Muscle Ca2 ATPase Pumps Ca2 Ions from the Cytosol into the Sarcoplasmic Reticulum

In skeletal muscle cells, Ca2+ ions are concentrated and stored in the sarcoplasmic reticulum (SR); release of stored Ca2+ ions from the SR lumen into the cytosol causes con

▲ FIGURE 7-7 Operational model of the Ca2+ ATPase in the SR membrane of skeletal muscle cells. Only one of the two catalytic a subunits of this P-class pump is depicted. E1 and E2 are alternative conformations of the protein in which the Ca2+-binding sites are accessible to the cytosolic and exoplasmic faces, respectively. An ordered sequence of steps (1 - 6|, as diagrammed here, is essential for coupling ATP hydrolysis and the transport of Ca2+ ions across the membrane. In the figure, traction, as discussed in Chapter 19. A P-class Ca2+ ATPase located in the SR membrane of skeletal muscle pumps Ca2 + from the cytosol into the lumen of the SR, thereby inducing muscle relaxation. Because this muscle calcium pump constitutes more than 80 percent of the integral protein in SR membranes, it is easily purified and has been studied extensively.

In the cytosol of muscle cells, the free Ca2+ concentration ranges from 10~7 M (resting cells) to more than 10~6 M (contracting cells), whereas the total Ca2+ concentration in the SR lumen can be as high as 10 2 M. However, two soluble proteins in the lumen of SR vesicles bind Ca2+ and serve as a reservoir for intracellular Ca2 + , thereby reducing the concentration of free Ca2+ ions in the SR vesicles and consequently the energy needed to pump Ca2+ ions into them from the cytosol. The activity of the muscle Ca2+ ATPase increases as the free Ca2+ concentration in the cytosol rises. Thus in skeletal muscle cells, the calcium pump in the SR membrane can supplement the activity of a similar Ca2+ pump located in the plasma membrane to assure that the cytosolic concentration of free Ca2+ in resting muscle remains below 1 ^M.

The current model for the mechanism of action of the Ca2+ ATPase in the SR membrane involves two conformational states of the protein termed E1 and E2. Coupling of

~P indicates a high-energy acyl phosphate bond; -P indicates a low-energy phosphoester bond. Because the affinity of Ca2+ for the cytosolic-facing binding sites in E1 is a thousandfold greater than the affinity of Ca2+ for the exoplasmic-facing sites in E2, this pump transports Ca2+ unidirectionally from the cytosol to the SR lumen. See the text and Figure 7-8 for more details. [See C. Toyoshima et al., 2000, Nature 405:647; P Zhang et al., 1998, Nature 392:835; and W. P Jencks, 1989, J. Biol. Chem. 264:18855.]

SR lumen

Membrane

Cytosol

Actuator domain

Phosphorylation domain

Nucleotide binding domain

Membrane

SR lumen

Cytosol

Actuator domain

Phosphorylation domain

Nucleotide binding domain

▲ FIGURE 7-8 Structure of the catalytic a subunit of the muscle Ca2+ ATPase. (a) Three-dimensional models of the protein in the E1 state based on the structure determined by x-ray crystallography. There are 10 transmembrane a helices, four of which (green) contain residues that site-specific mutagenesis studies have identified as participating in Ca2+ binding. The cytosolic segment forms three domains: the nucleotide-binding domain (orange), the phosphorylation domain (yellow), and the actuator domain (pink) that connects two of the membrane-spanning helices. (b) Hypothetical model of the pump in the E2 state, based on a

ATP hydrolysis with ion pumping involves several steps that must occur in a defined order, as shown in Figure 7-7. When the protein is in the E1 conformation, two Ca2+ ions bind to two high-affinity binding sites accessible from the cytosolic side and an ATP binds to a site on the cytosolic surface (step 1). The bound ATP is hydrolyzed to ADP in a reaction that requires Mg2 + , and the liberated phosphate is transferred to a specific aspartate residue in the protein, forming the high-energy acyl phosphate bond denoted by E1 ~ P (step 2). The protein then undergoes a conformational change that generates E2, which has two low-affinity Ca2 +-binding sites accessible to the SR lumen (step |3). The free energy of hydrolysis of the aspartyl-phosphate bond in E1 ~ P is greater than that in E2—P, and this reduction in free energy of the aspartyl-phosphate bond can be said to power the E1 n E2 conformational change. The Ca2+ ions spontaneously dissociate from the low-affinity sites to enter the SR lumen (step 4), following which the aspartyl-phosphate bond is hydrolyzed (step |5). Dephosphorylation powers the E2 n E1 conformational change (step 6), and E1 is ready to transport two more Ca2+ ions.

lower-resolution structure determined by electron microscopy of frozen crystals of the pure protein. Note the differences between the E1 and E2 states in the conformations of the nucleotide-binding and actuator domains; these changes probably power the conformational changes of the membrane-spanning a helices (green) that constitute the Ca2+-binding sites, converting them from one in which the Ca2+-binding sites are accessible to the cytosolic face (E1 state) to one in which they are accessible to the exoplasmic face (E2 state). [Adapted from C. Xu, 2002, J. Mol. Biol. 316:201, and D. Mcintosh, 2000, Nature Struc. Biol. 7:532.]

Much evidence supports the model depicted in Figure 7-7. For instance, the muscle calcium pump has been isolated with phosphate linked to an aspartate residue, and spectroscopic studies have detected slight alterations in protein conformation during the E1 n E2 conversion. The 10 membrane-spanning a helices in the catalytic subunit are thought to form the passageway through which Ca2+ ions move, and mutagenesis studies have identified amino acids in four of these helices that are thought to form the two Ca2+-binding sites (Figure 7-8). Cryoelectron microscopy and x-ray crystallography of the protein in different confor-mational states also revealed that the bulk of the catalytic subunit consists of cytosolic globular domains that are involved in ATP binding, phosphorylation of aspartate, and transduction of the energy released by hydrolysis of the as-partyl phosphate into conformational changes in the protein. These domains are connected by a "stalk" to the membrane-embedded domain.

All P-class ion pumps, regardless of which ion they transport, are phosphorylated on a highly conserved aspartate residue during the transport process. Thus the operational model in Figure 7-7 is generally applicable to all these ATP-powered ion pumps. In addition, the catalytic a subunits of all the P pumps examined to date have a similar molecular weight and, as deduced from their amino acid sequences derived from cDNA clones, have a similar arrangement of transmembrane a helices (see Figure 7-8). These findings strongly suggest that all these proteins evolved from a common precursor, although they now transport different ions.

Lower Your Cholesterol In Just 33 Days

Lower Your Cholesterol In Just 33 Days

Discover secrets, myths, truths, lies and strategies for dealing effectively with cholesterol, now and forever! Uncover techniques, remedies and alternative for lowering your cholesterol quickly and significantly in just ONE MONTH! Find insights into the screenings, meanings and numbers involved in lowering cholesterol and the implications, consideration it has for your lifestyle and future!

Get My Free Ebook


Post a comment