-C coupling is exclusively carried out by the system represented by the DHP- and ryanodine-binding proteins. Ca2+ release by inositol-l,4,5 trisphosphate, IP3, does not participate in the physiologic process (87). Perhaps the situation in cardiac muscle is not greatly different from that in skeletal muscle. CICR, not physiologically functioning in skelet­ al muscle, may also not play a substantial role in cardiac muscle (21). In smooth muscle, however, CICR appears to be utilized as an intracellular Ca2+ mobilization system (cf 21) together with the IP3 system.

Brief Comments on E-C Coupling Proteins To summarize, depolarization is transduced by the DHP-binding protein into a signal of a different nature, which is then transmitted to the ryanodine­ binding protein, composed of the foot part and the Ca2+ release channel. It is possible that this signal is a conformational change of the DHP-binding protein, the change which subsequently affects the foot region of ryanodine­ binding protein to open the Ca2+ release channel. The time course of E-C coupling is fairly fast, comparable to t�at of contractile processes that are controlled by the troponin system, a de­ repression-type device ( 13, 16). Since the derepression-type regulation is suitable for rapid processes, it is cQnceivable that E-C coupling is also operated by a derepression-type mechanism, i.e. the DHP-binding protein exerts an inhibitory effect on the foot in the resting state, .and its con­ formational change resulting from depolarization removes this inhibition and causes Ca2+ release. This mechanism is in accord with the postulation, by Chandler & his colleagues (7). The spatial arrangement of these two proteins has been schematically depicted by Numa & his associates (see Figure 6 in 81). We cannot deny that an additional protein(s) might be involved as a modulator (see above), but it is very attractive to, assume that this remarkable function, E-C coupling, is principall:y carried out by the two 'proteins.

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At this point we are aware of two puzzling facts. First, the matter concern­ ing DHP-binding protein: Ca antagonists were first recognized in the research on cardiac and smooth muscles, and there is no doubt that they block the entry of Ca2+ in these muscles. The DHP-binding protein in the T-system of skeletal muscle is a Ca2+ channel protein. It does not function as the Ca2+ channel, however, but as the voltage sensor, although Ca2+ may pass through it on depolarization. The dyad and subsurface cistern in cardiac muscle may perform a similar function to the triad of skeletal muscle. This means that the DHP-binding protein of cardiac muscle has a dual function; some molecules simply act as the Ca channel, but others act as the voltage sensor if they are confronted by the foot protein. The other enigma is that the channel for CICR, which has no role in the physiologic process of skeletal muscle, but is a good target of drugs, now appears to be identical with the physiologic Ca2+ -release channel; there still remains a possibility that the two are different proteins, but they are, at any rate, closely related to each other. Thus we meet another case where one protein has a dual function. This may sound unusual, but in pharmacology similar phenomena have been noticed. Some physiologic receptors are stimulated by drugs, e.g. baroreceptor in heart ventricle muscle by veratrine. Since it is not likely that such an agent as veratrine exists in the blood stream as a humoral agent, the drug action may have no physiologic meaning. It was natural to suppose that the receptor protein for such a drug must be different from that for a physiologic function. In view of the dual function of the ryanodine receptor, however, it is now conceivable that both receptors, physiologic and pharmacological, may reside in the same molecule. Then the question is whether the two processes, CICR and physiologic Ca2 + release, are independent of each other, and utilize different parts of the protein, or are inseparably related and use the same molecular structure. This question has general significance in unraveling the secret of how the proteins are constructed as a physiologic device. In this connection, there are several receptors on which drugs (including chemical transmitters) show marked pharmacological effects, but the physiologic roles of the receptors have not been identified. One such example is the acetylcholine receptor in the vascular system. Since there is no cholinergic nerve and acetylcholine-containing tissue adjacent to the vascular system, it is not likely that acetylcholine can reach it because of the abun­ dance of cholinesterase in the blood. Hence, there is little possibility that acetylcholine affects the vascular system under physiologic conditions. This is puzzling, but in view of the dual function, physiologic and pharmacologi­ cal, of the ryanodine-binding protein, we must consider that another function involved in a physiologic phenomenon may be present in this receptor.

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Addendum Ca2+ is taken up by the whole surface of the SR during relaxation. Then the rate of Ca2+ return to the cistern is a limiting factor in recycling of contrac­ tion. This rate in frog skeletal muscle now appears to be faster than once reported, i.e. 1.1 sec for half recovery at room temperature (78). It was noted that this time constant coincided with the off-rate of Ca2+ from parvalbumin (78), a strong Ca2+ -binding protein of undefined function abundantly present in amphibian and fish skeletal muscle (72). It is possible that parvalbumin in these muscles intervenes between the troponin and the SR on the path of Ca2+ back to the SR. In this connection Gillis (33) has proposed that parvalbumin facilitates the removal of Ca2+ from troponin by reducing the myoplasmic Ca2+ concentration, thus increasing the rate of relaxation after twitch and brief tetanus (cf. 33, 79). The decisive role of troponin C as the sole Ca2+ receptor protein was established on the basis of various experiments with isolated contractile protein systems (cf. 70), but there was no comparable work done with a fiber model such as glycerinated muscle fibers. This left room for the assertion that Ca2+ also might exert an effect on contractility through a route other than troponin. Recently, Ohtsuki & his colleagues (66) succeeded in removing troponin C perfectly from a glycerinated muscle fiber using cyclohexanedia­ minetetraacetic acid (COTA) and found that the fiber thus treated completely lost its contractility, which then could fully be restored by the addition of troponin C. This procedure also removed the myosin light chain 2 to a considerable extent, but troponin C by itself was enough to restore the original contractility; this may indicate that the light chain has a little, if any, role in the contractile machinery. ELEMENTAL AND VITAL PHYSIOLOGY OF MUSCLE Some Facets of Sliding Mechanism

1954 was a memorable year for muscle science because the proposal of the sliding mechanism was made by A. F. Huxley & Niedergerke (41) as well as by H. E. Huxley & 'Hanson (45). This ingenious idea was further sub­ stantiated and fully established mainly by the efforts of both Huxleys; two papers published late in the 1950s (40, 43) were particularly impressive and crucial. Since then, efforts of muscle scientists have concentrated on the precise mechanism of sliding. Remarkable progress in muscle research has recently been made in utilizing the optical microscope, the most classical and useful instrument in the biolog­ ical sciences. The long term effort of Oosawa's group t!J see the movement of a single actin filament is now realized using the filament labeled with fluores­ cent phalloidine (92). This technique combined with the method of

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binding myosin, or its subfragment, to a glass surface coated with nitrocellu­ lose (56), or hydrophobic substance (34), has brought in a new era in muscle research. One of the rewards was the clear answer to whether or not the entire structure of the myosin molecule, composed of a shaft (L-meromyosin), a hinge (subfragment-2)-, and two heads (subfragment-l, or S-O, is required for sliding. As first shown by Spudich's group, glass surface-bound S-I, the in­ tramolecular movement of which must be intensely restricted, can dislocate an actin filament with a speed comparable to that of myosin (half the velocity of the latter) (85, 35). Furthermore, a new technique developed by Yanagi­ da's group (55) has shown that the interaction of glass surface-bound S-1 inolecules and a single actin filament can produce a force almost the same as that of intact myosin (35). Thus it is now clear that S-I, the single head, can exhibit almost full activity in sliding and force generation. Another result is interesting but puzzling. It was thought that the formation of a transient but definite binding between one myosin head and one actin molecule, the cross-bridge formation, was the elemental reaction of muscle contraction, which thereby resulted in the breakdown of one ATP. Yanagida & his colleagues, however, showed that under specified conditions actin filaments in myofibrils slid a distance about 60 nm per ATP (91). Essentially the same phenomenon, i.e. sliding more than 100 nm per ATP, was also observed using glass surface-bound myosin (35). On the other hand Spudich's group (86) showed that the sliding distance with heavy meromyosin (HMM, composed of a hinge and two heads) was about 8 nm, which was compatible with the classical concept. The most marked difference in experimental conditions between the two groups was the temperature, 22°C for the former and 30°C for the latter. Since myosin once isolated is very heat labile, it is hoped that the two groups will repeat their experiments using the same (preferably a lower) temperature. Stoichiometry is one of the most fundamental concepts in chemistry, but it may not be an a priori principle if the matter concerns a process where the conversion of the form of energy is involved. This is particularly so when ATP is the source of energy, the amount of energy released from which being only around 10 kT, not much different from that of thermal agitation. In this connection the stoichiometry in the transport ATPase also might be a subject for reconsideration [e. g. a very high ratio of Ca to ATP in Ca transport of SR was reported (9)]. Another enigma is that no rotational movement of the myosin head of a frequency expected from the time course of sliding has been found (cf. 67). Recently a very rapid rotational motion on a ten microsecond scale during isometric contraction, the rate of which is almost the same as that in the

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relaxed state, has been noticed (1). The implication of this finding may be profound, but so far no clear explanation has been given. Thus the situation is exciting but perplexing, and this may not be the time to conclude what is right and what is wrong. Most likely something important is still missing.

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Physiology of Resting Muscle It is well known that if the intact fiber is stretched beyond a certain length, so-called passive tension develops, which has considerable size and can exceed maximum active tension. The origin of this tension had long been an enigma to physiologists. The fact that the Natori's fiber, or mechanically skinned fiber (68), showed essentially the same properties as intact fibers upon stretching (69), excluded the sarcolemma from the list of candidates responsible for the passive tension. Maruyama became deeply interested in this phenomenon and eventually found that a gigantic protein with an elastic nature was responsible; he named it connectin (63) and Wang confirmed this result and termed the protein titin (88). Connectin has an affinity for myosin, connecting a myosin filament to an adjacent Z-band (cf. 62). The molecular mass of a-connectin, perhaps the native form, is about 3,000 kd (54). It does not contain a helical structure, but does have considerable amounts of f3-sheet and f3-tum. An elegant study involving the removal of actin filament from a skinned fiber by gelsolin has visualized a connectin filament in the sarcomere and provided conclusive evidence that connectin is entirely responsible for the tension developed by resting muscle (31). If a muscle contracts in the body, its movement is constrained by antagonis­ tic muscles, which are more or less in the resting state. Thus the mechanical property of resting muscle is an important factor in body movement. It has also been shown that actin filaments, which have lost their way back because of overstretching in the resting state, can regain their original position upon activation (38). This means that the role of connectin is not confined to the resting state, but is also involved in the dynamic movement of muscle as an indispensable supporting player. Hence the study on connectin should be a new subject of exercise physiology. Since the physicochemical studies of such a gigantic molecule are a new subject of protein chemistry, and knowledge of the physiology of resting muscle is still in an immature stage, collaboration between physiologists and biochemists or biophysicists is essential. This may open a new field in muscle physiology. In addition to the physiologic aspects mentioned above, we must emphasize the extremely protease-sensitive nature of connectin (cf. 62). In various muscle diseases, connectin may be the first myoplasmic protein to be affected

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by proteolysis. This consideration is particularly important in elucidating the pathogenesis of progressive muscular dystrophy. Connectin is classified as one of the cytoskeleton proteins. It is localized in the myoplasm and retains an ordered arrangement of myofilaments so that contractile processes can be carried out without fail, but it has no contact with the protein group beneath the plasma membrane. I would like to propose the term endo- and exocytoskeleton by the analogy of endo- and exoskeleton. The endocytoskeleton is represented by connectin. a-Actinin, a Z-band protein (64), initially discovered as a superprecipitation-promoting factor (15), be­ longs to the category of the endocytoskeleton in skeletal and cardiac muscle, but to that of the exocytoskeleton in smooth muscle and non-muscle tissues in which this protein lies under the surface membrane (when we found a-actinin, I was totally fascinated with it because I thought it the third contractile protein following myosin and actin; its identification as a mere Z-band protein was thus a deep disappointment to me).

CONCLUDING REMARKS Muscle contraction is based on the interaction of two proteins, myosin and actin, in the presence of ATP. This interaction is a subtle device for che­ momechanical energy conversion. Its mechanism remains as fascinating a topic as ever. Now it appears that the interaction of two kinds of Ca2+ channel proteins, i.e. DHP- and ryanodine-binding proteins, is the key step in E-C coupling. This interaction involves a different mode of energy conversion from the above, and the essential feature of this conversion remains to be solved. The discovery of an endocytoskeleton protein, connectin, which is respon­ sible not only for the elasticity of resting muscle, but also for appropriate positioning of myofilaments in a vital state of muscle, seems to have opened a new aspect of muscle research. As a whole, muscle is still a very attractive subject. Perhaps we are at the beginning of a renewed and prosperous age in muscle science.

Literature Cited 1. Barnett, V. A., Thomas D. D. 1989. Microsecond rotational motion of spin­ labeled myosin heads during isometric muscle contraction. Biophys. J. 56:51723 2. Bernstein, J. 1902. Untersuchungen ZUI Thennodynamik der bioelektrischen Strome. Pflugers Arch. 92:521-67 3. Borsotto, M., Barhanin, J., Nonnan, R. I., Lazdunski, M. 1984. Purification of the dihydropyridine receptor of the volt-

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Excitation-contraction coupling and the mechanism of muscle contraction.

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