Sarcoplasmic Reticulum Membranes

FIGURE 5-31 Structure of skeletal muscle, (a) Muscle fibers consist of single, elongated, multinucleated cells that arise from the fusion of many precursor cells. Within the fibers are many myofibrils (only six are shown here for simplicity) surrounded by the membranous sarcoplasmic reticulum. The organization of thick and thin filaments in the myofibril gives it a striated appearance. When muscle contracts, the I bands narrow and the Z disks come closer together, as seen in electron micrographs of (b) relaxed and (c) contracted muscle. 

Drug Biological membranes (sarcoplasmic reticulum) Octanol-buffer... 

Figure 12. Three-dimensional structures of Na+-K+-ATPase and Ca2+-ATPase based on image reconstruction analysis of electron microscopic data obtained from two-dimensional membrane crystals, (a). Na+-K+-ATPase molecule consisting of one a-subunit and one p-subunit. The horizontal arrows indicate a tentative location of the membrane surfaces (upper arrow cytoplasmic surface lower arrow extracellular surface suggested membrane thickness 39 A). Data from Hebert et al., 1988. (b). Ca2+-ATPase molecule consisting only of a catalytic subunit. A tentative location of the transmembrane helices (M1-M10) is indicated. The cytoplasmic part (head) is pointing upwards. In this study, the membrane (sarcoplasmic reticulum) was found to be only 32 A thick (surfaces indicated by shaded areas). Modified from Toyoshima et al., 1993. 

The following types of bion ihbrane stacks were investigated by paraciystal-line methods nerve myelin retinal rod outer segment discs human erythrocyte ost membranes sarcoplasmic reticulum chromatophore membrane cytochrome oxidase model membranes photosynthetic membranes. " mitochondrial cristae membranes ... 

Very recent X-ray examination by Wilkins et al. (1971) of several membranes suspended in water has shown that their main structural feature is the lipid bilayer. The preparations examined included erythrocyte ghosts, nerve-ending membranes, sarcoplasmic reticulum, E. coli plasma membrane, and that of Halobacterium cutiru-brum. 

Nerve plasma membrane Sarcoplasmic reticulum Retinal rod disc membrane Chloroplast membrane... 

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

Another mechanism in initiating the contraction is agonist-induced contraction. It results from the hydrolysis of membrane phosphatidylinositol and the formation of inositol triphosphate (IP3)- IP3 in turn triggers the release of intracellular calcium from the sarcoplasmic reticulum and the influx of more extracellular calcium. The third mechanism in triggering the smooth muscle contraction is the increase of calcium influx through the receptor-operated channels. The increased cytosolic calcium enhances the binding to the protein, calmodulin [73298-54-1]. 

M FIGURE 10.14 The arrangement of Ca -ATPase in the sarcoplasmic reticulum membrane. Ten transmembrane segments are postulated on the basis of hydropathy analysis. 

The trigger for all musele eontraetion is an increase in Ca eoneentration in the vicinity of the muscle fibers of skeletal muscle or the myocytes of cardiac and smooth muscle. In all these cases, this increase in Ca is due to the flow of Ca through calcium channels (Figure 17.24). A muscle contraction ends when the Ca concentration is reduced by specific calcium pumps (such as the SR Ca -ATPase, Chapter 10). The sarcoplasmic reticulum, t-tubule, and sarcolemmal membranes all contain Ca channels. As we shall see, the Ca channels of the SR function together with the t-tubules in a remarkable coupled process. 

In the sarcoplasm of smooth muscle cells there is a membrane bound compartment usually referred to as the SR by analogy with skeletal muscle. However, it is not at all clear that the interior of these membrane-bound regions are continuous as they are in skeletal muscle. The primary properties of this system seem to be quite similar to those of the endoplasmic reticulum of many other cell types. In general, calcium is concentrated into the membrane-bound reticulum and then released to initiate the characteristic action of the cell. 

Sarcoplasmic reticulum Ca -channels. In many smooth muscle cells the rise of intracellular calcium which triggers contraction comes from the flow of calcium from the SR through Ca channels. In others, the SR contributes some unknown fraction of the triggering calcium relative to the amount which comes from the extracellular space through the plasma membrane Ca -channels. There are at least two kinds of Ca -channels in the SR. 

Voluntary muscle contraction is initiated in the brain-eliciting action potentials which are transmitted via motor nerves to the neuromuscular junction where acetylcholine is released causing a depolarization of the muscle cell membrane. An action potential is formed which is spread over the surface membrane and into the transverse (T) tubular system. The action potential in the T-tubular system triggers Ca " release from the sarcoplasmic reticulum (SR) into the myoplasm where Ca " binds to troponin C and activates actin. This results in crossbridge formation between actin and myosin and muscle contraction. 

Pathogenesis of MH is not completely understood. Skeletal muscle, however, is the one tissue in MH with proven abnormalities, and it is further thought that the basic defect that causes the syndrome lies in the calcium regulation system found within the myoplasm. For example, calcium transport function appears to be decreased in the sarcoplasmic reticulum, mitochondria, and sarcolemma. Thus, the suggestion has been made that MH is characterized by a generalized membrane defeet. 

TBT and TFT are membrane-active molecules, and their mechanism of action appears to be strongly dependent on organotin(IV) lipophilicity. They function as ionophores and produce hemolysis, release Ca(II) from sarcoplasmic reticulum, alter phosphatodylseiine-induced histamine release, alter mitochondrial membrane permeability and perturb membrane enzymes. Organotin(IV) compounds have been shown to affect cell signaling they activate protein kinase and increase free arachidonic acid through the activation of phospholipase... 

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum to over 100 in the plasma membrane. Most membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that has revolutionized their study. In the absence of SDS, few membrane proteins would remain soluble during electrophoresis. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 41-2. 

Purified membrane proteins or enzymes can be incorporated into these vesicles in order to assess what factors (eg, specific lipids or ancillary proteins) the proteins require to reconstitute their function. Investigations of purified proteins, eg, the Ca " ATPase of the sarcoplasmic reticulum, have in certain cases suggested that only a single protein and a single lipid are required to reconstitute an ion pump. 

In the sarcoplasm of resting muscle, the concentration of Ca + is 10 to 10 mol/L. The resting state is achieved because Ca + is pumped into the sarcoplasmic reticulum through the action of an active transport system, called the Ca + ATPase (Figure 49-8), initiating relaxation. The sarcoplasmic reticulum is a network of fine membranous sacs. Inside the sarcoplasmic reticulum, Ca + is bound to a specific Ca -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. 

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 Ca release channel (ryanodine receptor), perhaps by interaction between it and the dihydropyridine receptor (slow Ca voltage channel), which are shown in close proximity. Release of Ca from the Ca release channel into the cytosol initiates contraction. Subsequently, Ca is pumped back into the cisternae of the sarcoplasmic reticulum by the Ca ATPase (Ca pump) and stored there, in part bound to calsequestrin.

Fig. 2b. The appearance of two crystal forms shows that the protein in the membrane exists in equilibrium between the protomeric aj8 unit and oligomeric (aj8>2 forms. The high rate of crystal formation of the protein in vanadate solution shows that transition to the E2 form reduces the difference in free energy required for self association of the protein. This vanadate-method for crystallization has been very reproducible [34-36] and it also leads to crystalline arrays of Ca-ATPase in sarcoplasmic reticulum [37] and H,K-ATPase from stomach mucosa [38]. 

Information about the putative folding of the H,K-ATPase catalytic subunit through the membrane has been obtained by the combined use of hydropathy analysis according to the criteria of Kyte and Doolittle [51], identification of sites sensitive to chemical modification [46,48,50,52-55], and localization of epitopes of monoclonal antibodies [56]. The model of the H,K-ATPase catalytic subunit (Fig. 1) which has emerged from these studies shows ten transmembrane segments and contains cytosolic N- and C-termini [53]. This secondary structure of the catalytic subunit is probably a common feature of the catalytic subunits of P-type ATPases, since evidence supporting a ten a-helical model with cytosolic N- and C-termini has also been published recently for both Ca-ATPase of the sarcoplasmic reticulum and Na,K-ATPase [57-59]. 

Most living cells, including muscle, maintain the cytoplasmic Ca concentration at submicromolar levels, against steep gradients of [Ca ], both at the cell surface and across the endoplasmic reticulum membrane [17]. In the musele cell two membrane systems are primarily involved in this function the sarcoplasmic reticulum and the surface membrane. 

The Ca transport ATPase of sarcoplasmic reticulum is an intrinsic membrane protein of 110 kDa [8-11] that controls the distribution of intracellular Ca by ATP-dependent translocation of Ca " ions from the cytoplasm into the lumen of the sarcoplasmic reticulum [12-16],... 

The Ca transport ATPase of the surface membrane is a Ca -calmodulin-dependent enzyme of approximately 138-kDa mass that is structurally distinct from the sarcoplasmic reticulum Ca -ATPase, but shares with it some similarities in the mechanism of Ca translocation [2,3,34]. In both enzymes the Ca -dependent phosphorylation of an aspartyl-carboxyl-group by ATP leads to the formation of an acyl phosphate intermediate that provides the coupling between ATP hydrolysis and Ca translocation. 

This chapter will summarize recent developments on the structure of the Ca -ATPase of the sarcoplasmic reticulum with occasional references to the Ca -ATPases in the plasma membranes and endoplasmic reticulum of non-muscle cells. 

Our discussion here will concentrate on the various forms of the Ca " transport ATPases that occur in the sarcoplasmic reticulum of muscle cells of diverse fiber types and in the endoplasmic reticulum of nonmuscle cells (SERCA). The structure of these enzymes will be compared with the Ca transport ATPases of surface membranes (PMCA) [3,29-32,34] and with other ATP-dependent ion pumps that transport Na, K, andH [46,50-52]. 

The large intrinsic birefringence of the sarcoplasmic reticulum [143] and the polarized attenuated total reflectance FTIR spectroscopy data obtained on oriented films of sarcoplasmic reticulum [144] indicate that a sizeable portion of the secondary structural elements are arranged perpendicularly to the plane of the membrane in a manner reminiscent to the structure of bacteriorhodopsin [145-148]. 

There are indications for the existence of two ATP binding sites both in the sarcoplasmic reticulum [149] and in the plasma membrane Ca -ATPases [30]. These two ATP binding sites may be located at two TGD containing conserved sequences that are present in the ATP binding domain of PMCAl and 2, 90 residues apart [30,34]. 

As in the Ca -ATPase of sarcoplasmic reticulum, the predicted number of membrane spanning sequences in PMCAl and PMCA2 is even, with both N- and C-terminus located on the cytoplasmic side, but their actual number is uncertain and may be 10 or less [30]. 

The dimer chains of Ca -ATPase can also be observed by freeze-fracture electron microscopy [119,165,166,172-174], forming regular arrays of oblique parallel ridges on the concave P fracture faces of the membrane, with complementary grooves or furrows on the convex E fracture faces. Resolution of the surface projections of individual Ca -ATPase molecules within the crystalline arrays has also been achieved on freeze-dried rotary shadowed preparations of vanadate treated rabbit sarcoplasmic reticulum [163,166,173,175]. The unit cell dimensions derived from these preparations are a = 6.5 nm b = 10.7 nm and 7 = 85.5° [175], in reasonable agreement with earlier estimates on negatively stained preparations [88]. 

Blasie and his colleagues have determined the separate profile structures of the lipid bilayer and of the Ca transport ATPase molecule within the sarcoplasmic reticulum membrane to 11 A resolution by a combination of X-ray and neutron diffraction techniques [128,140,187-199]. 

A wide selection of monoclonal and polyclonal anti-Ca -ATPase antibodies have become available in recent years. Studies with these antibodies defined the localization of Ca " -ATPase in the sarcoplasmic reticulum of developing and mature skeletal muscles [60,262-270] and established a pattern of cross reactivity with various Ca -ATPase isoenzymes in the sarco(endo)plasmic reticulum [270-286] and in the plasma membrane [284,287-290] of skeletal, cardiac and smooth muscles. Antibodies have also proved useful in the quantitation of Ca -ATPase, both in muscles of diverse fiber types [291-294] and in COS-1 cells transfected with Ca -ATPase cDNA [97,103,126,127,129,215],... 

Antibody A52 with its epitope at residues 657-672 [129,139,274,275] inhibited the vanadate-induced crystallization of Ca " -ATPase and decreased the stability of preformed Ca " -ATPase crystals [285]. The vanadate-induced crystals arise by the association of the ATPase monomers into dimers (type A interaction), the dimers into dimer chains (type B interaction), and the dimer chains into 2-dimensional arrays (type C interaction). It is suggested that antibody A52 interferes with type B interactions, preventing the formation of dimer chains, without exerting major effect on the concentration of Ca -ATPase dimers in the membrane. The simplest interpretation of the destabilization of Ca -ATPase crystals by mAb A52 is that binding of the antibody to its antigenic site physically blocks the interaction between ATPase molecules [285]. Considering the large bulk of the antibody, such interference is not unexpected, yet only a few of the antibodies that bind to the Ca -ATPase in native sarcoplasmic reticulum interfered with crystallization. 

The A20 antibody did not bind significantly to native SR vesicles, but solubilization of the membrane with C Eg or permeabilization of the vesicles by EGTA exposed its epitope and increased the binding more than 20-fold [139], By contrast, the A52 antibody reacted freely with the native sarcoplasmic reticulum, while the A25 antibody did not react either in the native or in the C Eg solubilized or permeabilized preparations, and required denaturation of Ca " -ATPase for reaction, Clarke et al, [139] concluded that the epitope for A52 is freely exposed on the cytoplasmic surface, while the epitope for A20 was assigned to the luminal surface, where it became accessible to cytoplasmic antibodies only after solubilization or permeabilization of the membrane. The epitope for A25 is assumed to be on the cytoplasmic surface in a folded structure and becomes accessible only after denaturation. 

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