P

Ouabain and another steroid derivative, digitoxi-genin, are the active ingredients of digitalis, an extract of the leaves of the foxglove plant. (Ouabain is found in lower concentrations in a number of other plants, presumably serving to discourage herbivores.) Digitalis has been used to treat congestive heart failure since its introduction for that purpose (treatment of "dropsy") by the British physician William Withering in 1785. It strengthens heart muscle contractions without increasing the heart rate and thus increases the efficiency of the heart. Digitalis inhibits the efflux of Na+, raising the intracellular [Na+] enough to activate a Na+-Ca2 + antiporter in cardiac muscle. The increased influx of Ca2+ through this antiporter produces elevated cytoso-lic [Ca2+], which strengthens the contractions of the heart. The potency of ouabain in animals led to the suggestion (50 years ago) that this plant product might act by mimicking a normal regulator of the Na+K+ ATPase produced in animals, and it now appears that this may be so. Ouabain itself has been isolated from bovine adrenal glands and has been detected in the blood plasma and hypothalamus of mammals. ■

P-Type Ca2+ Pumps Maintain a Low Concentration of Calcium in the Cytosol

The cytosolic concentration of free Ca2+ is generally at or below 100 nm, far lower than that in the surrounding medium, whether pond water or blood plasma. The ubiquitous occurrence of inorganic phosphates (Pi and PPi) at millimolar concentrations in the cytosol necessitates a low cytosolic Ca2+ concentration, because inorganic phosphate combines with calcium to form relatively insoluble calcium phosphates. Calcium ions are pumped out of the cytosol by a P-type ATPase, the plasma membrane Ca2+ pump. Another P-type Ca2+ pump in the endoplasmic reticulum moves Ca2+ into the ER lumen, a compartment separate from the cytosol. In myocytes, Ca2+ is normally sequestered in a specialized form of endoplasmic reticulum called the sarcoplasmic reticu-lum. The sarcoplasmic and endoplasmic reticulum calcium (SERCA) pumps are closely related in structure and mechanism, and both are inhibited by the tumor-promoting agent thapsigargin, which does not affect the plasma membrane Ca2+ pump.

The plasma membrane Ca2+ pump and SERCA pumps are integral proteins that cycle between phos-phorylated and dephosphorylated conformations in a mechanism similar to that for Na+K+ ATPase (Fig. 11-37). Phosphorylation favors a conformation with a high-affinity Ca2+-binding site exposed on the cyto-plasmic side, and dephosphorylation favors one with a low-affinity Ca2+-binding site on the lumenal side. By this mechanism, the energy released by hydrolysis of ATP during one phosphorylation-dephosphorylation cycle drives Ca2+ across the membrane against a large electrochemical gradient.

FIGURE 11-38 Structure of the Ca2+ pump of sarcoplasmic reticulum. (PDB ID 1EUL) Ten transmembrane helices surround the path for Ca2+ movement through the membrane. Two of the helices are interrupted near the middle of the bilayer, and their nonhelical regions form the binding sites for two Ca2+ ions (green). The carboxylate groups of an Asp residue in one helix and a Glu residue in another are central to the Ca2+-binding sites. Three globular domains extend from the cytoplasmic side: the N (nucleotide-binding) domain has the binding site for ATP; the P (phosphorylation) domain contains the Asp351 residue (blue) that undergoes reversible phosphorylation, and the A (actuator) domain somehow mediates the structural changes that alter the Ca2+ affinity of the Ca2+-binding site and its exposure to cytoplasm or lumen. Note the long distance between the phosphorylation site and the Ca2+-binding site. There is strong evidence that during one transport cycle, the N domain tips about 20° to the right, bringing the ATP site close to Asp351, and that during each catalytic cycle the A domain twists by about 90° around the normal (perpendicular) to the membrane. These conformational changes must expose the Ca2+-binding site first on one side of the membrane, then on the other, changing the Ca2+ affinity of the site from high on the cytoplasmic side to lower on the lumenal side. A complete understanding of the coupling between phosphorylation and Ca2+ transport awaits determination of all the conformations involved in the cycle.

The Ca2+ pump of the sarcoplasmic reticulum, which comprises 80% of the protein in that membrane, consists of a single polypeptide (Mr ~ 100,000) that spans the membrane ten times and has three cytoplas-mic domains formed by loops that connect the transmembrane helices (Fig. 11-38). The two Ca2+-binding sites are located near the middle of the membrane bi-

layer, 40 to 50 A from the phosphorylated Asp residue characteristic of all P-type ATPases, so the effects of Asp phosphorylation are not direct. They must be mediated by conformational changes that alter the affinity for Ca2+ and open a path for Ca2+ release on the lu-menal side of the membrane.

The amino acid sequences of the SERCA pumps and the Na+K+ ATPase share 30% identity and 65% sequence similarity, and their topology relative to the membrane is also the same. Thus it seems likely that the Na+K+ ATPase structure is similar to that of the SERCA pumps and that all P-type ATPase transporters share the same basic structure.

F-Type ATPases Are Reversible, ATP-Driven Proton Pumps

The F-type ATPase active transporters play a central role in energy-conserving reactions in mitochondria, bacteria, and chloroplasts; we discuss that role in detail in our description of oxidative phosphorylation and pho-tophosphorylation in Chapter 19. The F-type ATPases catalyze the uphill transmembrane passage of protons driven by ATP hydrolysis ("F-type" originated in the identification of these ATPases as energy-coupling factors). The Fo integral membrane protein complex (Fig. 11-39; subscript o denoting its inhibition by the drug oligomycin) provides a transmembrane pore for protons, and the peripheral protein F1 (subscript 1 indicating that it was the first of several factors isolated from mitochondria) is a molecular machine that uses the energy

Type Atpases

FIGURE 11-39 Structure of the F^ ATPase/ATP synthase. F-type ATPases have a peripheral domain, F1, consisting of three a subunits, three ft subunits, one 8 subunit (purple), and a central shaft (the y subunit, green). The integral portion of F-type ATPases, Fo (yellow), has multiple copies of c, one a, and two b subunits. Fo provides a transmembrane channel through which about four protons are pumped (red arrows) for each ATP hydrolyzed on the ft subunits of F1. The remarkable mechanism by which these two events are coupled is described in detail in Chapter 19. It involves rotation of Fo relative to F1 (black arrow). The structures of VoV1 and AoA1 are essentially similar to that of FoF1, and the mechanisms are probably similar, too.

FIGURE 11-39 Structure of the F^ ATPase/ATP synthase. F-type ATPases have a peripheral domain, F1, consisting of three a subunits, three ft subunits, one 8 subunit (purple), and a central shaft (the y subunit, green). The integral portion of F-type ATPases, Fo (yellow), has multiple copies of c, one a, and two b subunits. Fo provides a transmembrane channel through which about four protons are pumped (red arrows) for each ATP hydrolyzed on the ft subunits of F1. The remarkable mechanism by which these two events are coupled is described in detail in Chapter 19. It involves rotation of Fo relative to F1 (black arrow). The structures of VoV1 and AoA1 are essentially similar to that of FoF1, and the mechanisms are probably similar, too.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment