Stimulation of Ca2+-ATPase Transport Activity by a Small-Molecule Drug

The sarco(endo)plasmic reticulum Ca²⁺−ATPase (SERCA) hydrolyzes ATP to transport Ca²⁺ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small-molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP-dependent Ca²⁺ translocation. The concentration dependence of the CDN1163 effect provided an EC₅₀=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca²⁺ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca²⁺ release into the SR lumen.     

The Effect of Different Metal Cation Binding on the Proton Pumping in Bacteriorhodopsin

The first section of this paper is a detailed summary of studies made by us and others on metal cations binding to deionized bacteriorhodopsin (dIbR) and its variants. Our studies include the luminescence experiments of Eu³⁺ binding to dIbR and potentiometric studies of Ca²⁺ binding to dIbR, to deionized bR mutants, to bacterioopsin, and to dIbR with its C-terminus removed. The results suggest the presence of two classes of binding sites, one class has two high-affinity constants, and one has one low-affinity constant. For Ca²⁺ binding, there is one metal cation in each of the two high-affinity sites which are coupled to the charged aspartates 85 and 212 (known to be in the retinal cavity) but not coupled to each other. The low-affinity class can accommodate 0–6 Ca²⁺ ions and most of them are bound to the surface. Mg²⁺ has a slightly smaller value for its binding constant to the highest-affinity site. Thus, one expects more Ca²⁺ than Mg²⁺ bound to the two high-affinity sites. In the second section, we summarize our recent study on the effect of metal cation charge density (Ca²⁺, Mg²⁺, Eu³⁺, Tb³⁺, Ho³⁺, Dy³⁺) on the kinetics of both Schiff base deprotonation and proton transport to the extracellular surface. For all metal cations, the apparent rate constant of the slow components of the deprotonation process is the same as that for the transport process at 22 °C. The temperature studies, however, show this apparent equality to be fortuitous and to result from cancellation of the contribution of the energy and entropy of activation. Thus, while the entropy of activation is positive for the deprotonation process, it is negative for the proton transport process. These kinetic parameters depend weakly on the charge density, but in an opposite sense for the two processes. These results suggest that the deprotonation is not the rate-limiting step for the proton transport process. A possible mechanism is proposed in which a hydrated metal cation is used to induce the deprotonation of the protonated Schiff base and to dissociate one of its H₂O molecules to donate the proton in the L → M process.     

Crosstalk among Calcium ATPases: PMCA,SERCA and SPCA in Mental Diseases

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca²⁺ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca²⁺, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca²⁺-ATPase (PMCA), sarco/endoplasmic Ca²⁺-ATPase (SERCA) and secretory pathway Ca²⁺-ATPase (SPCA) are considered efficient line of defense against abnormal Ca²⁺ rises. However, their role is not limited only to Ca²⁺ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein—calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca²⁺-ATPases to neuropathology, with a special emphasis on mental diseases.     

Sorcin Activates the Brain PMCA and Blocks the Inhibitory Effects of Molecular Markers of Alzheimer’s Disease on the Pump Activity

Since dysregulation of intracellular calcium (Ca²⁺) levels is a common occurrence in neurodegenerative diseases, including Alzheimer’s disease (AD), the study of proteins that can correct neuronal Ca²⁺ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca²⁺-ATPase (PMCA), a high-affinity Ca²⁺ pump, is functionally impaired in AD and is inhibited by amyloid-β peptide (Aβ) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca²⁺-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), and other modulators of intracellular Ca²⁺ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aβ and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aβ and tau on the pump, and counteracts the neurotoxicity of Aβ and tau by interacting with them.     

Anticancer Ruthenium(III) Complex KP1019 Interferes with ATP‐Dependent Ca2+ Translocation by Sarco‐Endoplasmic Reticulum Ca2+‐ATPase (SERCA)

Sarco‐endoplasmic reticulum Ca²⁺‐ATPase (SERCA), a P‐type ATPase that sustains Ca²⁺ transport and plays a major role in intracellular Ca²⁺ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium‐based anticancer drugs, namely KP1019 (indazolium [trans‐tetrachlorobis(1H‐indazole)ruthenate(III)]), NAMI‐A (imidazolium [trans‐tetrachloro(1H‐imidazole)(S‐dimethylsulfoxide)ruthenate(III)]) and RAPTA‐C ([Ru(η⁶‐p‐cymene)dichloro(1,3,5‐triaza‐7‐phosphaadamantane)]), and cisplatin (cis‐diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid‐supported membrane was measured after ATP or Ca²⁺ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP‐dependent translocation of Ca²⁺ ions. An IC₅₀ value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca²⁺ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019.     

Calcium in Red Blood Cells—A Perilous Balance

Ca²⁺ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca²⁺ dependent signalling during differentiation from precursor cells. Intracellular Ca²⁺ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca²⁺ and a powerful Ca²⁺ pump maintains intracellular free Ca²⁺ levels between 30 and 60 nM, whereas blood plasma Ca²⁺ is approximately 1.8 mM. Thus, activation of Ca²⁺ uptake has an impressive impact on multiple processes in the cells rendering Ca²⁺ a master regulator in RBCs. Malfunction of Ca²⁺ transporters in human RBCs leads to excessive accumulation of Ca²⁺ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca²⁺ transport pathways allows harnessing Ca²⁺ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca²⁺ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.     

Preparation and phosphorus recovery performance of porous calcium–silicate–hydrate

Porous calcium–silicate–hydrate was synthesized and used to recover phosphorus from wastewater. The principal objective of this study was to explore the phosphorus recovery performance of porous calcium–silicate–hydrate prepared by different Ca/Si molar ratios. Phosphorus recovery mechanism was also investigated via Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive Spectrum (EDS), Brunauer–Emmett–Teller (BET) and X-ray Diffraction (XRD). The law of Ca²⁺ release was the key of phosphorus recovery performance. Different Ca/Si molar ratios resulted in the changes of pore structures. The increase of specific surface area and the increase in concentration of Ca²⁺ release were well agreement together. The Ca/Si molar ratio of 1.6 for porous calcium–silicate–hydrate is more proper to recover phosphorus. The pore structure of porous calcium–silicate–hydrate provided a local condition to maintain a high concentration of Ca²⁺ release. Porous calcium–silicate–hydrate could release a proper concentration of Ca²⁺ and OH^? to maintain the pH values at 8.5–9.5. This condition was beneficial to the formation of hydroxyapatite. Phosphorus content of porous calcium–silicate–hydrate reached 18.64% after phosphorus recovery.     

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD⁺ on: (1) the Ca²⁺-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca²⁺-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca²⁺-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca²⁺-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD⁺ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca²⁺-uptake and the free Ca²⁺ concentration in the medium. NAD(H) suppressed the Ca²⁺-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca²⁺-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.     

The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F_OF₁ ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F₁ sector, it makes stable contacts with both F₁ and the peripheral stalk, ensuring the structural and functional coupling between F_O and F₁, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²⁺ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²⁺-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death.     

The functioning of the lipids and lipoproteins of sarcotubular membranes in calcium transport

In the intact muscle cell, an internal tubular membrane system called the sarcoplasmic reticulum (SR) plays an important role in the contraction-relaxation cycle by controlling the Ca⁺⁺ of the myoplasm; release of Ca⁺⁺ from the SR to myoplasm initiates contractile activity and sequestring Ca⁺⁺ in the SR by means of a transport system causes muscle to relax. Fragments of the SR with a vesicular structure can be isolated from muscle homogenate and these vesicles are able to vigorously transport Ca⁺⁺ from incubation media into the intravesicular space thus enabling study of Ca⁺⁺ transport under precisely defined in vitro conditions. A highly purified fraction of SR vesicles called SF₁ were prepared from rat muscle by means of density gradient centrifugation procedures. The role of SR lipid in Ca⁺⁺ transport was studied. SF₁ was treated in vitro with either phospholipase A or C or D or polyene antibiotics. The effect of essential fatty acid deficiency, induced in vivo, was also investigated. It was concluded that the only structural feature of SF₁-lipid involved in Ca⁺⁺ transport and the associated adenosine triphosphatase is the phosphoryl moiety of the phospholipids. Evidence was obtained which inplicated histidine residues of the SF₁ protein in this transport function. To study the role of SF₁ protein in this process in depth, the membranes were solubilized by a sodium dodecylsulfate system and made free of their lipid components. More than 95% of this protein is soluble in dilute salt solution; of this, more than 90% is composed of a protein fraction which can be isolated by gel filtration (called protein fraction-2). Protein fraction-2 contains large molecular aggregates of small polypeptide subunits of identical or nearly identical molecular weight. They contain solely N-terminal glycine and probably only C-terminal alanine. The significance of such a high percentage of similar polypeptide subunits in SR is discussed. One of five papers to be published from the Symposium “Lipid Transport” presented at the AOCS Meeting, New Orleans, April 1970.     

Computational Analyses of the AtTPC1 (Arabidopsis Two-Pore Channel 1) Permeation Pathway

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca²⁺ permeability, a conclusion derived from relative permeability analyses using the Goldman–Hodgkin–Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca²⁺, but not for K⁺, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca²⁺ as demonstrated in in silico mutagenesis experiments. Such a Ca²⁺-specific coordination site in the pore explains contradicting data for the relative Ca²⁺/K⁺ permeability and strongly suggests that the Ca²⁺ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K⁺ but not Ca²⁺ through the open channel.     

The Orai Pore Opening Mechanism

Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca²⁺ concentrations. The primary source of Ca²⁺ entry into the cell is mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Its action is stimulated in response to internal Ca²⁺ store depletion. The fundamental constituents of CRAC channels are the Ca²⁺ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca²⁺-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1–Orai1 machinery.     

An Extended Empirical Valence Bond Model for Describing Proton Mobility in Water

In order to study the microscopic nature of the hydrated proton and its transport mechanism, we have introduced a multistate empirical valence bond model, fitted to ab initio results. This model was applied to the study, at low computational cost, of the structure and dynamics of an excess proton in liquid water. The quantum character of the proton is included by means of an effective parametrization of the model using preliminary path-integral calculations. The mechanism of proton transfer is interpreted as the translocation of a special O–H⁺–O bond along the hydrogen network, i.e., a series of reactions of the form H₅O₂⁺ + H₂O ⇌ H₂O + H₅O₂⁺, rather than H₃O⁺ + H₂O → H₂O + H₃O⁺ as usually described. The translocation of the special bond can be described as a diffusion process with a jump time of 1 ps. A time-dependent correlation function analysis of the special pair relaxation yields two timescales, 0.3 and 3.5 ps. The first time is attributed to the interconversion between a delocalized (H₅O₂⁺-like) and a localized (H₉O₄⁺-like) form of the hydrated proton within a given special pair. The second one is the relaxation time of the special pair, including return trajectories. The computed diffusion constant, as well as the isotopic substitution effect, are in good agreement with experiment. The hydration structure around the excess proton is discussed in terms of various radial distribution functions around the water molecules involved in the special pair and those in the first solvation shell. The hydrogen-bond-dynamics which accompanies the translocation process is studied statistically. The “Moses mechanism” proposed by Noam Agmon for proton mobility in water is partially verified by our simulations.     

Interaction of heparin with Ca2+: A model study with a synthetic heparin-like hexasaccharide

The binding of Ca²⁺ to synthetic hexasaccharide 1 , containing the structural motifs of the regular region of heparin, has been investigated using NMR spectroscopy and molecular modeling. The NMR data of the calcium salt of 1 indicate the existence of specific Ca²⁺ binding, and molecular modeling results predict three different types of binding sites with different negative potential and preorganized geometry. The presence of Ca²⁺ does not seem to affect the overall helical structure of hexasaccharide 1 , although it seems to have a marked influence on the flexibility of the oligosaccharide backbone.     

Ginsenoside Rd Attenuates Mitochondrial Permeability Transition and Cytochrome c Release in Isolated Spinal Cord Mitochondria: Involvement of Kinase-Mediated Pathways

Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. However, the effects of Rd on spinal cord mitochondrial dysfunction and underlying mechanisms are still obscure. In this study, we sought to investigate the in vitro effects of Rd on mitochondrial integrity and redox balance in isolated spinal cord mitochondria. We verified that Ca²⁺ dissipated the membrane potential, provoked mitochondrial swelling and decreased NAD(P)H matrix content, which were all attenuated by Rd pretreatment in a dose-dependent manner. In contrast, Rd was not able to inhibit Ca²⁺ induced mitochondrial hydrogen peroxide generation. The results of Western blot showed that Rd significantly increased the expression of p-Akt and p-ERK, but had no effects on phosphorylation of PKC and p38. In addition, Rd treatment significantly attenuated Ca²⁺ induced cytochrome c release, which was partly reversed by antagonists of Akt and ERK, but not p-38 inhibitor. The effects of bisindolylmaleimide, a PKC inhibitor, on Rd-induced inhibition of cytochrome c release seem to be at the level of its own detrimental activity on mitochondrial function. Furthermore, we also found that pretreatment with Rd in vivo (10 and 50 mg/kg) protected spinal cord mitochondria against Ca²⁺ induced mitochondrial membrane potential dissipation and cytochrome c release. It is concluded that Rd regulate mitochondrial permeability transition pore formation and cytochrome c release through protein kinases dependent mechanism involving activation of intramitochondrial Akt and ERK pathways.     

A study of the electrochemical oxidation of hydrogen peroxide on a platinum rotating disk electrode in the presence of calcium ions using Michaelis-Menten kinetics and binding isotherm analysis

The present work demonstrates a potential suppression in the electrochemical signal of H₂O₂ oxidation due to the presence of Ca²⁺ ions. A mechanistic scheme was proposed to include a reversible interaction of Ca²⁺ ions with either the electrode surface binding sites (competitive) or the complex sites (non-competitive). The degree of inhibition was inspected by evaluating the kinetic currents as a function of [Ca²⁺] applying Koutecky-Levich kinetics. These observations were further supported with models based on enzyme kinetics such as Michaelis-Menten model applying Lineweaver-Burk plot along with non-linear least-square fitting analysis. The experimental results suggests that the strength of the complex binding sites decreases considerably with increasing [Ca²⁺] and that a single H₂O₂ molecule is required to combine with one available active binding site.     

Adsorption from aqueous solution by δ-manganese dioxide I. Adsorption of the alkaline-earth cations

The adsorption isotherms of M²⁺ ions (M = Mg, Ca, Sr or Ba) were determined at pH 7.0 and at different temperatures. The adsorbent, δ-MnO₂, was converted to the K⁺ form prior to adsorption and about 1.5 mol K⁺ ions were released per mol of M²⁺ ions adsorbed. The adsorption capacity at a given temperature increased in the series: Mg²⁺ < Ca²⁺ ≦ Sr²⁺ < Ba²⁺. This was explained by an ion exchange mechanism between hydrated ions: K⁺ ions in the outer Helmholtz layer and M²⁺ ions in the bulk of the solution. The radii of the hydrated ions decreased in the series: Mg²⁺ > Ca²⁺ > Sr²⁺ > Ba²⁺. The adsorption of M²⁺ ions at pH values below the point of zero charge (pH 3.3) was significant for Mg²⁺ ions only. Although adsorption was not strictly reversible, the results fitted the Langmuir isotherm and ‘apparent heats of adsorption’, Q, were calculated. The endothermic heats (Q = 20,18, 11 and 5 kJ mol^?1 for Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ adsorption respectively) indicated positive entropy contributions which are expected for the adsorption mechanism suggested. The decrease in Q down the alkaline-earth group was correlated to the entropy effects and to the hydration numbers of the cations.     

Calcium ion modulates protein release from chitosan‐hyaluronic acid poly electrolyte gel

Polyelectrolyte complex (PEC) of chitosan (CH) and hyaluronic acid (HA) are widely used for skin, cartilage, and bone tissue engineering. However, no reports are seen on their response at high ionic media, like increased Ca²⁺ where they are likely to be exposed in the form of bone constructs and the influence of these ions on modulating the release of incorporated entities such as drugs and growth factors. Here, we prepared freeze dried scaffolds of PEC of CH and HA (CH‐HA) and characterized them by FTIR, TGA, SEM, and ESEM. FITC conjugated BSA, designated as FA, was incorporated into the PEC to study the release properties in response to Ca²⁺. The swellability of CH‐HA and the extent of drug release from the matrix, FA loaded CH‐HA was studied in deionised water and aqueous Na⁺ and Ca²⁺ solutions. Swelling and drug release were high for the matrix in aqueous Ca²⁺ whereas it was remarkably low in water and Na⁺. Drug released was found to increase with concentrations of Ca²⁺ (0.02–1.0M) indicating that CH‐HA is a promising matrix for Ca²⁺ responsive delivery of agents to accelerate healing of bone cracks, which is known to release high amount of Ca²⁺. POLYM. ENG. SCI., 55:2089–2097, 2015. © 2015 Society of Plastics Engineers     

Heavy metals in cement phases: on the solubility of Mg, Cd, Pb and Ba in Ca3Al2O6

The compounds formed when the divalent cations Mg²⁺, Cd²⁺, Ba²⁺ and Pb²⁺ are present during the preparation of Ca₃Al₂O₆ have been studied using X-ray microanalysis and diffraction methods. The smaller Mg cations are found to partially substitute for Ca²⁺, and structural refinements show that Mg preferentially occupies the smaller six-coordinate sites in Ca_3−xMg_xAl₂O₆. When Ba is present, it preferentially occupies the larger eight- and nine-coordinate sites. X-ray microanalysis suggests that Pb and Cd are lost from the samples during the preparation process. The diffraction patterns show a small decrease in the lattice parameters, suggesting that a defect structure of the type Ca_3−x(vac)_xAl₂O₆ is formed. The distribution of products formed on hydration of the doped Ca_3−xM_xAl₂O₆ is found to be very different than that observed for the undoped material.