Characterization of a Ca2+-ATPase in osteoclast plasma membrane

Abstract

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain-sensitive Na+,K(+)-ATPase and 5'-nucleotidase) indicated that plasma membrane enrichment was 11.87-fold and 7.25-fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N-acetylglucosaminidase activities, respectively. SDS latency of Na+,K(+)-ATPase and 5'-nucleotidase activities of the isolated plasma membranes revealed that 43-50% of vesicles were sealed, with 10-16% in the inside-out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca2(+)-ATPase had a high-affinity (KCa = 0.22 microM; Vmax = 0.16 mumol/mg per min) and a low-affinity (KCa = 148 microM; Vmax = 0.37 mumol/mg per min) component. Calmodulin (0.12 microM) had no effect on Ca2(+)-ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high-affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca2+ and Mg2+ chelating agent, high-affinity Ca2(+)-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2(+)-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 microM; Vmax = 0.022 mumol/mg per min) and a low-affinity (Km = 43.8 microM; Vmax = 0.278 mumol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2(+)-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.

Role of regulator of G protein signaling proteins in bone

“…Calcium is an important signal for osteoclast motility downstream of tyrosine kinase signals (106). Osteoclasts, as calcium-mobilizing cells, have an active calcium regulatory system, including a membrane Ca 2+ -ATPase (107) and an endoplasmic Ca 2+ -ATPase (108). The basolateral membranes of osteoclasts are quite sensitive to elevated Ca 2+ ; cytosolic Ca 2+ rises, with cell– matrix detachment and cessation of bone resorption (109).…”

Calcium and bone disease

“…Maintenance of cellular and endosomal calcium is critical in most cells, and it is not surprising that in a calcium-handling cell like the osteoclast widely expressed calcium pumps are found [86]. Osteoclasts are sensitive to partial defects in calcium maintenance, and the heterozygotic mouse for SERCA-2 endosomal calcium ATPase had significantly increased bone mineral density [87].…”

Molecular mechanisms, physiological roles, and therapeutic implications of ion fluxes in bone cells: Emphasis on the cation‐Cl− cotransporters

“…As described in Figure 1 (bottom half) and accompanying legend, these ions are transported from the apical to basolateral side of osteoclasts through transcytotic vesicles (along with digested bone matrix) and mitochondria 2 (Kawahara et al, 2009;Zhao, 2012). As shown again in Figure 1 (bottom half), the presence of transport systems for Ca 2+ and PO 4 2− at the surface (Albano et al, 2015;Bekker & Gay, 1990;van der Eerden et al, 2005;Gupta et al, 1996;Khadeer et al, 2003;Kim et al, 2012;Moonga et al, 2001;Yan et al, 2011) of both membranes suggests that these ions are also recycled through protein-facilitated transepithelial routes and that their movements should thus be affected by the activity of KCC1 and/or other CCCs.…”

“… Model of ion transport in osteoclasts and contribution of KCC1. Serosal ion transport systems (from top to bottom): 1Cl − /1HCO 3 − exchanger AE2/SLC4A2 (Wu et al, 2008 ); K + channels (many types); Na + /K + ‐ATPase α1β2 (Francis et al, 2002 ; Makihira et al, 2011 ); 3Na + −1PO 4 2− cotransporter NaPi2a/SLC34A1 (Albano et al, 2015 ; Khadeer et al, 2003 ); 3Na + /1Ca 2+ exchanger NCX1 (Moonga et al, 2001 ); Ca 2+ ‐ATPase PMCA1 (Bekker & Gay, 1990 ; Kim et al, 2012 ). Apical ion transport systems (from top to bottom): vacuolar H + ‐ATPase ATP6V0a3d2V1B2C1 (Feng et al, 2009 ); 1H + /2Cl − exchanger CLCN7 (Kornak et al, 2001 ); Cl − channel; KCC1/SLC12A4 and KCC2/SLC12A5 (Kajiya et al, 2006 ); 1Na + −2PO 4 2− cotransporters PIT1/SLC20A1 and PIT2/SLC20A2 4 (Gupta et al, 1996 ); Ca 2+ channel TRPV5 (van der Eerden et al, 2005 ; Yan et al, 2011 ).…”