Anil K. Verma

Monoclonal antibodies to human erythrocyte membrane Ca++-Mg++ adenosine triphosphatase pump recognize an epitope in the basolateral membrane of human kidney distal tubule cells.

Human calcium transporting tissues were examined to determine whether they contained a protein similar to the Ca++-Mg++ adenosine triphosphatase (Ca++-Mg++ATPase) pump of the human erythrocyte membrane. Tissues were processed for immunoperoxidase staining using monoclonal antibodies against purified Ca++-Mg++ATPase. In human kidneys, specific staining was found only along the basolateral membrane of the distal convoluted tubules. Glomeruli and other segments of the nephron did not stain. Staining of erythrocytes in human spleen was readily observed. Human small intestine, human parathyroid, and human liver showed no antigens that crossreacted with the antibodies to Ca++-Mg++ATPase. Specific staining of distal tubule basolateral membranes from the kidney of a chimpanzee was also noted. Our experiments show, for the first time, that basolateral membranes of the human distal convoluted tubule contain a protein that is immunologically similar to the human erythrocyte Ca++-Mg++ATPase. These observations suggest that the cells of the distal convoluted tubules of human kidney may have a calcium pump similar to that of human erythrocyte membranes.

Protein kinase C activates the plasma membrane Ca2+ pump isoform 4b by phosphorylation of an inhibitory region downstream of the calmodulin-binding domain

The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as calmodulin. One of these regions is approximately coterminous with the calmodulin-binding domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the calmodulin-binding domain, and that the downstream inhibitory domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of calmodulin to the calmodulin-binding domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.

Calcium pump epitopes in placental trophoblast basal plasma membranes.

The syncytiotrophoblast represents the primary cellular barrier between maternal and fetal circulations in the placenta. Large amounts of Ca2+ are transported across this barrier by mechanisms that are not clearly understood. To further understand this phenomenon, we examined rat and human placenta by immunohistochemical and protein blotting techniques with a monoclonal antibody raised against the human erythrocyte plasma membrane Ca2+ pump. Immunohistochemistry with this antibody showed specific staining in the human placenta of the basal (fetal facing) surface of the syncytiotrophoblast. In the rat placenta, immunohistochemistry also showed specific staining of the innermost (fetal facing) layer of the trophoblast and the basal surface of the endoderm of the intraplacental yolk sac. In Western blots of placental homogenates and membranes, the monoclonal antibody bound to a 140,000-mol wt band, characteristic of Ca2+ pumps in other tissues. Western blots of isolated basal membranes showed more intense staining than isolated microvillous membranes, confirming the results of the immunohistochemistry. In addition, Ca2+ transport in basal membrane vesicles from human placenta was inhibited by polyclonal antibodies prepared against the erythrocyte Ca2+ pump. We conclude that basal (fetal facing) layers of human and rat placentas contain a high-affinity Ca2+ pump situated to transport Ca2+ from the maternal to the fetal circulation.

Cellular and segmental distribution of Ca2+-pump epitopes in rat intestine

We used a monoclonal antibody (5F10) specific for the human erythrocyte plasma membrane Ca++-pump to demonstrate the presence and distribution of Ca++-pump epitopes in rat intestine. In paraffin embedded tissue sections, antibody 5F10 binds to epitopes in the basolateral membranes of absorptive cells in rat duodenum and portions of jejunum but not ileum. Western blot analysis of intestinal mucosal proteins with antibody 5F10 shows binding of antibody to major bands of Mr ≈ 135,000 and Mr ≈ 72,000, and to lesser bands of Mr ≈ 125,000 and Mr ≈ 27,000. This pattern was seen in mucosal homogenates of rat duodenal and jejunal cells and to a lesser extent in ileal cells. The Mr ≈ 135,000 band corresponds to the molecular weight of Ca++-pumps in other tissues. The other bands correspond in size to known proteolytic fragments of the Ca++-pump. Slot-blot analysis of nitrocellulose immobilized mucosal homogenates shows binding of 5F10 to be greatest in duodenum and least in ileum. Ca++- transport studies by the everted gut sac technique show a correlation between vitamin D induction of active Ca++-transport and the segmental distribution of Ca++-pump epitopes.

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography

Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins.

Antibodies directed toward human erythrocyte Ca2+-ATPase: Effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources

Abstract Antibodies directed against purified human erythrocyte Ca2+-ATPase (purified according to a procedure modified from V. Niggli, J. T. Penniston, and E. Carafoli, 1979, J. Biol. Chem., 254, 9955–9958) were raised in rabbits. In competitive radioimmunoassay tests of immunological cross-reactivity, human erythrocyte Ca2+-ATPase shows a consistent pattern of immunological similarity to the Ca2+-ATPases derived from cell surface fractions of other species, such as rat and dog erythrocyte ghosts, rat corpus luteum plasma membranes, and rat brain synaptic plasma membranes. On the other hand, a purified Ca2+-ATPase preparation from rabbit skeletal muscle sarcoplasmic reticulum failed to show any immunological similarity to the human enzyme. The amount of Ca2+-ATPase protein in the erythrocyte ghosts was estimated to be about 0.6 μg/mg ghost protein, which was not too different from the calculated value of 1.2 ± 0.2 μg/mg ghost protein (mean ± SD, n = 6) based on the calmodulin binding studies of the erythrocyte ghosts. Anti-Ca2+-ATPase immunoglobulin G inhibited enzyme activity and calcium transport, showing that at least one subpopulation of antibodies can block the active site of the enzyme. The antibodies had no effect on the binding of calmodulin to erythrocyte membranes.

The Calcium Pump of the Plasma Membrane: Structure/Function Relationships

The Ca pump of the plasma membrane is an ATPase of the P-class (1–2), i.e.; it forms a phosphorylated intermediate during the reaction cycle and is inhibited by low concentrations of vanadate (see 3–4 for comprehensive reviews). Calmodulin stimulates the ATPase by direct interaction, shifting the Ca affinity of the enzyme from the normal Km value, of between 10 and 20 μM to values around 0.5 μM. The direct interaction with calmodulin has been exploited to purify the enzyme to essential homogeneity on a calmodulin affinity chromatography column (5). The purified enzyme has been shown to be fully competent functionally: it has the expected high affinity for Ca in the presence of calmodulin, it is sensitive to vanadate, and can be reconstituted as an ATP-dependent Ca-transporting system in liposomes. Work on the purified enzyme has permitted to establish that its Ca/ATP-stoichiometry is 1, and that protons are obligatorily exchanged for Ca in the transport reaction. Table 1 offers a summary of the properties of the ATPase: A comprehensive review on the properties of the purified enzyme has appeared in 1982 (6). Most of the work on the pump has so far been carried out on erythrocytes, but the enzyme has been detected, with essentially the same properties, in all plasma membranes so far examined, with the possible exception of liver. One interesting property of the pump, first established on heart plasma membranes(7) but later extended to the enzyme purified from heart sarcolemma and from erythrocytes (8) is the activation by a phosphorylation reaction mediated by the cAMP-dependent protein kinase.

Evidence against involvement of the human erythrocyte plasma membrane Ca2+-ATPase in Ca2+-dependent K+ transport

Two tests were performed to assess the relationship between the Ca2+-activated K+ channel and the Ca2+-pumping ATPase in human erythrocytes. Antibodies against the purified ATPase inhibited the ATPase in resealed erythrocytes, but had no effect on the K+ channel (as assessed by Rb+ efflux). Reconstituted liposomes containing the purified active Ca2+-pumping ATPase showed no Ca2+-activated Rb+ influx. Both of these results suggest that some molecule other than the Ca2+-ATPase is responsible for the K+ channel.

Effects of affinity-purified antibodies on the Ca2+ pumping ATPase of erythrocyte membranes

Antibodies raised in rabbits against the purified erythrocyte membrane Ca2+ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg2+-ATPase activity. In such cases, the antibodies inhibited the Mg2+-ATPase as well as the Ca2+-ATPase. This is consistent with the hypothesis that a portion of the Mg2+-ATPase activity of ghosts is derived from the Ca2+-ATPase. When nitrophenylphosphatase activity was observed, both Mg2+ - and Ca2+-stimulated activities were observed. Only the Ca2+ activity was inhibited by the antibodies, confirming that this activity is due to the Ca2+ pump, and suggesting that the Mg2+-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca2+-ATPases had no effect on the Na+-K+-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na+-K+-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na+-K+-ATPase.In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca2+-ATPase, while antibodies added to the outside had no significant effect.

Replacement of Val674 by Pro increases the sensitivity of the plasma membrane Ca2+ pump to inhibition by Mg2+.

A cDNA encoding a plasma membrane Ca2+ pump mutant V674P(ct120) was constructed and expressed in COS-1 cells. Immunoblots of transfected COS-1 membranes showed that the V674P(ct120) and the wild-type hPMCA4b(ct120) proteins were expressed at similar levels. The change of Val674 to Pro reduced the activity of the hPMCA4b(ct120) to an extent similar to that observed previously in the full-length Ca2+ pump (Adamo et al. (1995) J. Biol. Chem. 270, 30111-30114). Despite its lower activity, the apparent affinity for Ca2+ of the V674P(ct120) enzyme was at least as high as that of hPMCA4b(ct120), indicating that substitution of Val674 by Pro did not impair the interaction of the enzyme with Ca2+. The sensitivity of the V674P(ct120) enzyme to inhibition by vanadate was not significantly different from that of the hPMCA4b(ct120), supporting the idea that the mutation did not alter the equilibrium between E2-E1. The study of the Mg2+ dependency of the Ca2+ transport showed that the V674P(ct120) mutant reached maximum activation at 100 microM Mg2+ in contrast with 500 microM in the hPMCA4b(ct120). Furthermore, while at 2 mM Mg2+ the hPMCA4b(ct120) showed no sign of inhibition, the activity of the mutant decreased to less than 50% of the maximum activity observed at 100 microM Mg2+. These results indicate that the decrease in the activity observed upon substitution of Val674 by Pro was due to a higher sensitivity to Mg2+ as inhibitor.