Plasma Membrane Mg2 Research Articles

SLC41A1 Mg2+ transport is regulated via Mg2+-dependent endosomal recycling through its N-terminal cytoplasmic domain

SLC41A1 (solute carrier family 41, member A1) is a recently described vertebrate member of the MgtE family of Mg(2+) transporters. Although MgtE transporters are found in both prokaryotic and eukaryotic organisms, and are highly conserved, little is known about the regulation of their Mg(2+) transport function. In the present study, we have shown that endogenous SLC41A1 transporter expression is post-transcriptionally regulated by extracellular Mg(2+) in TRPM7 (transient receptor potential cation channel, subfamily M, member 7)-deficient cells, suggesting that SLC41A1 transporters underlie a novel plasma membrane Mg(2+) transport function. Consistent with this conclusion, structure-function analyses of heterologous SLC41A1 transporter expression demonstrate that SLC41A1 transporters exhibit the same plasma membrane orientation as homologous bacterial MgtE proteins, are capable of complementing growth of TRPM7-deficient cells only when the Mg(2+) transporting pore is intact, and require an N-terminal cytoplasmic domain for Mg(2+)-dependent regulation of lysosomal degradation and surface expression. Taken together, our results indicate that SLC41A1 proteins are a central component of vertebrate Mg(2+) transport systems, and that their Mg(2+) transport function is regulated primarily through an endosomal recycling mechanism involving the SLC41A1 N-terminal cytoplasmic domain.

Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development

Magnesium (Mg(2+)) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg(2+) transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg(2+) influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg(2+). Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg(2+) concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg(2+) or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg(2+) transport system.

SLC41A2 encodes a plasma-membrane Mg2+ transporter

The TRPM7 (transient receptor potential melastatin 7) ion channel has been implicated in the uptake of Mg2+ into vertebrate cells, as elimination of TRPM7 expression through gene targeting in DT40 B-lymphocytes renders them unable to grow in the absence of supplemental Mg2+. However, a residual capacity of TRPM7-deficient cells to accumulate Mg2+ and proliferate when provided with supplemental Mg2+ suggests the existence of Mg2+ uptake mechanism(s) other than TRPM7. Evaluation of the expression of several members of the SLC41 (solute carrier family 41) family, which exhibit homology with the MgtE class of prokaryotic putative bivalent-cation transporters, demonstrated that one, SLC41A2 (solute carrier family 41 member 2), is expressed in both wild-type and TRPM7-deficient DT40 cells. Characterization of heterologously expressed SLC41A2 protein indicated that it is a plasma-membrane protein with an N-terminus-outside/C-terminus-inside 11-TM (transmembrane)-span topology, consistent with its functioning as a trans-plasma-membrane transporter. In contrast with a previous report of ion-channel activity associated with SLC41A2 expression in oocytes, investigation of whole cell currents in SLC41A2-expressing DT40 cells revealed no novel currents of any type associated with SLC41A2 expression. However, expression of SLC41A2 in TRPM7-deficient cells under the control of a doxycycline-inducible promoter was able to conditionally enhance their net uptake of 26Mg2+ and conditionally and dose-dependently provide them with the capacity to grow in the absence of supplemental Mg2+, observations strongly supporting a model whereby SLC41A2 directly mediates trans-plasma-membrane Mg2+ transport. Overall, our results suggest that SLC41A2 functions as a plasma-membrane Mg2+ transporter in vertebrate cells.

Leishmania Plasma Membrane Mg2+-ATPase Is a H+/K+-Antiporter Involved in Glucose Symport: STUDIES WITH SEALED GHOSTS AND VESICLES OF OPPOSITE POLARITY

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.

A comparison of the effects of ?-ketotriazole, MCPA and carbaryl on the plasma membrane Mg2+-ATPase activity and lipid composition of rice shoots (Oryza sativa)

The effect of γ-ketotriazole, MCPA and carbaryl on the lipid composition and ATPase activity associated with plasma membrane fractions from rice (Oryza sativa cv Bahia) shoots was investigated. With γ-ketotriazole and MCPA treatments the relative amount of δ5-avenasterol (%) was reduced in the plasma membrane, whereas with γ-ketotriazole a reduction was also found in the sitosterol content, expressed as a percentage of the total free sterol composition. The fatty acyl chain length of phosphatidylcholine fractions from MCPA-treated plants was also reduced. The plasma membrane Mg2+-ATPase activity was only stimulated in MCPA-treated plants. No changes were observed in lipid composition or ATPase activity with carbaryl treatment.

Control of the transmembrane phospholipid distribution in eukaryotic cells by aminophospholipid translocase.

The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport of phospholipid takes place in some organelles such as chromaffin granules. On the other hand, the phospholipid flippase of rat liver endoplasmic reticulum does not require ATP and has a low lipid specificity. The biological implications of these phospholipid flippases are discussed.

Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae.

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.

Covalent phosphorylation of the Mg2+-dependent ATPase of yeast plasma membranes.

Phosphorylation by [gamma-32P]ATP of proteins associated with the plasma membrane of Saccharomyces cerevisiae has been studied both in vivo and in vitro. Although at least nine proteins are labeled in vivo, there is only one major protein labeled in vitro. This species with an apparent molecular weight of 114,000 has been identified as the plasma membrane Mg2+-ATPase. Phosphorylation of this enzyme occurs exclusively on serine residues. This is the first report that the proton-translocating ATPase of fungal plasma membranes is subject to phosphorylation by a protein kinase.

Potassium transport coupled to ATP hydrolysis in reconstituted proteoliposomes of yeast plasma membrane ATPase.

Potassium transport coupled to ATP hydrolysis has been reconstituted in proteoliposomes using a highly purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe. The ATPase activity in the incorporated enzyme was strongly stimulated (2.2-fold) by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP). The H+/K+ exchanger nigericin (in the presence of K+) stimulated 1.6-fold the ATPase activity. When both ionophores were added together, the stimulation was increased up to 2.7-fold. When a potassium concentration gradient (high K+ in) was applied to the proteoliposome membrane, a significant drop in the CCCP-stimulated ATPase activity was observed. Inversion of the K+ concentration gradient (high K+ out) did not decrease the stimulation by CCCP. High Na+ in also decreased the stimulation induced by CCCP in the absence but not in the presence of external K+. However, high Li+ in had no effect. Direct potassium efflux from the proteolyposomes was detected upon addition of MgATP using a selective K+ electrode. The ATP-dependent potassium efflux was abolished in CCCP and/or nigericin-pretreated proteoliposomes. However, during steady state ATP hydrolysis, a transient and small K+ efflux was observed upon addition of a CCCP pulse. I propose that the plasma membrane Mg2+-dependent ATPase in yeast cells not only carries out electrogenic H+ ejection but also drives the uptake of potassium via a voltage-sensitive gate which is closed in the absence and open in the presence of the membrane potential.

Electrogenic proton translocation coupled to ATP hydrolysis by the plasma membrane Mg2+-dependent ATPase of yeast in reconstituted proteoliposomes.

The purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe was incorporated in liposomes using a cholate-dialysis method. The ATPase activity of the incorporated enzyme was stimulated by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone and to a much lower extent of the K+-ionophore valinomycin in the presence of potassium. The K+/H+ exchanger nigericin (plus K+) did not stimulate ATPase activity, whereas the combined addition of both nigericin plus valinomycin was strongly stimulatory. The incorporated ATPase activity was controlled by the generated electrochemical H+ gradient since only conditions which collapse both the membrane potential and the pH gradient stimulated fully the ATPase activity of the incorporated enzyme. Direct measurement of proton movement with a pH glass electrode showed a fast and transient proton entry into the proteoliposomes upon addition of MgATP in the presence of the charge-compensating cation K+ (plus valinomycin). Moreover, during the steady state ATP hydrolysis, a H+ entry was again observed when the membrane potential was collapsed upon addition of valinomycin in the presence of K+. These data demonstrate that the plasma membrane ATPase of yeast cells is involved in electrogenic H+ translocation coupled to ATP hydrolysis since the purified enzyme incorporated in the liposomes is virtually free of mitochondrial F1F0-ATPase contaminant.

Calcium ion-dependent adenosine triphosphatase activity and plasma-membrane phosphorylation in the human neutrophil

A plasma-membrane fraction was isolated from a post-nuclear extract of human neutrophils by centrifugation through a linear sucrose density gradient. This fraction exhibited a Ca2+-dependent adenosine triphosphatase (ATPase) activity that could be differentiated from mitochondrial or myosin ATPase and from plasma-membrane Mg2+-dependent ATPase. When assayed in the presence of [gamma-32P]ATP, the Ca2+-dependent ATPase reaction resulted in the formation of an acid-resistant hydroxylamine-sensitive bond between the gamma-[32P] phosphate group and a membrane protein subunit with an apparent mol.wt. of 135000. Half-maximal activating effect of Ca2+ was found at 82nM and 0.18 microM for the ATPase and the formation of the 32P-membrane complex respectively. Generation of the phosphorylated product attained the steady state at 0 degrees C by about 30s, and was rapidly reversed by ADP. These results suggest that the Ca2+-activated ATPase reaction occurs through the formation of a phosphoprotein intermediate, similar to that described for some Ca2+-dependent ATPase enzymes associated with Ca2+ transport. The possibility thus exists that the neutrophil Ca2+-dependent ATPase catalyses a process of Ca2+ extrusion from the cell, thereby participating in the regulation of several Ca2+-dependent neutrophil functions.

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.

The Stimulation of Adipocyte Plasma Membrane Magnesium Ion-stimulated Adenosine Triphosphatase by Insulin and Concanavalin A

SUMMARY These studies were designed to determine whether insulin affected the ATPase system of the adipocyte plasma mem- brane. The hormone was found to stimulate the Mg2+- ATPase significantly at concentrations as low as 5 microunits per ml and reaching maximum stimulation between 50 to 100 microunits per ml. The effect occurred at the earliest time point measured (2.0 min) and was linear over a 20-min incubation. The maximal mean dBerence in specific activity caused by 100 microunits of insulin per ml was 1.10 f 0.11 pmoles of Pi per mg of protein per 10 min in 25 experiments and was a 12.1% increase over the basal mean specific activity of 9.82 f 0.38. Insulin did not stimulate the sodium and potassiti ion-activated Mg2+ATPase of the plasma membrane nor the Mg2+ATPase of either the adipo- cyte mitochondrial or microsomal fractions. Epinephrine had no effect on the plasma membrane Mg2+ATPase. Pre- incubation of adipocytes with insulin prior to preparation of the plasma membranes or preincubation of the membranes prior to assay did not prevent the insulin stimulation of the Mg2+ATPase. The basal enzyme activity of insulin-pre- treated membranes or of membranes from insulin-pre- treated cells was signi&antly lower than the corresponding controls. Oligomycin and sodium azide significantly lowered the basal Mg2+ATPase specific activity and prevented the insulin stimulation of the enzyme but neither agent blocked the binding of insulin to the plasma membranes. Con- canavalin A mimicked the effect of insulin on the plasma membrane Mg2+ATPase but produced a greater magnitude of change and could overcome the inhibition caused by oligo- mycin and sodium azide. The data suggest that insulin stimulates the Mg2+ATPase after interacting with the mem- brane receptor but the role of this phenomenon in the mech- anism of insulin action remains to be determined. Insulin is known to cause a set

Chromium occurrence and function in biological systems.

An extensive review of the biogeochemistry of chromium is presented. Topics dealt with in detail include: the chemistry of chromium, its occurrence in nature, chromium's interactions with proteins, nucleic acids, bacteria, yeasts and red blood cells, the biochemical aspects of low-chromium states, the effects of chromium on life span, growth, and survival, the mode and site of action in biological systems, the metabolism (including absorption, distribution, excretion, and toxicology) the effects of chromium's chemical form on biological activity, chromium nutrition in man, and the effects of chromium supplementation in man). 265 references, 12 figures, 8 tables.