Changes In The Conformation Of The Head Of Myosin Drive Muscle Contraction

How can hydrolysis of ATP produce macroscopic movement? Muscle contraction essentially consists of the cyclic attachment and detachment of the S-1 head of myosin to the F-actin filaments. This process can also be referred to as the making and breaking of cross-bridges. The attachment of actin to myosin is followed by con-formational changes which are of particular importance in the S-1 head and are dependent upon which nu-cleotide is present (ADP or ATP). These changes result in the power stroke, which drives movement of actin filaments past myosin filaments. The energy for the power stroke is ultimately supplied by ATP, which is hydrolyzed to ADP and P;. However, the power stroke itself occurs as a result of conformational changes in the myosin head when ADP leaves it.

The major biochemical events occurring during one cycle of muscle contraction and relaxation can be represented in the five steps shown in Figure 49-6:

(1) In the relaxation phase of muscle contraction, the S-1 head of myosin hydrolyzes ATP to ADP and P;, but these products remain bound. The resultant ADP-Pi-myosin complex has been energized and is in a so-called high-energy conformation.

(2) When contraction of muscle is stimulated (via events involving Ca2+, troponin, tropomyosin, and actin, which are described below), actin becomes accessible and the S-1 head of myosin finds it, binds it, and forms the actin-myosin-ADP-Pi complex indicated.

(3) Formation of this complex promotes the release of Py which initiates the power stroke. This is followed by release of ADP and is accompanied by a large conformational change in the head of myosin in relation to its tail (Figure 49-7), pulling actin about 10 nm toward the center of the sarcomere. This is the power stroke. The myosin is now in a so-called low-energy state, indicated as actin-myosin.

(4) Another molecule of ATP binds to the S-1 head, forming an actin-myosin-ATP complex.

(5) Myosin-ATP has a low affinity for actin, and actin is thus released. This last step is a key component of relaxation and is dependent upon the binding of ATP to the actin-myosin complex.

Figure 49-5. The decoration of actin filaments with the S-1 fragments of myosin to form "arrowheads."

(Courtesy of JA Spudich.)

Figure 49-5. The decoration of actin filaments with the S-1 fragments of myosin to form "arrowheads."

(Courtesy of JA Spudich.)

Figure 49-6. The hydrolysis of ATP drives the cyclic association and dissociation of actin and myosin in five reactions described in the text. (Modified from Stryer L: Biochemistry, 2nd ed. Freeman, 1981.)

Figure 49-7. Representation of the active cross-bridges between thick and thin filaments. This diagram was adapted by AF Huxley from HE Huxley: The mechanism of muscular contraction. Science 1969;164:1356. The latter proposed that the force involved in muscular contraction originates in a tendency for the myosin head (S-1) to rotate relative to the thin filament and is transmitted to the thick filament by the S-2 portion of the myosin molecule acting as an inextensible link. Flexible points at each end of S-2 permit S-1 to rotate and allow for variations in the separation between filaments. The present figure is based on HE Huxley's proposal but also incorporates elastic (the coils in the S-2 portion) and stepwise-shortening elements (depicted here as four sites of interaction between the S-1 portion and the thin filament). (See Huxley AF, Simmons RM: Proposed mechanism of force generation in striated muscle. Nature [Lond] 1971;233:533.) The strengths of binding of the attached sites are higher in position 2 than in position 1 and higher in position 3 than position 2. The myosin head can be detached from position 3 with the utilization of a molecule of ATP; this is the predominant process during shortening. The myosin head is seen to vary in its position from about 90° to about 45°, as indicated in the text. (S-1, myosin head; S-2, portion of the myosin molecule; LMM, light meromyosin) (see legend to Figure 49-4). (Reproduced from Huxley AF: Muscular contraction. J Physiol 1974; 243:1. By kind permission of the author and the Journal of Physiology.)

Another cycle then commences with the hydrolysis of ATP (step 1 of Figure 49-6), re-forming the high-energy conformation.

Thus, hydrolysis of ATP is used to drive the cycle, with the actual power stroke being the conformational change in the S-1 head that occurs upon the release of

ADP. The hinge regions of myosin (referred to as flexible points at each end of S-2 in the legend to Figure 49-7) permit the large range of movement of S-1 and also allow S-1 to find actin filaments.

If intracellular levels of ATP drop (eg, after death), ATP is not available to bind the S-1 head (step 4 above), actin does not dissociate, and relaxation (step 5) does not occur. This is the explanation for rigor mortis, the stiffening of the body that occurs after death.

Calculations have indicated that the efficiency of contraction is about 50%; that of the internal combustion engine is less than 20%.

Tropomyosin & the Troponin Complex Present in Thin Filaments Perform Key Functions in Striated Muscle

In striated muscle, there are two other proteins that are minor in terms of their mass but important in terms of their function. Tropomyosin is a fibrous molecule that consists of two chains, alpha and beta, that attach to F-actin in the groove between its filaments (Figure 49-3). Tropomyosin is present in all muscular and muscle-like structures. The troponin complex is unique to striated muscle and consists of three polypeptides. Troponin T (TpT) binds to tropomyosin as well as to the other two troponin components. Troponin I (Tpl) inhibits the F-actin-myosin interaction and also binds to the other components of troponin. Troponin C (TpC) is a calcium-binding polypeptide that is structurally and functionally analogous to calmodulin, an important calcium-binding protein widely distributed in nature. Four molecules of calcium ion are bound per molecule of troponin C or calmodulin, and both molecules have a molecular mass of 17 kDa.

Ca21 Plays a Central Role in Regulation of Muscle Contraction

The contraction of muscles from all sources occurs by the general mechanism described above. Muscles from different organisms and from different cells and tissues within the same organism may have different molecular mechanisms responsible for the regulation of their contraction and relaxation. In all systems, Ca2+ plays a key regulatory role. There are two general mechanisms of regulation of muscle contraction: actin-based and myosin-based. The former operates in skeletal and cardiac muscle, the latter in smooth muscle.

Actin-Based Regulation Occurs in Striated Muscle

Actin-based regulation of muscle occurs in vertebrate skeletal and cardiac muscles, both striated. In the gen eral mechanism described above (Figure 49-6), the only potentially limiting factor in the cycle of muscle contraction might be ATP. The skeletal muscle system is inhibited at rest; this inhibition is relieved to activate contraction. The inhibitor of striated muscle is the tro-ponin system, which is bound to tropomyosin and F-actin in the thin filament (Figure 49-3). In striated muscle, there is no control of contraction unless the tropomyosin-troponin systems are present along with the actin and myosin filaments. As described above, tropomyosin lies along the groove of F-actin, and the three components of troponin—TpT, TpI, and TpC—are bound to the F-actin-tropomyosin complex. TpI prevents binding of the myosin head to its F-actin attachment site either by altering the conformation of F-actin via the tropomyosin molecules or by simply rolling tropomyosin into a position that directly blocks the sites on F-actin to which the myosin heads attach. Either way prevents activation of the myosin ATPase that is mediated by binding of the myosin head to F-actin. Hence, the TpI system blocks the contraction cycle at step 2 of Figure 49-6. This accounts for the inhibited state of relaxed striated muscle.

The Sarcoplasmic Reticulum Regulates Intracellular Levels of Ca2' in Skeletal Muscle

In the sarcoplasm of resting muscle, the concentration of Ca2+ is 10-8 to 10-7 mol/L. The resting state is achieved because Ca2' is pumped into the sarcoplasmic reticulum through the action of an active transport system, called the Ca2+ ATPase (Figure 49-8), initiating relaxation. The sarcoplasmic reticulum is a network of fine membranous sacs. Inside the sarcoplasmic reticu-lum, Ca2+ is bound to a specific Ca2+-binding protein designated calsequestrin. The sarcomere is surrounded by an excitable membrane (the T tubule system) composed of transverse (T) channels closely associated with the sarcoplasmic reticulum.

When the sarcolemma is excited by a nerve impulse, the signal is transmitted into the T tubule system and a Ca2+ release channel in the nearby sarcoplasmic reticulum opens, releasing Ca2+ from the sarcoplasmic reticulum into the sarcoplasm. The concentration of Ca2+ in the sarcoplasm rises rapidly to 10-5 mol/L. The Ca2+-binding sites on TpC in the thin filament are quickly occupied by Ca2+. The TpC-4Ca2+ interacts with TpI and TpT to alter their interaction with tropomyosin. Accordingly, tropomyosin moves out of the way or alters the conformation of F-actin so that the myosin head-ADP-P; (Figure 49-6) can interact with F-actin to start the contraction cycle.

The Ca2+ release channel is also known as the ryanodine receptor (RYR). There are two isoforms of this

Sarcolemma

Dihydropyridine receptor

Ca2+ release channel

Cisterna

Calsequestrin

Sarcolemma

Dihydropyridine receptor

Ca2+ release channel t

Ca2+

T tubule

Calsequestrin t

Ca2+

Ca2+

Calsequestrin

Sarcomere

Figure 49-8. Diagram of the relationships among the sarcolemma (plasma membrane), a T tubule, and two cisternae of the sarcoplasmic reticulum of skeletal muscle (not to scale). The T tubule extends inward from the sarcolemma. A wave of depolarization, initiated by a nerve impulse, is transmitted from the sarcolemma down the T tubule. It is then conveyed to the Ca2+ release channel (ryanodine receptor), perhaps by interaction between it and the dihydropyridine receptor (slow Ca2+ voltage channel), which are shown in close proximity. Release of Ca2+ from the Ca2+ release channel into the cytosol initiates contraction. Subsequently, Ca2+ is pumped back into the cisternae of the sarcoplasmic reticulum by the Ca2+ ATPase (Ca2+ pump) and stored there, in part bound to calsequestrin.

receptor, RYR1 and RYR2, the former being present in skeletal muscle and the latter in heart muscle and brain. Ryanodine is a plant alkaloid that binds to RYR1 and RYR2 specifically and modulates their activities. The Ca2+ release channel is a homotetramer made up of four subunits of kDa 565. It has transmembrane sequences at its carboxyl terminal, and these probably form the Ca2+ channel. The remainder of the protein protrudes into the cytosol, bridging the gap between the sar-coplasmic reticulum and the transverse tubular membrane. The channel is ligand-gated, Ca2+ and ATP working synergistically in vitro, although how it operates in vivo is not clear. A possible sequence of events leading to opening of the channel is shown in Figure 49-9. The channel lies very close to the dihydropyridine receptor (DHPR; a voltage-gated slow K type

Depolarization of nerve

Depolarization of skeletal muscle i

Depolarization of the transverse tubular membrane r

Charge movement of the slow Ca2+ voltage channel (DHPR) of the transverse tubular membrane

Opening of the Ca2+ release channel (RYR1)

Figure 49-9. Possible chain of events leading to opening of the Ca2+ release channel. As indicated in the text, the Ca2+ voltage channel and the Ca2+ release channel have been shown to interact with each other in vitro via specific regions in their polypeptide chains. (DHPR, dihydropyridine receptor; RYR1, ryanodine receptor 1.)

Ca2+ channel) of the transverse tubule system (Figure 49-8). Experiments in vitro employing an affinity column chromatography approach have indicated that a 37-amino-acid stretch in RYR1 interacts with one specific loop of DHPR.

Relaxation occurs when sarcoplasmic Ca2+ falls below 10-7 mol/L owing to its resequestration into the sarcoplasmic reticulum by Ca2+ ATPase. TpC.4Ca2+ thus loses its Ca2+. Consequently, troponin, via interaction with tropomyosin, inhibits further myosin head and F-actin interaction, and in the presence of ATP the myosin head detaches from the F-actin.

Thus, Ca2+ controls skeletal muscle contraction and relaxation by an allosteric mechanism mediated by TpC, Tpl, TpT, tropomyosin, and F-actin.

A decrease in the concentration of ATP in the sar-coplasm (eg, by excessive usage during the cycle of contraction-relaxation or by diminished formation, such as might occur in ischemia) has two major effects: (1) The Ca2+ ATPase (Ca2+ pump) in the sarcoplasmic reticulum ceases to maintain the low concentration of Ca2+ in the sarcoplasm. Thus, the interaction of the myosin heads with F-actin is promoted. (2) The ATP-depen-dent detachment of myosin heads from F-actin cannot occur, and rigidity (contracture) sets in. The condition of rigor mortis, following death, is an extension of these events.

Muscle contraction is a delicate dynamic balance of the attachment and detachment of myosin heads to F-actin, subject to fine regulation via the nervous system.

Table 49-1 summarizes the overall events in contraction and relaxation of skeletal muscle.

Mutations in the Gene Encoding the Ca2+ Release Channel Are One Cause of Human Malignant Hyperthermia

Some genetically predisposed patients experience a severe reaction, designated malignant hyperthermia, on exposure to certain anesthetics (eg, halothane) and depolarizing skeletal muscle relaxants (eg, succinyl-choline). The reaction consists primarily of rigidity of skeletal muscles, hypermetabolism, and high fever. A high cytosolic concentration of Ca2+ in skeletal muscle is a major factor in its causation. Unless malignant hyperthermia is recognized and treated immediately, patients may die acutely of ventricular fibrillation or survive to succumb subsequently from other serious complications. Appropriate treatment is to stop the anesthetic and administer the drug dantrolene intravenously. Dantrolene is a skeletal muscle relaxant that acts to inhibit release of Ca2+ from the sarcoplasmic reticulum into the cytosol, thus preventing the increase of cytosolic Ca2+ found in malignant hyperthermia.

Table 49-1. Sequence of events in contraction and relaxation of skeletal muscle.1

Steps in contraction

(1) Discharge of motor neuron

(2) Release of transmitter (acetylcholine) at motor endplate

(3) Binding of acetylcholine to nicotinic acetylcholine receptors

(4) Increased Na+ and K+ conductance in endplate membrane

(5) Generation of endplate potential

(6) Generation of action potential in muscle fibers

(7) Inward spread of depolarization along T tubules

(8) Release of Ca2+ from terminal cisterns of sarcoplasmic reticulum and diffusion to thick and thin filaments

(9) Binding of Ca2+ to troponin C, uncovering myosin binding sites of actin

(10) Formation of cross-linkages between actin and myosin and sliding of thin on thick filaments, producing shortening

Steps in relaxation

(1) Ca2+ pumped back into sarcoplasmic reticulum

(2) Release of Ca2+from troponin

(3) Cessation of interaction between actin and myosin

Reproduced, with permission, from Ganong WF: Review of Medical Physiology, 21st ed. McGraw-Hill, 2003.

Malignant hyperthermia also occurs in swine. Susceptible animals homozygous for malignant hyperthermia respond to stress with a fatal reaction (porcine stress syndrome) similar to that exhibited by humans. If the reaction occurs prior to slaughter, it affects the quality of the pork adversely, resulting in an inferior product. Both events can result in considerable economic losses for the swine industry.

The finding of a high level of cytosolic Ca2+ in muscle in malignant hyperthermia suggested that the condition might be caused by abnormalities of the Ca2+ ATPase or of the Ca2+ release channel. No abnormalities were detected in the former, but sequencing of cDNAs for the latter protein proved insightful, particularly in swine. All cDNAs from swine with malignant hyperthermia so far examined have shown a substitution of T for C1843, resulting in the substitution of Cys for Arg615 in the Ca2+ release channel. The mutation affects the function of the channel in that it opens more easily and remains open longer; the net result is massive release of Ca2+ into the cytosol, ultimately causing sustained muscle contraction.

The picture is more complex in humans, since malignant hyperthermia exhibits genetic heterogeneity. Members of a number of families who suffer from malignant hyperthermia have not shown genetic linkage to the RYR1 gene. Some humans susceptible to malignant hyperthermia have been found to exhibit the same mutation found in swine, and others have a variety of point mutations at different loci in the RYR1 gene. Certain families with malignant hypertension have been found to have mutations affecting the DHPR. Figure 49-10 summarizes the probable chain of events in malignant hyperthermia. The major promise of these findings is that, once additional mutations are detected, it will be possible to screen, using suitable DNA probes, for individuals at risk of developing malignant hyperthermia during anesthesia. Current screening tests (eg, the in vitro caffeine-halothane test) are relatively unreliable. Affected individuals could then be given alternative anesthetics, which would not endanger their lives. It should also be possible, if desired, to eliminate malignant hyperthermia from swine populations using suitable breeding practices.

Another condition due to mutations in the RYR1 gene is central core disease. This is a rare myopathy presenting in infancy with hypotonia and proximal muscle weakness. Electron microscopy reveals an absence of mitochondria in the center of many type I (see below) muscle fibers. Damage to mitochondria induced by high intracellular levels of Ca2+ secondary to abnormal functioning of RYR1 appears to be responsible for the morphologic findings.

Figure 49-10. Simplified scheme of the causation of malignant hyperthermia (MIM 145600). At least 17 different point mutations have been detected in the RYR1 gene, some of which are associated with central core disease (MIM 117000). It is estimated that at least 50% of families with members who have malignant hyperthermia are linked to the RYR1 gene. Some individuals with mutations in the gene encoding DHPR have also been detected; it is possible that mutations in other genes for proteins involved in certain aspects of muscle metabolism will also be found.

Figure 49-10. Simplified scheme of the causation of malignant hyperthermia (MIM 145600). At least 17 different point mutations have been detected in the RYR1 gene, some of which are associated with central core disease (MIM 117000). It is estimated that at least 50% of families with members who have malignant hyperthermia are linked to the RYR1 gene. Some individuals with mutations in the gene encoding DHPR have also been detected; it is possible that mutations in other genes for proteins involved in certain aspects of muscle metabolism will also be found.

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