Among the most important Ca

Among the most important Ca regulatory mechanisms are the Plasma Membrane Calcium ATPase (PMCA) and Sarcoplasmic Reticulum Calcium ATPase (SERCA). These pumps belong to the family of P-ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. PMCA and SERCA use the ATP hydrolysis as a source of energy to transport calcium from cytoplasm to the extracellular medium or the reticulum lumen, respectively. PMCA is more regulated than SERCA, and through its interaction with regulatory, targeting and signaling proteins, regulates both global Ca homeostasis and spatially defined Ca signaling []. Detailed structural information about PMCA is currently lacking. Its little abundance (approximately 0.1% of the total protein in the membrane) and the presence of several isoforms hindered efforts to produce suitable crystals for X-ray diffraction. Although PMCA has not been crystallized yet, the structures of several reaction intermediates of SERCA have been resolved (, []). Its membrane-buried region is made up of 10 membrane spanning helices and is connected to a large cytoplasmic headpiece, which is further separated into three distinct domains, denoted A (“actuator”), P (“phosphorylation”), and N (“nucleotide binding”). However, unlike SERCA, PMCA is highly regulated by calmodulin (CaM), which activates this protein by binding to an auto-inhibitory region [] and changes the conformation of the pump from an inhibited state to an activated one [, , ]. The current kinetic model for PMCA and SERCA proposes that enzymes exist in two main conformations: and . After the binding of intracellular Ca to high affinity sites, can be phosphorylated by ATP with formation of the intermediate P. The conformational transition to P leads to the release of Ca to the opposite side of the membrane. The dephosphorylation of P to and a new conformational transition to allow a new pump Picrotoxin [,]. Magnesium is necessary for optimal catalysis of PMCA [] and SERCA []. This ion accelerates the  →  [,] and the P → P transitions []. Several ligands have been used for studying intermediates (P) in these pumps. In PMCA, lanthanum (La) is known to prevent the Mg- dependent transition P → P, fixing the pump in P [,] while vanadate has been used for mimicking the P state []. Furthermore, beryllium fluoride (BeFx) [], magnesium fluoride (MgFx) [,] and aluminum fluoride (AlFx) [] were useful in SERCA to elucidate the mechanism of ATP hydrolysis, because these phosphate analogues bind to the phosphorylation site mimicking the different steps of the P dephosphorylation []. In the presence of calcium and ADP, aluminum fluoride stabilizes the P analogue conformation []. In this work, we study the effect of aluminum on PMCA purified from human erythrocytes -about 90% PMCA4 and 10% PMCA1 []- and SERCA isolated from skeletal muscle -mostly SERCA1 []-. Our results show that Al can bind quickly and strongly to both pumps inhibiting the Ca-ATPase activity. This binding is affected by Ca in SERCA, but not in PMCA. In SERCA, Al prevents the phosphorylation, while in PMCA it prevents the dephosphorylation of the pump. Our results suggest that, in the presence of calcium and ATP, Al fixes PMCA in an P analogue conformation. This is the first evidence of an inhibitor that stabilizes an P analogue conformation with the phosphate covalently bound. Materials and methods
Results
Discussion Although with a different stoichiometry and regulation [35,36,38], PMCA and SERCA are P-ATPases that transport Ca2+ against the electrochemical gradient using the energy of ATP hydrolysis. They form phosphorylated intermediates during their reaction cycles. Phosphorylation, transition between phosphoenzyme conformers and dephosphorylation steps depend on the binding of Mg2+. Our results show that Al3+ stabilizes a phosphorylated intermediate of PMCA whereas it binds to a dephosphorylated intermediate in SERCA. Both PMCA [39] and SERCA [40] bind ATP with high affinity in the absence of Ca2+. However, Ca2+ binding is required for the enzyme activation, even if Mg2+ is present. This is because Ca2+ is required for ATP γ-phosphate to reach the Asp residue, which allows the phosphorylation of the enzyme [40]. Therefore, the fact that in PMCA the EAl complex becomes phosphorylated suggests that the pump acquires the correct conformation. Following a similar line of argument, the conformation of the EAl complex in SERCA is clearly not suitable for phosphorylation. This conclusion and the protective effect produced by Ca2+ suggest that Al3+ could bind in SERCA to the Ca2+ binding site.