Magnesium enhances aurintricarboxylic acid's inhibitory action on the plasma membrane Ca2+-ATPase.

Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.

Conformational changes during the reaction cycle of plasma membrane Ca2+-ATPase in the autoinhibited and activated states.

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity. Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2·Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

Epigallocatechin 3-gallate inhibits the plasma membrane Ca2+-ATPase: effects on calcium homeostasis.

Flavonoids are natural compounds responsible for the health benefits of green tea. Some of the flavonoids present in green tea are catechins, among which are: epigallocatechin, epicatechin-3-gallate, epicatechin, catechin and epigallocatechin-3-gallate (EGCG). The latter was found to induce apoptosis, reduce reactive oxygen species, in some conditions though in others it acts as an oxidizing agent, induce cell cycle arrest, and inhibit carcinogenesis. EGCG also was found to be involved in calcium (Ca2+) homeostasis in excitable and in non-excitable cells. In this study, we investigate the effect of catechins on plasma membrane Ca2+-ATPase (PMCA), which is one of the main mechanisms that extrude Ca2+ out of the cell. Our studies comprised experiments on the isolated PMCA and on cells overexpressing the pump. Among catechins that inhibited PMCA activity, the most potent inhibitor was EGCG. EGCG inhibited PMCA activity in a reversible way favoring E1P conformation. EGCG inhibition also occurred in the presence of calmodulin, the main pump activator. Finally, the effect of EGCG on PMCA activity was studied in human embryonic kidney cells (HEK293T) that transiently overexpress hPMCA4. Results show that EGCG inhibited PMCA activity in HEK293T cells, suggesting that the effects observed on isolated PMCA occur in living cells.

E2P-like states of plasma membrane Ca2+­ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues.

The plasma membrane Ca2+­ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues BeFx, AlFx and MgFx inhibit PMCA Ca2+­ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca2+­ATPase inhibition by AlFx and BeFx depended on Mg2+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell.

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.

Aluminum inhibits the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases by different mechanisms.

Aluminum (Al3+) is involved in the pathophysiology of neurodegenerative disorders. The mechanisms that have been proposed to explain the action of Al3+ toxicity are linked to changes in the cellular calcium homeostasis, placing the transporting calcium pumps as potential targets. The aim of this work was to study the molecular inhibitory mechanism of Al3+ on Ca2+-ATPases such as the plasma membrane and the sarcoplasmic reticulum calcium pumps (PMCA and SERCA, respectively). These P-ATPases transport Ca2+ actively from the cytoplasm towards the extracellular medium and to the sarcoplasmic reticulum, respectively. For this purpose, we performed enzymatic measurements of the effect of Al3+ on purified preparations of PMCA and SERCA. Our results show that Al3+ is an irreversible inhibitor of PMCA and a slowly-reversible inhibitor of SERCA. The binding of Al3+ is affected by Ca2+ in SERCA, though not in PMCA. Al3+ prevents the phosphorylation of SERCA and, conversely, the dephosphorylation of PMCA. The dephosphorylation time courses of the complex formed by PMCA and Al3+ (EPAl) in the presence of ADP or ATP show that EPAl is composed mainly by the conformer E2P. This work shows for the first time a distinct mechanism of Al3+ inhibition that involves different intermediates of the reaction cycle of these two Ca2+-ATPases.

Selectivity of plasma membrane calcium ATPase (PMCA)-mediated extrusion of toxic divalent cations in vitro and in cultured cells.

In the recent years, the toxicity of certain divalent cations has been associated with the alteration of intracellular Ca2+ homeostasis. Among other mechanisms, these cations may affect the functionality of certain Ca2+-binding proteins and/or Ca2+ pumps. The plasma membrane calcium pump (PMCA) maintains Ca2+ homeostasis in eukaryotic cells by mediating the efflux of this cation in a process coupled to ATP hydrolysis. The aim of this work was to investigate both in vitro and in cultured cells if other divalent cations (Sr2+, Ba2+, Co2+, Cd2+, Pb2+ or Be2+) could be transported by PMCA. Current results indicate that both purified and intact cell PMCA transported Sr2+ with kinetic parameters close to those of Ca2+ transport. The transport of Pb2+ and Co2+ by purified PMCA was, respectively, 50 and 75% lower than that of Ca2+, but only Co2+ was extruded by intact cells and to a very low extent. In contrast, purified PMCA-but not intact cell PMCA-transported Ba2+ at low rates and only when activated by limited proteolysis or by phosphatidylserine addition. Finally, purified PMCA did not transport Cd2+ or Be2+, although minor Be2+ transport was measured in intact cells. Moreover, Cd2+ impaired the transport of Ca2+ through various mechanisms, suggesting that PMCA may be a potential target of Cd2+-mediated toxicity. The differential capacity of PMCA to transport these divalent cations may have a key role in their detoxification, limiting their noxious effects on cell homeostasis.

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb(+).

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K2)Mg-MgF4, an E2∙Pi-like state capable to occlude K(+) (or Rb(+)). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb(+) with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1-E2 conformational transition and the kinetics of Rb(+) exchange between the medium and the E2(Rb2)Mg-MgF4 state. Our results show that both in the absence and in the presence of Rb(+), simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg-MgF4 complex, that is unable to shift to E1 upon addition of Na(+). The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg-MgF4 formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb(+) caused by Mg(2+). Na(+)-induced release of Rb(+) from E2(Rb2)Mg-MgF4 occurs at the same rate as from E2(Rb2) but is insensitive to ADP. The rate of Rb(+) occlusion into the E2Mg-MgF4 state is 5-8 times lower than that described for the E2Mg-vanadate complex. Since the E2Mg-MgF4 and E2Mg-vanadate complexes represent different intermediates in the E2-P→E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.

Conformational changes produced by ATP binding to the plasma membrane calcium pump.

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Calcium occlusion in plasma membrane Ca2+-ATPase.

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 µM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.