Functional and molecular characterization of proteins involved in Mg2+ and K+ homeostasis

Transportprozesse durch Zellmembranen sind ein herausforderndes und faszinierendes Forschungsgebiet. Mrs2p und sein bakterielles Homolog CorA gehören zu einer großen Familie von Mg2+ Transportern. Charakteristische Eigenschaften der Mitglieder dieser Familie sind zwei Transmembrandomänen (TMs) im C-terminalen Bereich der Proteine und das hochkonservierte Motiv G-M-N am Ende der TM 1. Um weitere Einblicke in die molekularen Mechanismen der Regulation dieser Transporter zu bekommen, wurden Mutationsanalysen an Mitgliedern der CorA/MRS2 Familie durchgeführt. Bei CorA von Thermotoga maritima konnte die Schlüsselrolle der Aminosäure Leu294 als Schleuse des Kanals bestätigt werden. Mutationen an dieser Position haben eine starke Auswirkung auf die Fähigkeit des Ionenkanals die Pore zu schließen und sind letal für die Zelle. Im Gegensatz dazu deuten unsere Ergebnisse mit MRS2 darauf hin, dass die Regulation des Ionentransports in den eukaryotischen Vertretern der Familie wesentlich komplexer ist. Wir haben eine vergleichende Strukturanalyse der Kristallstrukturen von T. maritima CorA und der N-terminalen Domäne von Saccharomyces cerevisiae Mrs2p durchgeführt. Es konnten Aminosäuren identifiziert werden, die höchstwahrscheinlich am Schließen des Kanals beteiligt sind. Unsere Ergebnisse zeigen, dass Met309 in Mrs2p und Leu294 in CorA eine gleichwertige Rolle in der Regulation des Ionenflusses spielen. Mutationen an dieser Position führen aber nicht zu einer vollständigen Deregulierung des Schließmechanismus, wie es bei Leu294 von CorA beobachtet wurde. Eine zweite Schleuse (von Val315 gebildet) wurde ebenfalls identifiziert - scheint aber für die Kontrolle des Ionentransports eine untergeordnete Rolle zu spielen. Durch strukturbasierende Sequenzanalyse konnte auch eine potentielle Kationenbindungsstelle identifiziert werden, die als Messfühler der intramitochondrialen Mg2+ Konzentration fungiert. Die Beteiligung der hochkonservierten Aminosäure Asp97 an der Bildung einer Kationenbindungsstelle konnte experimentell aber nicht eindeutig gezeigt werden. Zusätzlich wurde das hochkonservierte G-M-N Motiv von Mrs2p mittels Zufallsmutagenese analysiert. In früheren Arbeiten wurde gezeigt, dass sogar konservative Substitutionen einzelner Aminosäuren in diesem Motiv nicht toleriert werden und die Transportaktivität von CorA und Mrs2p stark minimieren. Eine Serie von Mutanten zeigt aber noch immer Transportaktivität, obwohl die Sequenzen überraschend stark von der Sequenz G-M-N abweichen. Die Selektivität dieser Mutanten für Mg2+ war aber reduziert. Diese Ergebnisse lassen darauf schließen, dass das G-M-N Motiv eine zentrale Rolle für die Substratselektivität der CorA/Mrs2p Familie spielt. In der vorliegenden Arbeit wurde auch die Funktion von Lpe10p, dem Homolog von Mrs2p in Hefe, genauer untersucht. Die Deletion von LPE10 oder MRS2 führt in beiden Fällen zu einem Wachstumsdefekt auf nicht-fermentierbaren Kohlenstoffquellen. Im Gegensatz zur Deletion von MRS2 führt die Deletion von LPE10 aber zusätzlich zu einer starken Reduktion des mitochondrialen Membranpotentials. Lpe10p alleine kann keinen Mg2+ selektiven Ionenkanal bilden, es bildet aber Heterooligomere mit Mrs2p, die zu einer Reduktion der Leitfähigkeit des Mrs2p Kanals führen. Unsere Ergebnisse zeigen, dass die Interaktion zwischen den beiden Proteinen eine entscheidende Rolle für die mitochondriale Mg2+ Homöostase in S. cerevisiae spielt. Im Vergleich zu Lpe10p oder CorA, besitzt Mrs2p einen ungewöhnlich langen C-Terminus. Wir haben eine ortspezifische Mutagenese am nicht-konservierten, positiv geladenem KRRRK Motiv durchgeführt und eine C-terminale Verkürzung des Mrs2 Proteins hergestellt. Während das KRRRK Motiv selbst keine entscheidende Rolle für die Transportaktivität des Kanals spielt, führt die größere Deletion am C-Terminus zu eine starken Reduktion der Funktionalität des Kanals. Im zweiten Teil dieser Arbeit wurden zwei Mitglieder der MDM38/LETM1 Familie aus Hefe charakterisiert. In vorangegangenen Studien wurde Mdm38p bereits als essentieller Faktor im mitochondrialen K+/H+ Austausch in S. cerevisiae beschrieben. In einer genomweiten Suche nach Suppressoren, wurden die Proteine Mrs7p and Ydl183cp als starke Suppressoren der MDM38 Deletion identifiziert. Während Mrs7p eine große Ähnlichkeit zu Mdm38p aufweist, scheint Ydl183c ein nicht verwandtes Protein zu sein. Da beide Proteine vermutlich nur eine Transmembrandomäne besitzen, ist es relativ unwahrscheinlich, dass sie den aktiven K+/H+ Austauscher alleine bilden. Mdm38p/Letm1 und Mrs7p wurden als Teil von Proteinkomplexen mit hohem Molekulargewicht in der inneren Mitochondrienmembran nachgewiesen. Diese Beobachtung lässt darauf schließen, dass diese Proteine Homomultimere formen oder- noch wahrscheinlicher- als Cofaktoren mit dem bisher unbekannten K+/H+ Austauscher assozieren. Um diese Fragestellung zu klären, wurde versucht Interaktionspartner von Mrs7p zu identifizieren, mit der Erwartung den verantwortlichen Antiporter zu finden.Transport processes across cellular membranes have been a challenging and fascinating field of research for the past 50 years. Mrs2p and its bacterial homolog CorA belong to a large family of Mg2+ transporters. Common characteristics of members of this family are two transmembrane domains (TMs) in the C-terminal part of the protein and the highly conserved G-M-N motif at the end of TM 1. In order to obtain new insights into the molecular mechanisms of regulation of these transporters, mutational analyses on members of the CorA/MRS2 family were performed. For CorA from Thermotoga maritima we confirmed the key gating role of residue Leu294 by forming a mechanical barrier for ion permeation. Mutations at this position strongly affect the ability of the channel to close the ion conduction pathway, which is eventually lethal for the cell. In contrast, our studies on MRS2 suggest that the regulation of Mg2+ transport is more complex in eukaryotic members of this family. We performed a comparative analysis of the crystal structure of T. maritima CorA and the structure of the N-terminal domain of Saccharomyces cerevisiae Mrs2p, including the pore forming TM 1. We thereby identified amino acid residues most likely involved in gating of the channel. Our results show that in Mrs2p, Met309 is the equivalent to Leu294 in CorA and fulfils a similar function in gating the channel. However, mutations at this position do not lead to a complete deregulation of the closing mechanism as observed for Leu294 in CorA. A second gate formed by Val315 was identified but appears to be less important in controlling ion translocation. By structure-based sequence analysis, we identified a potential cation binding site putatively involved in sensing the intramitochondrial Mg2+ concentration. However, our attempts to experimentally demonstrate the involvement of the highly conserved amino acid Asp97 in formation of a cation binding site did not yield a clear result. We also performed a random mutational analysis of the highly conserved G-M-N motif of Mrs2p. It has been shown that even conservative single amino acid substitutions in this motif abolish the transport activity of CorA and Mrs2p. Surprisingly, we identified a series of mutants with sequences completely different from G-M-N, but still able to transport Mg2+. However, these mutants exhibited reduced selectivity for Mg2+. Our results suggest that the G-M-N motif plays a central role for the substrate specificity in the CorA/Mrs2p family. Besides Mrs2p the genome of S. cerevisiae encodes a homologous protein, namely Lpe10p. In this study Lpe10p was investigated in more detail. Deletion of LPE10 or MRS2 similarly leads to impaired growth on non-fermentable carbon sources. However, in contrast to deletion of MRS2, deletion of LPE10 results in a strong reduction of the mitochondrial membrane potential. Lpe10p alone cannot form a Mg2+-selective channel but it is able to hetero-oligomerize with Mrs2p and thereby reduces the conductance of the Mrs2p channel. Our results indicate that the interplay between the two proteins is important to maintain the mitochondrial Mg2+ homeostasis in S. cerevisiae. Compared to Lpe10p or CorA, S. cerevisiae Mrs2p has an exceptionally long C-terminus. We performed site-directed mutagenesis on the non-conserved positively charged KRRRK motif and created a C-terminally truncated version of Mrs2p. While the KRRRK motif does not appear to be crucial for the function, large deletions in the C-terminal part of the protein strongly affect the transport activity of the channel. The second part of this thesis focuses on the biochemical characterization of the two yeast members of the MDM38/LETM1 family. Mdm38p has previously been characterized as an essential factor for mitochondrial K+/H+ exchange in S. cerevisiae. In a genome-wide suppressor screen, the proteins Mrs7p and Ydl183cp were identified as strong suppressors of the MDM38 deletion. While Mrs7p exhibits a high sequence similarity to Mdm38p and Letm1, Ydl183c appears to be an unrelated protein. Since these proteins only have one predicted transmembrane domain, it is unlikely that they form the K+/H+ exchanger. Mdm38p/Letm1 and Mrs7p were found to form high molecular weight complexes in the inner mitochondrial membrane suggesting that they form homo-multimers or rather associate as a cofactor with the so far unidentified K+/H+ exchanger. To tackle this question, we aimed here at identifying the interaction partners of Mrs7p, and expected to find the antiporter among them

Effects of butyrate− on ruminal Ca2+ transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels

The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate− were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate− (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate− stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4)

The bovine TRPV3 as a pathway for the uptake of Na+, Ca2+, and NH4+

Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia

The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport

Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed

Solute carrier 41A3 encodes for a mitochondrial Mg2+ efflux system

The important role of magnesium (Mg(2+)) in normal cellular physiology requires flexible, yet tightly regulated, intracellular Mg(2+) homeostasis (IMH). However, only little is known about Mg(2+) transporters of subcellular compartments such as mitochondria, despite their obvious importance for the deposition and reposition of intracellular Mg(2+) pools. In particular, knowledge about mechanisms responsible for extrusion of Mg(2+) from mitochondria is lacking. Based on circumstantial evidence, two possible mechanisms of Mg(2+) release from mitochondria were predicted: (1) Mg(2+) efflux coupled to ATP translocation via the ATP-Mg/Pi carrier, and (2) Mg(2+) efflux via a H(+)/Mg(2+) exchanger. Regardless, the identity of the H(+)-coupled Mg(2+) efflux system is unknown. We demonstrate here that member A3 of solute carrier (SLC) family 41 is a mitochondrial Mg(2+) efflux system. Mitochondria of HEK293 cells overexpressing SLC41A3 exhibit a 60% increase in the extrusion of Mg(2+) compared with control cells. This efflux mechanism is Na(+)-dependent and temperature sensitive. Our data identify SLC41A3 as the first mammalian mitochondrial Mg(2+) efflux system, which greatly enhances our understanding of intracellular Mg(2+) homeostasis

Optimizing adipogenic transdifferentiation of bovine mesenchymal stem cells: a prominent role of ascorbic acid in FABP4 induction

Adipocyte differentiation of bovine adipose-derived stem cells (ASC) was induced by foetal bovine serum (FBS), biotin, pantothenic acid, insulin, rosiglitazone, dexamethasone and 3-isobutyl-1-methylxanthine, followed by incubation in different media to test the influence of ascorbic acid (AsA), bovine serum lipids (BSL), FBS, glucose and acetic acid on transdifferentiation into functional adipocytes. Moreover, different culture plate coatings (collagen-A, gelatin-A or poly-L-lysine) were tested. The differentiated ASC were subjected to Nile red staining, DAPI staining, immunocytochemistry and quantitative reverse transcription PCR (for NT5E, THY1, ENG, PDGFRα, FABP4, PPARγ, LPL, FAS, GLUT4). Nile red quantification showed a significant increase in the development of lipid droplets in treatments with AsA and BSL without FBS. The presence of BSL induced a prominent increase in FABP4 mRNA abundance and in FABP4 immunofluorescence signals in coincubation with AsA. The abundance of NT5E, ENG and THY1 mRNA decreased or tended to decrease in the absence of FBS, and ENG was additionally suppressed by AsA. DAPI fluorescence was higher in cells cultured in poly-L-lysine or gelatin-A coated wells. In additional experiments, the multi-lineage differentiation potential to osteoblasts was verified in medium containing ß-glycerophosphate, dexamethasone and 1,25-dihydroxyvitamin D3 using alizarin red staining. In conclusion, bovine ASC are capable of multi-lineage differentiation. Poly-L-lysine or gelatin-A coating, the absence of FBS, and the presence of BSL and AsA favour optimal transdifferentiation into adipocytes. AsA supports transdifferentiation via a unique role in FABP4 induction, but this is not linearly related to the primarily BSL-driven lipid accumulation

Overexpression of Na+/Mg2+ exchanger SLC41A1 attenuates pro-survival signaling

The Na+/Mg2+ exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg2+ efflux system, and its overexpression decreases [Mg2+]intracellular. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg2+]i impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan® RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg2+]extracellular in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr308 and/or Ser473 and of Erk1/2 on Thr202/Tyr204 in the presence of 0 or 1 mM (physiological) Mg2+ in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg2+]i in the presence of [Mg2+]e = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB – Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg2+]e (and consequently also [Mg2+]i) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells

The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro

The ability of enteropathogenic Escherichia coli (EPEC) to express virulence factor genes and develop attaching and effacing (AE) lesions is inhibited in acidic environmental conditions. This inhibition is due to the activation of transcription factor GadX, which upregulates expression of glutamic acid decarboxylase (Gad). Gad, in turn, produces γ-aminobutyric acid (GABA), which was recently shown to have a beneficial effect on the jejunal epithelium in vitro due to increased mucin-1 levels. In the present study, we sought to test whether forced GadX activation/overexpression abolishes virulence associated features of EPEC and provokes increased GABA production. EPEC strains were isolated from diarrheic pigs and submitted to activation of GadX by acidification as well as gadX overexpression via an inducible expression vector plasmid. GABA concentrations in the growth medium, ability for adhesion to porcine intestinal epithelial cells (IPEC-J2) and virulence gene expression were determined. Growth in acidified media led to increased GABA levels, upregulated gadA/B expression and downregulated mRNA synthesis of the bacterial adhesin intimin. EPEC strains transformed with the gadX gene produced 2.1 to 3.4-fold higher GABA levels than empty-vector controls and completely lost their ability to adhere to IPEC-J2 cells and to induce actin accumulation. We conclude that intensified gadX activation can abolish the ability of EPEC to adhere to the intestinal epithelium by reducing the expression of major virulence genes

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations

The G-M-N motif determines ion selectivity in the yeast magnesium channel Mrs2p

The highly conserved G-M-N motif of the CorA-Mrs2-Alr1 family of Mg2+ channels has been shown to be essential for Mg2+ transport. We performed random mutagenesis of the G-M-N sequence of Saccharomyces cerevisiae Mrs2p in an unbiased genetic screen. A large number of mutants still capable of Mg2+ influx, albeit below the wild-type level, were generated. Growth complementation assays, performed in media supplemented with Ca2+ or Co2+ or Mn2+ or Zn2+ at varying concentrations, lead to identification of mutants with reduced growth in the presence of Mn2+ and Zn2+. We hereby conclude that (1) at least two, but predominantly all three amino acids of the G-M-N motif must be replaced by certain combinations of other amino acids to remain functional, (2) replacement of any single amino acid within the G-M-N motif always impairs the function of Mrs2p, and (3) we show that the G-M-N motif determines ion selectivity, likely in concurrence with the negatively charged loop at the entrance of the channel thereby forming the Mrs2p selectivity filter