Monika Schweigel

Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria

Steady‐state concentrations of mitochondrial Mg2+ previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg2+ transport protein CorA suggested a role for Mrs2p in Mg2+ influx into the organelle, other functions in cation homeostasis could not be excluded. Making use of the fluorescent dye mag‐fura 2 to measure free Mg2+ concentrations continuously, we describe here a high capacity, rapid Mg2+ influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Δψ and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases influx rates 5‐fold, while the deletion of the MRS2 gene abolishes this high capacity Mg2+ influx. Mg2+ efflux from isolated mitochondria, observed with low Δψ only, also requires the presence of Mrs2p. Cross‐linking experiments revealed the presence of Mrs2p‐containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo‐ oligomers. Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.

Pathophysiology of Grass Tetany and other Hypomagnesemias: Implications for Clinical Management

Magnesium is an essential mineral with many physiologic and biochemical functions. Surprisingly, Mg homeostasis is not regulated by a hormonal feedback system, but simply depends on inflow (absorption) from the gastrointestinal tract and outflow (endogenous secretion, requirement for milk production, uptake by tissues). Any surplus (inflow greater than outflow) is excreted via urine. Conversely, if the outflow (mainly milk secretion and endogenous loss) exceeds inflow, hypomagnesemia occurs because of the lack of hormonal mechanisms of homeostasis. The major reason for insufficient inflow is a reduced absorption of Mg from the forestomachs. Recent studies from our laboratory and data from the literature permit the proposal of a putative transport model for the secondary active transport of Mg across the rumen epithelium. This model includes two uptake mechanisms across the luminal membrane (PD-dependent and PD-independent) and basolateral extrusion via a Na/Mg exchange. The well-known negative interaction between ruminal K concentration and Mg absorption can be explained on the basis of this model: an increase of ruminal K depolarizes the potential difference of the luminal membrane, PDa, and as the driving force for PD-dependent (or K-sensitive) Mg uptake. Because Na deficiency causes an increase of K concentration in saliva and ruminal fluid, Na deficiency should be considered a potentially important risk factor. The data obtained from in vitro and in vivo studies on the association of Mg transport, changes of ruminal K concentration, and PDa are extensive and confirm the model, if the ruminal Mg concentrations are below 2 to 3 mM. It is further proposed by the model that the PD-independent Mg uptake mechanism is primarily working at high ruminal Mg concentration (above 2 mM). Mg absorption becomes more and more independent of ruminal K with increasing Mg concentration, which can be considered as an explanation for the well-known prophylaxis of hypomagnesemia by increasing oral Mg intake. Fermentation products, NH4+ and SCFA, influence Mg absorption. The possible meaning regarding the pathogenesis of hypomagnesemia is not quite clear. A sudden increase of ruminal NH4+ should be avoided, because high NH4+ concentrations transiently reduce Mg absorption. The most prominent signs of hypomagnesemia are excitations and muscle cramps, which are closely correlated with the Mg concentration in the CSF. It is suggested that the clinical signs are caused by spontaneous activation of neurons in the CNS at low Mg concentrations, which leads to tetany. Prophylactic measures are discussed in context with the known effects on ruminal Mg absorption.

SLC41A1 Is a Novel Mammalian Mg2+ Carrier

The molecular biology of mammalian magnesium transporters and their interrelations in cellular magnesium homeostasis are largely unknown. Recently, the mouse SLC41A1 protein was suggested to be a candidate magnesium transporter with channel-like properties when overexpressed in Xenopus laevis oocytes. Here, we demonstrate that human SLC41A1 overexpressed in HEK293 cells forms protein complexes and locates to the plasma membrane without, however, giving rise to any detectable magnesium currents during whole cell patch clamp experiments. Nevertheless, in a strain of Salmonella enterica exhibiting disruption of all three distinct magnesium transport systems (CorA, MgtA, and MgtB), overexpression of human SLC41A1 functionally substitutes these transporters and restores the growth of the mutant bacteria at magnesium concentrations otherwise non-permissive for growth. Thus, we have identified human SLC41A1 as being a bona fide magnesium transporter. Most importantly, overexpressed SLC41A1 provide HEK293 cells with an increased magnesium efflux capacity. With outwardly directed Mg2+ gradients, a SLC41A1-dependent reduction of the free intracellular magnesium concentration accompanied by a significant net decrease of the total cellular magnesium concentration could be observed in such cells. SLC41A1 activity is temperature-sensitive but not sensitive to the only known magnesium channel blocker, cobalt(III) hexaammine. Taken together, these data functionally identify SLC41A1 as a mammalian carrier mediating magnesium efflux.

Mg2+ transport in sheep rumen epithelium: evidence for an electrodiffusive uptake mechanism

The potential difference (PD)-dependent component of transcellular Mg(2+) uptake in sheep rumen epithelium was studied. Unidirectional (28)Mg(2+) fluxes were measured at various transepithelial PD values, and the unidirectional mucosal-to-serosal (28)Mg(2+) flux (J(Mg)(ms)) was correlated with the PD across the apical membrane (PD(a)) determined by mucosal impalement with microelectrodes. PD(a) was found to be -54 +/- 5 mV, and J(Mg)(ms) was 65.9 +/- 13.8 nmol. cm(-2). h(-1) under short-circuit conditions. Hyperpolarization of the ruminal epithelium (blood-side positive) depolarized PD(a) and, most noticeably, decreased J(Mg)(ms). Further experiments were performed with cultured ruminal epithelial cells (REC). With the aid of the fluorescence probe mag-fura 2, we measured the intracellular free Mg(2+) concentration ([Mg(2+)](i)) of isolated REC under basal conditions at various extracellular Mg(2+) concentrations ([Mg(2+)](e)) and after alterations of the transmembrane voltage. Basal [Mg(2+)](i) was 0.54 +/- 0.08 mM. REC suspended in media with [Mg(2+)](e) between 0.5 and 7.5 mM showed an increase in [Mg(2+)](i) that was dependent on [Mg(2+)](e) and that exhibited a saturable component (Michaelis-Menten constant = 1.2 mM; maximum [Mg(2+)](i) = 1.26 mM). Membrane depolarization with high extracellular K(+) (40, 80, or 140 mM K(+)) and the K(+) channel blocker quinidine (50 and 100 microM ) resulted in a decrease in [Mg(2+)](i). On the other hand, hyperpolarization created by K(+) diffusion (intracellular K(+) concentration > extracellular K(+) concentration) in the presence of valinomycin induced a 15% increase in [Mg(2+)](i). None of the manipulations had any effect on intracellular Ca(2+) concentration and intracellular pH. The results support the assumption that the membrane potential acts as a principal driving force for Mg(2+) entry in REC and suggest that the pathway for this electrodiffusive Mg(2+) uptake across the luminal membrane is a channel or a carrier.The potential difference (PD)-dependent component of transcellular Mg2+uptake in sheep rumen epithelium was studied. Unidirectional28Mg2+fluxes were measured at various transepithelial PD values, and the unidirectional mucosal-to-serosal28Mg2+flux ([Formula: see text]) was correlated with the PD across the apical membrane (PDa) determined by mucosal impalement with microelectrodes. PDa was found to be -54 ± 5 mV, and [Formula: see text] was 65.9 ± 13.8 nmol ⋅ cm-2 ⋅ h-1under short-circuit conditions. Hyperpolarization of the ruminal epithelium (blood-side positive) depolarized PDa and, most noticeably, decreased [Formula: see text]. Further experiments were performed with cultured ruminal epithelial cells (REC). With the aid of the fluorescence probe mag-fura 2, we measured the intracellular free Mg2+ concentration ([Mg2+]i) of isolated REC under basal conditions at various extracellular Mg2+ concentrations ([Mg2+]e) and after alterations of the transmembrane voltage. Basal [Mg2+]iwas 0.54 ± 0.08 mM. REC suspended in media with [Mg2+]ebetween 0.5 and 7.5 mM showed an increase in [Mg2+]ithat was dependent on [Mg2+]eand that exhibited a saturable component (Michaelis-Menten constant = 1.2 mM; maximum [Mg2+]i= 1.26 mM). Membrane depolarization with high extracellular K+ (40, 80, or 140 mM K+) and the K+ channel blocker quinidine (50 and 100 μM ) resulted in a decrease in [Mg2+]i. On the other hand, hyperpolarization created by K+ diffusion (intracellular K+ concentration > extracellular K+ concentration) in the presence of valinomycin induced a 15% increase in [Mg2+]i. None of the manipulations had any effect on intracellular Ca2+ concentration and intracellular pH. The results support the assumption that the membrane potential acts as a principal driving force for Mg2+ entry in REC and suggest that the pathway for this electrodiffusive Mg2+ uptake across the luminal membrane is a channel or a carrier.

Splice-variant 1 of the ancient domain protein 2 (ACDP2) complements the magnesium-deficient growth phenotype of Salmonella enterica sv. typhimurium strain MM281

Evidence arguing for the existence of genes encoding for proteins directly involved in the transport of Mg2+ through the cytoplasmic membrane have accumulated over the last few years. Gene ACDP2 (ancient conserved domain protein 2; old name CNNM2, cyclin M2) is one such gene. ACDP2 is a distant homologue of the bacterial gene corC, which is known to be involved in cobalt resistance. We have previously demonstrated that the over-expression of the human Mg2+ carrier SLC41A1 partly complements the Mg2+-dependent growth deficiency of Salmonella strain MM281 (triple disruptant in genes: mgtA, mgtB and corA) cultivated in media containing growth non-permissive [Mg2+]e. We have used the same approach to examine whether over-expressed human ACDP2 has a similar efficacy to complement growth deficiency of the MM281 strain in media containing growth non-permissive [Mg2+]e. Two splicing variants of the ACDP2 gene have been tested. Here, we show that over-expressed isomorph 1 is efficient in restoring growth of the MM281 strain in media containing growth non-permissive [Mg2+]e, whereas isomorph 2 is not. Therefore, we conclude that ACDP2sp.v.1 is a functional Mg2+-transporting entity per se. Our conclusion is supported by the measurable Mg2+ influx seen in MM281 bacteria over-expressing ACDP2sp.v.1 but not in MM281 bacteria over-expressing ACDP2sp.v.2 or in cells transformed with the empty vector.

Cellular Mg2+ Transport and Homeostasis: An Overview

Magnesium plays a vital role as a cofactor for many enzymes, as a binding partner of nucleotides, and in stabilizing nucleic acids and membranes. It acts as a modulator of ion channels, and it affects many other cellular processes such as neuromuscular excitability, secretion of hormones, and it antagonizes the actions of Ca2+, to name a few effects.1–4 Mg2+ deficiency was found to be associated with hypertension, ischemic heart disease, infl ammation, eclampsia, diabetes, cystic fibrosis, and in the establishment of human immunodeficiency virus 1 (HI V-1) reservoirs.5–10 Several disease phenotypes have been shown to be due to inherited disorders of Mg2+ homeostasis.11–15 Therefore, the regulation of extracellular and intracellular magnesium levels by transmembrane and transepithelial transport processes is critical for numerous cellular and organ functions.

The vacuolar-type H+-ATPase in ovine rumen epithelium is regulated by metabolic signals.

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H+-ATPase (vH+-ATPase) activity was investigated. Using fluorescent spectroscopy, basal pHi of REC was measured to be 7.3 ± 0.1 in HCO3−-free, glucose-containing NaCl medium. MI induced a strong pHi reduction (−0.44 ± 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A (−0.30 ± 0.03 versus −0.21 ± 0.03 pH units). Treatment with foliomycin, a specific vH+-ATPase inhibitor, decreased REC pHi by 0.21 ± 0.05 pH units. After MI induction, this effect was nearly abolished (−0.03 ± 0.02 pH units). In addition, membrane-associated localization of vH+-ATPase B subunit disappeared. Metabolic control of vH+-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability.