Tissue magnesium loss during prolonged hypokinesia in magnesium supplemented and unsupplemented rats

This study aims at showing that during hypokinesia (HK) tissue magnesium (Mg2+) content decreases more with higher Mg2+ intake than with lower Mg2+ intake and that Mg2+ loss increases more with higher than lower tissue Mg2+ depletion due to inability of the body to use Mg2+ during HK. Studies were conducted on male Wistar rats during a pre-HK period and a HK period. Rats were equally divided into four groups: unsupplemented vivarium control rats (UVCR), unsupplemented hypokinetic rats (UHKR), supplemented vivarium control rats (SVCR) and supplemented hypokinetic rats (SHKR). SVCR and SHKR consumed 42 mEq Mg2+ per day. The gastrocnemius muscle and right femur bone Mg2+ content decreased significantly, while plasma Mg2+ level and urine and fecal Mg2+ loss increased significantly in SHKR and UHKR compared with their pre-HK values and their respective vivarium controls (SVCR and UVCR). However, muscle and bone Mg2+ content decreased more significantly and plasma Mg2+ level, and urine and fecal Mg2+ loss increased more significantly in SHKR than in UHKR. The greater tissue Mg2+ loss with higher Mg2+ intake and the lower tissue Mg2+ loss with lower Mg2+ intake shows that the risk of higher tissue Mg2+ depletion is directly related to the magnitude of Mg2+ intake. The higher Mg2+ loss with higher tissue Mg2+ depletion and the lower Mg2+ loss with lower Mg2+ tissue depletion shows that the risk of greater Mg2+ loss is directly related to the magnitude of tissue Mg2+ depletion. It was concluded that tissue Mg2+ depletion increases more when the Mg2+ intake is higher and that Mg2+ loss increases more with higher than lower tissue Mg2+ depletion indicating that during prolonged HK the tissue Mg2+ depletion is not due to the Mg2+ shortage in food but to the inability of the body to use Mg2+.     

An ATP-dependent Na+/Mg2+ countertransport is the only mechanism for Mg extrusion in squid axons

The components of magnesium efflux in squid axons have been studied under internal dialysis and voltage clamp conditions. The present report rules out the existence of an ATP-dependent, Nao- and Mgo-independent Mg2+ efflux (ATP-dependent Mg2+ pump) leaving the Mg2+-Na+ exchange system as the only mechanism for Mg2+ extrusion. The main features of the Mg2+ efflux are: (1) The efflux is completely dependent on ATP. (2) The efflux can be activated either by external Na+ (forward Mg2+-Na+ exchange) or external Mg2+ (Mg2+-Mg2+ exchange). (3) The mobility of the Mg2+ exchanger in the Na+o-loaded form is greater than that in the Mg2+-loaded one. (4) In variance with the Na+-Ca2+ exchange mechanism, Mg2+-Mg2+ exchange is not activated by external monovalent cations. (5) ATP gamma S replaces ATP in activating Mg2+-Na+ exchange suggesting that a phosphorylation/dephosphorylation process regulates this transport mechanism.     

Characterization of Na+/Mg2+ antiport by simultaneous 28Mg2+ influx

During net Mg2+ efflux from Mg2+-preloaded chicken erythrocytes, which occurs via Na+/Mg2+ antiport, 28Mg2+ is taken up intracellularly. Km of 28Mg2+ influx amounted to 1 mM. In Na+-free medium Vmax of 28Mg2+ influx was increased and Km was reduced to 0.2 mM. 28Mg2+ influx was noncompetitively inhibited by amiloride as was found for Na+/Mg2+ antiport. The results indicate that, extracellularly, Mg2+ can compete with Na+ for common binding sites of the Na+/Mg2+ antiporter, resulting in 28Mg2+-24Mg2+ exchange. The rate of Mg2+ exchange depends on extracellular Na+ and on the rate of net Mg2+ efflux.     

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.     

The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.     

Characterization of Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes

Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes was determined from the increase of extracellular Mg2+ concentration or decrease of intracellular Mg2+ content, as measured by means of atomic absorption spectrophotometry. Mg2+ efflux was specifically combined with the uptake of Na+ at a stoichiometric ratio of 2Na+:1Mg2+, indicating electroneutral Na+/Mg2+ antiport. Na+/Mg2+ antiport depended on intracellular ATP and was inhibited by amiloride and quinidine, but was insensitive to strophanthin. Net Mg2+ efflux was only occurring at increased concentration of intracellular Mg2+ ([Mg2+]i), and stopped when the physiological Mg2+ content was reached. Intracellular Mg2+ acted cooperatively with a Hill coefficient of 2.4, which may indicate gating of Na+/Mg2+ antiport at increased [Mg2+]i. At increased intracellular Na+ concentration, Na+ competed with intracellular Mg2+ for Mg2+ efflux and Na+ could leave the rat erythrocyte via this transport system. Na+/Mg2+ antiport was working asymmetrically with respect to extra- and intracellular Na+ and Mg2+, and did not perform net Mg2+ uptake.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.     

Quantitation of cation binding to wheat germ ribosomes: influences on subunit association equilibria and ribosome activity.

The binding of Mg2+, spermine, and spermidine to wheat germ ribosomes was quantitated following equilibrium dialysis. The Mg2+ binding data demonstrate that Mg2+ and K+ compete for binding to the ribosomes. Mg2+ binding saturates at approximately 0.56 positive charges per phosphate (+/P). The Mg2+, spermine and spermidine binding data indicate that either polyamine replaces Mg2+ upon binding to the ribosomes. Mg2+ and polyamine binding combined saturates at approximately 0.29 +/P under the conditions reported. When a critical number of Mg2+ ions are replaced by either polyamine, the activity of the ribosomes falls dramatically. Ribosomal subunit association increases with the degree of phosphate charge neutralization due to the binding of Mg2+. Total charge neutralization during subunit association by Mg2+ and polyamine binding combined, is much less than that achieved by Mg2+ alone.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Activity changes of three nucleolytic enzymes during the life cycle of Saccharomyces cerevisiae

Conditions were established for the assay of three nucleolytic enzymes: a Mg2+-independent endoribonuclease, a Mg2+-dependent endonuclease, and a Mg2+-dependent 5'-exonuclease in Saccharomyces cerevisiae cell extracts. The changes in the activities of these enzymes were determined throughout the life cycle of the organism. As the cells progressed from the exponential to the stationary growth phase, the specific activities of the Mg2+-independent endoribonuclease and of the Mg2+-dependent 5'-exonuclease increased, whereas the Mg2+-dependent endonuclease decreased. During sporulation the Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease increased several-fold over the first 10 h, but, since a similar increase was seen in nonsporulating control cells, the increases did not appear to be related to sporulation. However, the specific activity of the Mg2+-dependent endonuclease showed a sporulation-related increase during the first 3 h of sporulation, with a subsequent decline to very low levels. The specific activity of this enzyme increased again during germination to the levels seen in exponential phase cells. The Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease showed little change during germination of the ascospores. The high specific activity of the Mg2+-independent endoribonuclease during periods of nutrient deprivation is in agreement with the proposed role for this enzyme in the degradation of rRNA under these conditions.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Kinetics of Mg2+ flux into rat liver mitochondria.

Unidirectional fluxes of Mg2+ across the limiting membranes of rat liver mitochondria have been measured in the presence of the respiratory substrate succinate by means of the radioisotope 28Mg. Rates of both influx and efflux of Mg2+ are decreased when respiration is inhibited. A linear dependence of the reciprocal of the Mg2+ influx rate on the reciprocal of the Mg2+ concentration is observed. The apparent Km for Mg2+ averages about 0.7 mM. N-Ethyl-maleimide, an inhibitor of transmembrane phosphate-hydroxyl exchanges, enhances the observed pH dependence of Mg2+, influx. In the presence of MalNEt, the apparent Vmax of Mg2+ influx is greater at pH 8 than at pH 7, and there is a linear dependence of the Mg2+ influx rate on the external OH- concentration. The K+ analogue Tl+ inhibits Mg2+ influx, while La3+, an inhibitor of mitochondrial Ca2+ transport, has no effect on Mg2+ influx. Mg2+ competitively inhibits the flux of K+ into rat liver mitochondria. The mechanism(s) mediating mitochondrial Mg2+ and K+ fluxes appear to be similar in their energy dependence, pH dependence, sensitivity to Tl+, and insensitivity to La3+.     

Matrix free Mg2+ changes with metabolic state in isolated heart mitochondria

The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored by using the fluorescent probe furaptra (mag-fura-2). Beef heart mitochondria respiring in a KCl medium in the absence of external Mg2+ maintain free matrix Mg2+ near 0.50 mM. Addition of Pi under these conditions decreases free Mg2+ by 0.12-0.17 mM depending on the substrate. This decrease in free Mg2+ appears to reflect changing ligand availability in the matrix. The decrease is prevented when the Pi transporter is blocked by mersalyl. Addition of ADP to initiate state 3 respiration causes a marked increase in free matrix Mg2+ (0.1-0.2 mM) that persists as long as ATP formation is taking place; free Mg2+ then returns to the base level. This cyclic change is blocked by oligomycin and carboxyatractyloside and appears to reflect to a large extent the decrease in matrix Pi that accompanies oxidative phosphorylation. Exchange of external ADP for matrix ATP may also contribute to the increase in free matrix Mg2+. Addition of an uncoupler promotes anion efflux and increases free matrix Mg2+. Similar changes in free Mg2+ on addition of Pi, ADP, or uncoupler are seen when extramitochondrial Mg2+ is buffered from 0.5 to 2 mM, but the basal free matrix Mg2+ increases as external Mg2+ concentration increases in this range. Free matrix Mg2+ also increases when total mitochondrial Mg2+ is increased by respiration-dependent uptake in the presence of Pi. It is concluded that matrix free Mg2+ changes significantly with changing ligand availability and that such changes may contribute to the regulation of Mg2(+)-sensitive matrix enzymes and membrane transporters.     

Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5 degrees, 15 degrees, and 25 degrees C. The two sites in a molecule were equivalent on Mg2(+)-Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25 degrees C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin on Ca2+ and Mg2+ binding and Mg2(+)-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236-2240]. Although no major conformational changes were noted between Ca2(+)- and Mg2(+)-bound forms of toad parvalbumin, the conformational difference was larger in Ca2+ (or Mg2+) binding to site 1 than site 2. This may indicate that the metal-free form is much less stable than any form with Ca2+ (or Mg2+) bound at one site at least. On Mg2(+)-Ca2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former indicates the sequestering of nonpolar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2(+)-Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2(+)-Ca2+ exchange.     

PIP2 activates TRPV5 and releases its inhibition by intracellular Mg2+

The transient receptor potential type V5 channel (TRPV5) is a Ca2+-selective TRP channel important for epithelial Ca2+ transport. Intracellular Mg2+ causes a fast voltage-dependent block of the TRPV5 channel by binding to the selectivity filter. Here, we report that intracellular Mg2+ binding to the selectivity filter of TRPV5 also causes a slower reversible conformational change leading to channel closure. We further report that PIP2 activates TRPV5. Activation of TRPV5 by PIP2 is independent of Mg2+. Yet, PIP2 decreases sensitivity of the channel to the Mg2+-induced slow inhibition. Mutation of aspartate-542, a critical Mg2+-binding site in the selectivity filter, abolishes Mg2+-induced slow inhibition. PIP2 has no effects on Mg2+-induced voltage-dependent block. Thus, PIP2 prevents the Mg2+-induced conformational change without affecting Mg2+ binding to the selectivity filter. Hydrolysis of PIP2 via receptor activation of phospholipase C sensitizes TRPV5 to the Mg2+-induced slow inhibition. These results provide a novel mechanism for regulation of TRP channels by phospholipase C-activating hormones via alteration of the sensitivity to intracellular Mg2+.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.     

Intracellular and extracellular concentrations of Na+ modulate Mg2+ transport in rat ventricular myocytes

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25 degrees C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24 mM Mg2+ either in the presence of 106 mM Na+ plus 1 mM ouabain (Na+ loading) or in the presence of only 1.6 mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140 mM Na+ and 1 mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31 mM and 47 mM after Na+ loading for 1 and 3 h, respectively, and decreased to approximately 0 mM after 3 h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at approximately 40 mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130 mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.