390

Review Article Calmodulin and its roles in skeletal Michael P. Walsh i'H D muscle function

The purpose of this review is to describe the importance Calcium ions are recognized to be important intra- of calmodutin as a mediator of the effects of calcium ions cellular messengers involved in a host of physiolog- in living systems, particularly in the process of skeletal ical responses to nervous and hormonal stimuli, the . effects of Ca 2+ being mediated by specific Ca 2+- Calmodulin is a low molecular weight, acidic, calcium binding ) The second messenger con- binding which mediates the Ca2+ regulation of cept in relation to calcium ions is summarized in a wide range of physiological processes throughout Figure 1. eukaryotic organisms. At low free Ca2+ concentrations, An appropriate stimulus leads to an elevation of such as exist in resting muscle sarcoptasm, calmodulin cytosolic Ca 2+ concentration, this Ca 2+ coming exists in the Ca2+-free form in which state it does not from intraeellular stores or the extraeellular space. generally interact with a target protein_ Following an This cytosolic Ca 2+ then interacts with one or more appropriate stimulus, the free Ca2§ concentration rises calcium-binding proteins, depending on the tissue whereupon Ca2+ binds to calmodulin which undergoes a under consideration. These calciproteins form a conforn~ional change enabling it to interact with a target family of structurally homologous proteins, ex- protein(s). The overall result of this protein-protein amples of which are calmodulin (the subject of this interaction is aphysiological effect, e.g., Ca2+ binding to article), C (which is a component of the calmodulin in allows it to interact with primary Ca z§ regulatory mechanism in striated and activate light chain which catalyzes muscles), mid parvalbmnins (the soluble relaxing the phasphorylation of myosin. This reaction results in factors of fast-twitch ). The resultant contraction of the smooth muscle. Recent studies have Ca2+-calcium binding protein complex is then implicated calmodulin in the Ca2+ control of three capable of interaction with one or more target eneymes in skeletal muscle: phosphorytose kinase, myosin proteins, again depending on the tissue under light chain kinase and a of the sarco- consideration, to form a ternary complex of Ca 2+- plasmic reticulum. Various classes of drugs, btcluding calcium binding protein-target protein. The effect is certain local anaesthetics, have been shown to affect generally to convert the target protein (which is calmodulin-depeadent processes. It is likely ttmt the often an ) from an inactive to an active state. effects of such drugs result from their interaction with The end-result of the targel protein activation is a catmodulin. physiological event which may be the immediate consequence of target protein activation or may Key words result from a series of intermediate reactions (usually MUSCLE, SKELETAL: calcium, ealmodulin, protein ) triggered by the target , myosin , protein activation. . An obvious example to quote is contraction of skeletal muscle fibers in response to nervous stimula- tion which leads to an elevation of sarcoplasmic Fmm the Department of Medical Biochemistry, Faculty Ca 2§ concentration. These Ca 2§ ions interact with of Medicine, University of Calgary, Calgary, Alberta inducing a conformational change in the T2N 1N4. calcium binding protein which is transmitted to

CAN ANAESTH SOC I 1983 t 30:4 / pp 390-398 Walsh: CALMODULIN 391

ways they are affected in disease conditions or Stimulus following administration of drugs or anaesthetics. t For this reason, it is hoped that this review will be t t useful and interesting to anaesthetists and other clinicians. t"ytosotic [Ca 2§ ] I"

Ca 2+BP --~ Slructural aspects Calmodulin is a monomeric, globular protein of Ca2§ - Ca2+Bp molecular weight 417,000 daltons. It is a highly acidic protein (pI = 4.0-4.3) due to a high content (Inaetive) (-35 per cent) of aspartate and glutamate residues. Calmodulins lack tryptophan and, usually, cys- Ca2. - Ca2"BP-TP(active) teine. A high ratio of phenylalanine:tyrosine (com- I ! ! monly 8:2) gives rise to a characteristic UV absorp- ! tion spectrum which is atypical of common globular Physiological event proteins. Most calmodulins contain a single residue of the unusual amino acid, ~-N-trimethyllysine, Ca2+Bp = calcium binding protein which presumably arises from a post-translational TP = target protein. methylafion of a residue. The amino-terminus of calmodulin is blocked by an N-acetyl group. FIGURE I Seeond messengerconcept. The amino acid sequences of nine calmodulins ranging from human brain to Tetrahymena pyri- neighbouring subunits of the troponin complex formis (a ciliated protozoan) have been completed causing a movement of into the aetin or almost completed, The primary structure of groove. This permits -myosin interaction and calmodulin is remarkably conserved throughout contraction of the muscle at the expense of ATP. 2 evolution, suggesting the importance of the entire Of the numerous calcium binding proteins studied molecule in the diverse functions of this calcium to date, calmodulin is the most widespread. It has binding protein. been purified to homogeneity from a wide range of Calmodulin is capable of binding 4 Ca 2§ ions specie~ and tissues from higher vertebrates (includ- ing man) through the invertebrates, higher plants, fungi, slime molds and unicellular organisms. 3 TABLE Diversity of ealmodulin function Calmodulin has been identified in every tissue and CMmodulin-dependent species of examined; it has not, however, Physiological role been found in prokaryotes. In keeping with its wide- Cyclic nucleotide phospho- spread distribution, ealmodulin exhibits a great diesterase Cyclic nucleotide metabolism diversity of function as shown in the Table. Calmo- Adenylate cyclase dulin was originally identified as the Ca 2+- (Ca2+ - Mg2+) ATPases Ca 2+ transport dependent activator of bovine heart and brain cyclic kirtase Smooth muscle contraction nucleotide phosphodiesterases by Cheung in the and non-muscle motility Phospho~lase kinase Glycogen metabolism U.S. 4 and Kalduchi in Japan) Soon afterwards it Other eaimodulin-dependent e.g. neurotransmitrer release was shown to activate brain adenylate cyclase, also in a Ca2+-dependent manner. 6 Calmodulin has PhospholipaseA2 Platelet aggregation since been implicated in the regulation of a host of Dynein ATPase Ciliary and flagellar motility other key enzymes and physiological processes Other calmodulin-regulated processes (Table). Insulin secretion from pancreatic 13 cells Because of its widespread functional importance, Pancreatic enzyme secretion knowledge of ealmodulin and its roles in physio- Intestinal secretion logical processes is fundamental to eventual under- Platelet release reaction Platelet adhesion end plug formation standing of how living systems operate and in what 392 CANADIAN ANAESTHETISTS' SOCIETY /OURNAL per molecule with affinities in the range of -0.2- 3 txM. Thus in resting cells calmodulin will release 4Ca 2§ + CaM its Ca 2§ while in excited cells it will be saturated with Ca~§ . This provides the basis for calmodulin's roles in biological regulation. The 4 Ca2+ binding 1L sites in calmodulin and the individual Ca2+ coordi- Ca4~§ .CaM hating ligands have been predicted on the basis of the x-ray crystallographic sa-ucture of the homolo- gous calcium binding protein, carp , Ca~ 2+ "CAM* and the known amino acid sequence of calmodulin. The calmodulin molecule can be divided into four (inactive) approximately equal parts, each of which contains one Ca 2+ binding site. t Each of these four domains Ca,2+,CaM*,TE consists of an or-helical segment, followed by a Ca 2+ binding loop, followed by another ct helical Jt segment. Not surprisingly, the four domains exhibit Ca42 § C aM*.TE * (inactive) a considerable degree of sequence homology. *Denotes an activeconformation. Mechanism of action CaM = calmodulin. TE = target enzyme. An essential phase in the mechanism of activation of enzymes by ealmodulin is a conformational FIGURE 2 Mechanismof activationof target enzymesby change induced in calmodulin by I~inding of Ca z+ . ca]madulin. This results in exposure of a site(s) which can interact with the target enzyme. A considerable amount of evidence has been provided in support of this Ca2+-induced conformationat change. 3 Upon block the interaction of calmodulin with its target binding Ca:+, calmodulin becomes a more com- protein; calmodulin binds 2 moles of triltuoperazine/ pact, globular structure and a hydrophobic site mole with high affinity (Kn = 1.5 ~,M) only in the becomes exposed on the surface of the molecule. presence of Ca 2+. Levin and Weiss B made the This hydrophohic site is helieved to be involved in interesting observation that the phosphodiesterase the interaction of calmodulin with its target pro- inhibitory effects of a series of phenothiazines teins. Figure 2 illustrates the mechanism whereby correlated with their clinical effectiveness as anti- calmodulin activates most of its target enzymes. psychotic drugs. Furthermore, calmodulin-depen- dent pnosphodiesterase was inhibited by other Drug blndlng chemical classes of antipsychofics: bulyrophenones, The possibility that certain classes of drugs may thioxanthenes and diphenylbutylpiperidines. No exert their pharmacological effects via calmodulin significant inhibition by antidepressants (amitripty- originated with the observation that antipsychotic line and desipramine) or anxiolytics (medazepam agents (specifically phenothiazines) inhibit the ac- and chlordiazepoxide) was observed. Little or no tivities of ealmodulin-dependent rat brain adenylate inhibition was seen with known phosphodiesterase cyclase and cyclic nucleotide phosphodiesterase. 7 inhibitors (theophylline and papaverine) or other The inhibitory effect of antipsychofics on phos- centrally acting drugs (, (+)-Iysergic phodiesterase was shown to result from binding of acid diethylamide, pentobarhital and morphine). the drugs to calmodulin. Antipsychoties are now Phenothiazines have been widely used in recent known to inhibit a number of other calmodulin- years to implicate calmodulin in various Ca2§ dependent enzymes and processes including myosin dePendent cellular processes. For example, pheno- light chain kinase, and plate- thiazines inhibit both myosin phosphorylation and let phospholipase A2. These inhibitory effects can actin-aetivated myosin Mg 2§ ATPase activity in all be explained on the basis of interaction between smooth muscle actomyosin, and tension develop- the drug and the CaZ+-calmodulin complex so as to ment in smooth muscle fibers. These observations Walsh: CALMODUL|N 393

(1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-13- PhK + Ca t+ (2,4-dichlorobenzyloxy)phenethyl] imidazolinium chloride), local anaesthetics (dibucaine, QX572, ll tetracaine and phenacaine) and other drugs with local anaesthetic-like properties (mepacrine, pro- PhK' Ca2+ pranolol and (SKF525A)). In some cases, direct interaction between the drug and calmodulin has been demonstrated, e.g., felodipine (an anti- hypertensive agent). It is conceivable that potent Phos b Phos a inhalation agents such as halothane, which is known to cause release of Ca 2+ from the sareo- plasmic reticulura, may exert their effects by direct interaction with calmodulin. Glycogen GIP Calmodulin in skeletal muscle FIGURE 3 Role of ca|modulin-dependemphosphorylase Calmodulm has, to date, been implicated in three kina~e[rt regulationof glycogenmetabolism in skeletalmuscle. processes in skeletal, muscle: (I) regulation of the enzyme phosphorylase kinase, a key enzyme in glycogen metabolism; (2) regulation of the enzyme myosin light chain kinase, which may play a role in provided supportive evidence that Ca 2+ , calmodulin- modulating actin-myosin interactions in skeletal dependent phosphorylation of myosin plays a cen- muscle; and (3) activation of a skeletal muscle tral role in the regulation of smooth muscle contrac- sareoplasmic reticulum (SR) phosphorylating sys- tion (see below). Similarly, ATP-dependent trans- tem which may be involved in controlling the port of Ca 2+ out of red blood cells by the (Ca2+ + release of Ca 2+ from the SR. The remainder of this Mg2+)ATPase was inhibited by phenothiazines and article will be concerned with each of these mecha- butyrophenones consistent with the proposed role nisms in turn. of ealmodulin in activation of this Ca2+ transport system. Trifluoperazine has been shown to inhibit 1 Phosphorytase kinase both -induced insulin release and - Phosphorylase kinase functions to mobilize gly- induced glucagon release from isolated, perfused cogen via the conversion of an inactive form of rat pancreas. These processes were also inhibited b to the active form, phos- by N-(6-aminohexyl)-5-chloro-1- naphthalenesul- phorylase a. Phosphorylase kinase is a tetramer of fonamide (W-7), a calmodulin antagonist which is four different subunits, i.e., it has the structure chemically unrelated to phenothiazines. (ct~/8)4. The molecular weights of the individual It should be noted, however, that extreme caution subunits are: ct 145,000, 13 128,000, 'y 45,000, B must be exercised when interpreting the effects 16,500. 9,1~The molecule, therefore, has an overall of ealmodnlin antagonists on biological systems. molecular weight of 1.3 • 10 6 daltons. It was Phenothiazines do exhibit Ca2+-dependent interac- known for several years that phosphorylase kinase tion with eaiciproteins other than calmodulin, e.g., activity is regulated by Ca 2+ , but it was only in 1978 troponin C and brain S-100b. Furthermore, pheno- that Cohen et al) ~ identified the B subunit as thiazines and butyrophenones have been shown to calmodulin; this subunit presumably confers Ca2+- stabilize membranes in some systems. It is, there- sensitivity to the ertzyme. Phosphorylase kinase fore, not possible to conclude that ealmodulin is differs from other calmodulin-dependent enzymes involved in the regulation of a specific physiologi- in that the B subunit (calmodulin) is tightly bound cal process solely on the basis of the observed within the phosphorylase kinase complex in the effects of antipsychotics on the system. Such an absence of Ca2+ . The role of calmodulin-dependent approach can lend only supportive evidence. phosphorylase kinase in the regulation of glycogen Recently, other classes of drugs have been shown metabolism in skeletal muscle is summarized in to affect calmodulin-dependent processes: R24571 Figure 3. 394 CANADIAN ANAESTHETISTS' SOCIETY JOURNAL

LCj alkali light chains or two LC~ alkali light GSK-2 + Ca 2§ chains. The myosin molecule contains a globular head region and a long rod-like tail. The tail region is composed exclusively of heavy chains while the globular head contains portions of the heavy chains GSK.2.Ca2+ and both pairs of light chains. The actin binding site and ATPase activity are both associated with the globular head region. The association of the tail portions of many myosin molecules forms the body GSa GSb of the thick filament. One region of the molecule protrudes from the bulk of the thick filament to form a cross-bridge which can interact with the thin actin filaments. This cross-bridge formation between the UDP-gluco~e Glycogen thick and thin filaments forms the basis of the contractile mechanism, which is believed to occur FIGURE4 Roleof calmodulin in inhibitingglycogen synthesis. according to the well-known sliding filament-cross- bridge cycling model. 12,13 It has been known for many years that the Binding of Ca2+ to the ~ subunit of phosphoryl- contractile state of skeletal muscle is regulated by ase kinase (PhK) activates the enzyme which then the level of sarcoplasmic Ca 2+ mediated by the catalyzes the phosphorylation of inactive phos- troponin system. 2 The possibility that a secondary phorylase b (phos b) to active phosphorylase a. The calcium regulatory system may exist in skeletal activated phosphorylase initiates the breakdown of muscle arose with the discovery by Perry and glycogen to glucose providing the energy required co-workers 14 that skeletal muscle contains a Ca 2+- for muscle contraction. dependent myosin light chain kinase (MLCK) At the same time, the polymerization of glucose which catalyzes the phosphorylation of a specific to glycogen is inhibited (Figure 4). Ca2+ ions bind to serine residue on the DTNB light chain of myosin. ealmodulin which activates the enzyme glycogen It is now well-established that MLCK is a cal- synthase kinase-2(GSg-2). This enzyme has recently modulin-dependent enzyme, i s been identified as phosphorylase kinase itselfI ~ and While myosin phosphorylation is not a prerequi- it catalyzes the phosphorylation of glycogen syn- site for contraction of striated muscles, it is widely thase (GS), i.e., the conversion of GSa of GSb. In believed to be essential for actin-myosin interaction this case the phosphorylation inactivates the enzyme in smooth muscle and various nonmuscle motile which would otherwise initiate the polymerization systems.16 The central role of myosin phosphoryla- of glucose and thereby make it unavailable as a tion in the regulation of smooth muscle contraction source of energy for muscle contraction. In sum- is summarized in Figure 5. mary, calcium ions, via ealmodulin-dependent acti- In the resting muscle, the level of sarcoplasmic vation of phosphorylase kinase, activate glycogen Ca 2§ is low (<10-7 M) and myosin exists in the breakdown and inhibit glycogen synthesis and so nonphosphorylated state, in which form it does not provide the energy to support muscle contraction. interact with actin. The trigger for contraction is an increase in sareoplasmie Ca2+ concentration to 2 Myosin light chain kinase -5 t~M. This Ca~+ binds to calmodulin which can Skeletal muscle myosin is a hexamer composed of then interact with the inactive MLCK apoenzyme to two heavy chains (Mr = 200,000 each) and two form an active ternary complex composed of Ca z+- pairs of light chains, the so-called alkali light ealmodulin-MLCK. This active enzyme catalyzes chains, LC1 and LC3 (Mr = 22,500 and 16,500, the phosphorylation of the 20,000-dalton light respectively) and the DTNB or phosphorylatable chain of smooth muscle myosin. The phosphorylated light chain, LC2. A given myosin molecule of myosin so formed is then capable of interaction with skelelal muscle consists of the two heavy chains, actin, the result being contraction of the muscle. two phosphorylatable light chains, and either two Relaxation occurs essentially by a reversal of these Walsh: CALMODULIN 395

Stimulus I I I t Ca2++ CaM

Ca~2+.CAMIt 1 ~ MLCK( inactive ) Ca2 § (active)

! ATP I ADP ~- i ~ Cotltraetlon ATP

Relaxation

+ Pi

Phosphalase(s)

FIGURE 5 Roleof myosin phosphorylationin regulationof smooth musclecontraction,

reactions following a return of the sarcoplasmic Post-tetanic potentiation of the peak twitch ten- Ca 2~ concentration to resting values. MLCK re- sion refers to the observation that the maximum turns to the inactive state so that no further tension developed in a muscle increases (or is phosphorylation of myosin occurs. The myosin that potentiated) following rapid frequency stimula- is already phosphorylated is dephosphorylated by tions. Manning and Stul117 measured the extent of one or more and the muscle relaxes. myosin LC2 phosphorylation at rest, during an As mentioned above, while this is believed to be isometric tetanic contraction, and following relaxa- the mechanism of regulation of smooth muscle tion. The phosphate content of LC2 in the resting contraction and nonmuscle motility, it does not muscle was 0.1 mole phosphate/mole LC 2. This represent the primary regulatory system in striated value did not change significantly during a one- muscles. That role is achieved by the troponin second tetamc contraction. However, within 10-20 system. It is, however, widely believed that myosin seconds following muscle relaxation, the phosphate phosphorylation plays a secondary, modulatory content had increased to 0.65-0.75 mole phosphate/ role in striated muscles, in some way affecting the mole LC2. There was also a transient increase in kinetics of actin-myosin interaction. Recent work peak twitch tension following the tetanic stimula- from several laboratories has implicated myosin tion: a direct correlation was observed between phosphorylation in two physiological phenomena post-tetardc potentiation and myosin LC2 phosphate related to skeletal muscle contraction: (1)post- content. Subsequently, Klug et al. 18 demonstrated tetanic potentiation of peak twitch tension; and (2) a that low frequency stimulation, which approxi- decrease in energy utilization during prolonged mates in rive conditions much more than does a isometric contractions. These will now be con- tetanic contraction of rat gastrocnemius, similarly sidered in turn. resulted in potentiation of isometric twitch tension. 396 CANADIAN ANAESTHETISTS' SOCIETY JOURNAL

This potentiation was temporally correlated to the branes of fast skeletal muscle; and (2) SR vesicles level of myosin LC2 phosphorylation. contain calmedulin-dependent protein kinase activity Crow and Kushmerick~9 recently provided evi- directed against endogenous substrates (SR pro- dence that myosin phosphorylation in mouse ex- teins). Chiesi and Carafolizj demonstrated cal- tensor digitorum longus muscles causes a decrease medulin-dependentphosphorylation of SR proteins in the energy cost for isometric force maintenance. of Mr = 57,000, 35,000, 20,000, and 13-15,000. The level of myosin LC2 phosphorylation increased The 57,000-dalton protein represented the major during a prolonged tetanus from ~0.1 mole phos phosphorylated substrate, the remainder being rela- phatelmole LCz in the unstimulated muscle to a tively minor. Campbell and MacLennan22 similarly maximum of 0.55 mole phosphate/mole LC2. The observed a major phosphorylated SR protein of M, energy cost for tension maintenance decreased = 60,000 and a minor substrate of Mr = 20,000. during the tetanus to -50 per cent of its initial Based on the known functions of the SR, one could value. A nonlinear correlation was observed be- postulate a role for such a calmodulin-dependent tween the reduction in energy cost for isometric phosphorylating system in regulation of Ca2+ up- tetani seen in prolonged stimulations and the extent take, regutation of Ca 2+ storage, or regulation of of myosin LC2 phosphorylation. Ca 2+ release from the SR. Calmodulin-dependent The data of Cooke et al. zo support these findings. phosphorylation of SR proteins was observed to They investigated the effect of myosin phosphoryla- have no effect on Ca 2+ transport or ATPase activity tion (using ATP) or thiophosphorylation (using the of SR vesicles. It is, therefore, unlikely that ATP analog, ATPyS (adenosine 5'-0(3-thiotriphos- calmodulin is involved in the regulation of Ca~§ phate)) on actomyosin ATPase activity in glycerin- uptake activity. There is no reason to believe this ated rabbit psoas fibers, and lightly calmodulin-dependent phosphorylating system is cross-linked myofibrils. Fifty to eighty per cent involved in the regulation of Ca 2+ storage in the SR, thiophosphorylation of myosin was achieved in a function which has been assigned to . fibers incubated with MLCK, calmodulin and ATP3,S The calmodulin-dependentphosphorylating system which resulted in an approximately 50 per cent may, therefore, be involved in the long-term regula- decrease in aetomyosin ATPase. The appropriate tion of Ca2"~ release from the SR during excitation. controls were not thiophosphorylated and exhibited Campbell and MacLennanz2 have proposed that normal ATPase activity. The isometric tension was calmodulin-dependent phosphorylation of the unaffected by thiophosphorylation. Phosphoryla- 60,000-dalton SR protein controls a Ca 2§ release tion or thiophosphorylation of myosin in lightly channel in the SR. When this protein is dephosphor- cross-linked myofibrils (which were incapable of ylated, the Ca2+ release channel is open. As Ca 2§ shortening) similarly caused an approximately 50 flows out it binds to calmodulin stimulating phos- per cent reduction in ATPase activity. The ATPase phorylation of the 60,O00-dalton protein which activity of non-cross-linkedmyofibrils, on the other closes a gate in the channel. Other Ca2-~ release hand, was unaffected by myosin phosphorylation. channels in the SR membrane are believed to be They concluded that myosin phosphorylation in controlled by proton gradients, the two regulatory skeletal muscle fibers is a mechanism which modu- mechanisms operating synergistically to control lates the rate of ATP hydrolysis and that the Ca2§ release from the SR. expression of this modulation requires an intact A 22,000-dalton protein, , has filament array. The possible relationship between been identified in cardiac SR membranes and shown the role of myosin phosphorylation in decreasing to be tightly associated with the CaZ*-transport energy utilization and in post-tetanic potentiation is ATPase. Cardiac phospholamban is phosphorylated unknown. by both cyclic AMP-dependent protein kinase and a calcium, ca]medulin-dependent protein kinase. 2a ~5 3 SR protein-phosphorylating system These phosphorylations have been implicated in the Two recent publications2f'zz have implicated control of the Caz+-transport ATPase in the heart. calmodulin in a protein-phosphorylating system of Phospholamban has not, however, been demon- skeletal muscle SR. The major findings reported strated in skeletal muscle SR. were: (1) calmodulin is associated with SR mem- Wa[sh: CALMODULIN 397

Concluding remarks References Calmodulin plays a central role in the regulation by 1 Kreuinger RH. Structure and evolution of calcium- Ca 2§ of diverse biological processes, including modulated proteins. CRC Crit Rev Biochem 1980; muscle contraction. Three calmodulin-dependent g: 119-74. enzymes have been recognized in skeletal muscle: 2 Weber A, Murray JM. Molecular control mecha- phosphorylase kinase, myosin light chain kinase nisms in muscular contraction. Physio] Roy 1973; and an SR protein kinase. It is conceivable, and 53: 612-73. ind~:d likely, that other calmodulin-dependent 3 Walsh MP, Hartshorne DJ. The Biochemistry of enzymes remain to be identified in skeletal muscle. Smooth Muscle (N.L. Stephens, ed.) CRC Press, In this context, Grand and Perry26 have observed in press. two calmodulin-binding proteins (Mr = 150,000 4 Cheung WY. Cyclic3',5'-nucleotide phosphodies- and 61,000) of unknown function in rabbit skeletal terase. Demonstration of an activator. Biochem muscle. Future efforts will be centred on complete Biophys Res Commun 1970; 533-8. elucidation of the role of myosin phosphorylation 5 Kakiuchi S, Yamazaki R, Nakajima H. Properties of and the involvement of caImodulin in Ca t+- a heat-stable phosphodiesterase activating factor induced Ca 2+ release from the SR, in addition to isolated from brain extracts. Studies on cyclic the identification of new calmodulin-dependent 3',5'-nucleotide phosphodiesterase U. Proc Jap enzymes in skeletal muscle. It is hoped that the Acad 1970; 46: 587-92. acquisition of such knowledge will be accompanied 6 Brostrom CO, Huang YC, Breckenridge B. McL, by elucidation of the effects (at the molecular level), WolffDJ. Identification of a calcium-binding pro- and mechanisms of action, of anaesthetics and other tein as a calcium.dependent regulator of brain agents which may be exerted at the level Qf adenylate cyclase. Pmc Natl Acad Sci (USA) 1975; calmodulin. 72: 64-8. 7 Uzunov P, Weiss B. Effects of phenothiazine tran- Acknowledgements quilizers on the cyclic 3' ,Y-adenosine monophos- The author is grateful to Dr. Beverley Britt for photo system of rat brain. Neuropharmacology 1971; suggesting that this review be written and for many 10: 697-708. helpful suggestions; to Dr. Keith Brownell for 8 Levin RM, Weiss B. Mechanism by which psycho- fruitful discussions. tropic drugs inhibit adenosine cyclic 3',5'-mono- phosphate phosphodiesterase in brain. Mol Phar- macol 1976; 12: 581-98. 9 Cohen P. The subunit structure of rabbit-skeletal- muscle phosphorylase kinase, and the molecular basis of its activation reactions. Eur J Biochem 1973; 34: 1-14. 10 Cohen P, Burchell A, Poulkes JG, Cohen PTW, Vanaman TC, Nairn AC. Identification of the cal- dam-dependent modulator protein as the fourth sub- unit of rabbit skeletal muscle phosphorylase kinase. FEBS Letters 1978; 92: 287-93. 11 Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-2 and phosphorylase kinase are the same enzyme. Ear J Biochem 1979; 100: 339-47, 12 Huxley AF. Muscle structure and the.odes of con- traction. Prog Biophys Mol Biol 1957; 7: 257-318. 13 Huxley HE, Hanson J, Changes in the cross- striations of muscle during contraction and stretch and their structural interpretation. Nature 1954; 173: 973-6. 14 Perrie WT, Smillie LB, Perry SV. A phosphoryl- 398 CANADIAN ANAESTHETISTS' SOCIEI'Y JOURNAL

ated light-chain r of myosin from skeletal R~sum~ muscle. Biochem J 1973; 135: 151-64. Celte revue vise c1 d~crire l' importance de la cabnoduline 15 Walsh MP. Calmodu]in-dependent myosin light comme m#diateur des effets des ions calciques clans les chain kinases. Cell Calcium 1981; 2: 333-52. systdmes biologiques, surtout dans le processus de la 16 Walsh MP, Hartshorne DJ. Actomyosin of smooth contraction musculaire squelettique. muscle in Calcium and Cell Function (W.Y. La calmoduline est une protdine acide de foible poids Cheung, ed.) 1982; 3: 223-69. mol~culaire, tiant le calcium, qui agit comme m6diateur 17 Manning DR, Stull JT. Myosin light chain phos- dane la r~gulatian du calcium pour une vari~t~ de phorylation and phosphorylase a activity in rat ex- processus physiobagiques d' organismes eukariotiques. A tensor digitorum Iongus muscle. Biochem Biophys une basse concentration de calcium libre, cetle existant Res Commun 1979; 90: 164-70. glans le sarcoplasme du muscle au repos, la catmoduline 18 Klug GA, Botterman DR, Stull JT. The effect of low est sous forme libre, non li~e au calcium, forme darts frequency stimulation on myosin light chain phos- laqueUe elle ne peut g~.n~ralement pas rdagir avec une phorylation in skeletal muscle. J Biol Chem 1982; prot~ine cible. 257: 4688-90. Aprds un stimulus appropri~, la concentration du 19 Crow MT, Kushmerick MJ, Myosin light chain calcium libre s'~Idve jusqu'd ce que celui-ci se lie ~ la phosphorylation is associated with a decrease in the calmoduline qui subit alors un changement de configura- energy cost for contraction in fast twitch mouse tion la rendant apte d r~agir avec une ou des prot~ine(s) muscle. 3 Biol Chem 1982; 257: 2121-4. cible(s). Le rdsultat final de cette interaction prot~ine- 20 Cooke R, Franks K, Stull JT. Myosin phosphoryl- prot~ine est un effet physiologique: par exempte, le ation regulates the ATPase activity of permeable calcium libre se liant d la calmoduline du muscle lisse ltti skeletal muscle fibers. FEBS Letters 1982; 144: permet de r~agir avecla myosine kinase d chafne l~g&e 33-7. qui catalyse la phosphorisation de la myo.~ine. Cette 21 Chiesi M, Carafoti E. The regulation of Ca2+ trans- r~action amdne la contraction du muscle lisse. port by fast skeletal muscle sarcoplasmic retieulum. Des ~tudes r~centes ant imptiqud la calmoduline dane Role of calmodulin and of the 53,000-dalton glyco- he contr~le calcique de trois enzymes du muscle squeletti- protein..r Biol Chem 1982; 257: 984-91. que: la phosphotylase-kinase, la myosine-kanase ~ chafne 22 Campbell KP, MacLennan DH. A calmodulin- t~gdre et la protdine-kinose du r#ticulum sarcoplasmi- dependent protein kinase system from skeletal que. On a d~montrd que des mddicaments dont certains muscle sareoplasmic reticulum. Phosphorylation of anesth6siques locaux, affectent lee proce.~sus d~pendant a 60,000-dalton protein. J Biol Chem 1982; 257: de la cabnoduline probablement par une inter-rdaetion 1238-46. avec celle-cL 23 Tada M, girchberger MA, Katz AM. Phosphoryla- tion of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-mono- phosphate-dependem protein kinase. J Biot Chem 1975; 250: 2640-7. 24 LePeuch CJ, Haier J, Demaitle JG. Concerted regulation of cardiac sarcoplasmic reticul~m calcium transport by cyclic adenosine monophosphate de- pendent and ealcium-calmodulin-dependent phos- phorylations. Biochemistry 1979; 18: 5150-7. 25 Kirchberger MA, Antonetz T. Calmodulin-mediated regulation of calcium transport and (Ca 2+ + Mg2+)- activated ATPase activity in isolated cardiac sarco- plasmic reticulum. J Biol (2hem 1982; 257: 5685-91. 26 Grand RJA, Perry SV. Calmodulin-binding proteins from b~ain and other tissues. Biochem I 1979; 183: 285-95.