Functional characteristics and therapeutic potential of SLC41 transporters.

Magnesium (Mg2+) plays an important role in various cellular functions such as protein synthesis, DNA stability, energy metabolism, enzyme and channel activities, and muscle contractility. Therefore, intracellular Mg2+ concentration is tightly regulated by multiple Mg2+ transporters and channels. So far, various candidate genes of Mg2+ transporters have been identified, and the research on their structure and function is currently in progress. The Solute Carrier 41 (SLC41) family, which is related to the bacterial Mg2+ transporter/channel MgtE, comprises three isoforms of SLC41A1, SLC41A2, and SLC41A3. Based on recent studies, SLC41A1 is thought to mediate Mg2+ influx or Na+-dependent Mg2+ efflux across the plasma membrane, whereas SLC41A2 and SLC41A3 may mediate Mg2+ fluxes across either the plasma membrane or organellar membranes. Intriguingly, SLC41A1 variants have been identified in patients with Parkinson's disease (PD) and nephronophthisis-related ciliopathies. Further genetic analyses reveal the association of SLC41A1 polymorphisms with PD risks. This review highlights the recent advances in the understanding of the molecular and functional characteristics of SLC41 family towards its therapeutic and diagnostic applications.

Alzheimer's Disease-Associated SNP rs708727 in SLC41A1 May Increase Risk for Parkinson's Disease: Report from Enlarged Slovak Study.

SLC41A1 (A1) SNPs rs11240569 and rs823156 are associated with altered risk for Parkinson's disease (PD), predominantly in Asian populations, and rs708727 has been linked to Alzheimer's disease (AD). In this study, we have examined a potential association of the three aforementioned SNPs and of rs9438393, rs56152218, and rs61822602 (all three lying in the A1 promoter region) with PD in the Slovak population. Out of the six tested SNPs, we have identified only rs708727 as being associated with an increased risk for PD onset in Slovaks. The minor allele (A) in rs708727 is associated with PD in dominant and completely over-dominant genetic models (ORD = 1.36 (1.05-1.77), p = 0.02, and ORCOD = 1.34 (1.04-1.72), p = 0.02). Furthermore, the genotypic triplet GG(rs708727) + AG(rs823156) + CC(rs61822602) might be clinically relevant despite showing a medium (h ≥ 0.5) size difference (h = 0.522) between the PD and the control populations. RandomForest modeling has identified the power of the tested SNPs for discriminating between PD-patients and the controls to be essentially zero. The identified association of rs708727 with PD in the Slovak population leads us to hypothesize that this A1 polymorphism, which is involved in the epigenetic regulation of the expression of the AD-linked gene PM20D1, is also involved in the pathoetiology of PD (or universally in neurodegeneration) through the same or similar mechanism as in AD.

Dietary Mg2+ Intake and the Na+/Mg2+ Exchanger SLC41A1 Influence Components of Mitochondrial Energetics in Murine Cardiomyocytes.

Cardiomyocytes are among the most energy-intensive cell types. Interplay between the components of cellular magnesium (Mg) homeostasis and energy metabolism in cardiomyocytes is poorly understood. We have investigated the effects of dietary Mg content and presence/functionality of the Na+/Mg2+ exchanger SLC41A1 on enzymatic functions of selected constituents of the Krebs cycle and complexes of the electron transport chain (ETC). The activities of aconitate hydratase (ACON), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (KGDH), and ETC complexes CI-CV have been determined in vitro in mitochondria isolated from hearts of wild-type (WT) and Slc41a1-/- mice fed a diet with either normal or low Mg content. Our data demonstrate that both, the type of Mg diet and the Slc41a1 genotype largely impact on the activities of enzymes of the Krebs cycle and ETC. Moreover, a compensatory effect of Slc41a1-/- genotype on the effect of low Mg diet on activities of the tested Krebs cycle enzymes has been identified. A machine-learning analysis identified activities of ICDH, CI, CIV, and CV as common predictors of the type of Mg diet and of CII as suitable predictor of Slc41a1 genotype. Thus, our data delineate the effect of dietary Mg content and of SLC41A1 functionality on the energy-production in cardiac mitochondria.

SNPs rs11240569, rs708727, and rs823156 in SLC41A1 Do Not Discriminate Between Slovak Patients with Idiopathic Parkinson's Disease and Healthy Controls: Statistics and Machine-Learning Evidence.

Gene SLC41A1 (A1) is localized within Parkinson's disease-(PD)-susceptibility locus PARK16 and encodes for the Na+/Mg2+-exchanger. The association of several A1 SNPs with PD has been studied. Two, rs11240569 and rs823156, have been associated with reduced PD-susceptibility primarily in Asian populations. Here, we examined the association of rs11240569, rs708727, and rs823156 with PD in the Slovak population and their power to discriminate between PD patients and healthy controls. The study included 150 PD patients and 120 controls. Genotyping was performed with the TaqMan® approach. Data were analyzed by conventional statistics and Random Forest machine-learning (ML) algorithm. Individually, none of the three SNPs is associated with an altered risk for PD-onset in Slovaks. However, a combination of genotypes of SNP-triplet GG(rs11240569)/AG(rs708727)/AA(rs823156) is significantly (p < 0.05) more frequent in the PD (13.3%) than in the control (5%) cohort. ML identified the power of the tested SNPs in isolation or of their singlets (joined), duplets and triplets to discriminate between PD-patients and healthy controls as zero. Our data further substantiate differences between diverse populations regarding the association of A1 polymorphisms with PD-susceptibility. Lack of power of the tested SNPs to discriminate between PD and healthy cases render their clinical/diagnostic relevance in the Slovak population negligible.

Insulin Modulates the Na+/Mg2+ Exchanger SLC41A1 and Influences Mg2+ Efflux from Intracellular Stores in Transgenic HEK293 Cells.

BACKGROUND: Magnesium deficiency is a common complication of diabetes with an unclear molecular background. OBJECTIVE: We investigated the effect of the insulin (INS)-signaling pathway (ISP) on the regulation of Mg(2+) efflux (Mg(2+)E) conducted by solute carrier family 41, member A1 (SLC41A1; activated by protein kinase A) in transgenic human embryonic kidney (HEK) 293 cells. METHODS: HEK293 cells overexpressing SLC41A1 were loaded with the Mg(2+) fluorescent indicator mag-fura-2 and Mg(2+). Measurements of Mg(2+)E were conducted in Mg(2+)-free buffer by using fast-filter fluorescence spectrometry. We examined the effects of INS, inhibitors of ISP or p38 mitogen-activated protein kinase (p38 MAPK), an activator of adenylate cyclase (ADC), and their combinations on SLC41A1-attributed Mg(2+)E. RESULTS: The application of 400 µU/mL INS inhibited SLC41A1-mediated Mg(2+)E by up to 50.6% compared with INS-untreated cells (P < 0.001). Moreover, INS evoked the early onset of Mg(2+) release from intracellular stores. The application of 0.1 µM wortmannin or 10 µM zardaverine (both ISP inhibitors) restored SLC41A1 Mg(2+)E capacity in the presence of INS to the same levels in INS-untreated cells. The simultaneous application of 10 µM forskolin, an ADC activator, and INS resulted in a reduction of Mg(2+)E of up to 59% compared with untreated cells (P < 0.001), which was comparable to that in cells treated with INS alone. Inhibition of p38 MAPK with 10 µM SB 202190 (SB) in the absence of INS resulted in a decrease (P < 0.001) of SLC41A1-dependent Mg(2+)E (by up to 49%) compared with Mg(2+)E measured in untreated cells. Simultaneous exposure of cells to SB and INS had a stronger inhibitory effect on SLC41A1 activity than INS alone (P < 0.05). CONCLUSIONS: INS affects intracellular Mg(2+) concentration in transgenic HEK293 cells by regulating SLC41A1 activity (via ISP) and by influencing the compartmentalization and cellular distribution of Mg(2+). In addition, p38 MAPK activates SLC41A1 independently of INS action.

SLC41A1 knockdown inhibits angiotensin II-induced cardiac fibrosis by preventing Mg(2+) efflux and Ca(2+) signaling in cardiac fibroblasts.

Na(+)/Mg(2+) exchanger plays an important role in cardiovascular system, but the molecular mechanisms still largely remain unknown. The Solute Carrier family 41A1 (SLC41A1), a novel Mg(2+) transporter, recently was found to function as Na(+)/Mg(2+) exchanger, which mainly regulates the intracellular Mg(2+) ([Mg(2+)]i) homeostasis. Our present studies were designed to investigate whether SLC41A1 impacts on the fibrogenesis of cardiac fibroblasts under Ang II stimulation. Our results showed that quinidine, a prototypical inhibitor of Na(+)/Mg(2+) exchanger, inhibited Ang II-induced cardiac fibrosis via attenuating the overexpression of vital biomarkers of fibrosis, including connective tissue growth factor (CTGF), fibronectin (FN) and α-smooth muscle actin (α-SMA). In addition, quinidine also decreased the Ang II-mediated elevation of concentration of intracellular Ca(2+) ([Ca(2+)]i) and extrusion of intracellular Mg(2+). Meanwhile, silencing SLC41A1 by RNA interference also impaired the elevation of [Ca(2+)]i, [Mg(2+)]i efflux and the upregulation of CTGF, FN and α-SMA provoked by Ang II. Furthermore, we found that Ang II-mediated activation of NFATc4 translocation decreased in SLC41A1-siRNA cells. These results support the notion that rapid extrusion of intracellular Mg(2+) is mediated by SLC41A1 and provide the evidence that the intracellular free Ca(2+) concentration is influenced by extrusion of intracellular Mg(2+) which facilitates fibrosis reaction in cardiac fibroblasts.

SLC41 transporters--molecular identification and functional role.

The solute carrier family 41 (SLC41) encompasses three members A1, A2, and A3. Based on their distant homology to the bacterial Mg²âº channel MgtE, all have been linked to Mg²âº transport. There is only very limited knowledge on the molecular biology and exact functions of SLC41A2 and SLC41A3. SLC41A1 is ubiquitously expressed and data on its functional and molecular properties, regulation, complex-forming ability, and spectrum of binding partners are available. SLC41A1 was recently identified as being the Na⁺/Mg²âº exchanger (NME)-a predominant Mg²âº efflux system. Mg²âº-dependent and hormonal regulation of NME activity is now known to depend on the intracellular N terminus of SLC41A1 that is involved in Mg²âº sensing and contains phosphorylation sites for protein kinase (PK) A and PKC. Data showing a link between SLC41A1 and human disorders such as Parkinson's disease, nephronophthisis (induced by the null mutation c.698G>T in renal SLC41A1), and preeclampsia make the protein a candidate therapeutic target.

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.

SLC41A1, a Na+/Mg2+ exchanger, is downregulated during exercise.

Serum magnesium (Mg) levels are closely controlled through a variety of Mg transporters and ionic channels during physiological conditions. These levels have been shown to increase during exercise. However, the effect of Mg transporter expression during exercise remains to be determined. The purpose of this study was to examine the gene expression of SLC41A1, a Na+/Mg2+ exchanger, during exercise. In the present study, male C57BL/6JNarl mice (n=16, 8 weeks old) were subjected to 3 h forced exercise on a treadmill. The mice in the control and Mg groups were injected with saline and Mg (MgSO4, 90 mg/kg, intraperitoneal), respectively. Blood samples were obtained at three time points: prior to, following and 24 h after exercise. The gene expression levels of SLC41A1 were significantly downregulated to 23.6±4.6 and 12.6±10.2% following exercise in the control and Mg groups, respectively. The expression levels returned to the basal levels 24 h after exercise in the two groups and there was no significant difference found between the two groups. The downregulated role of SLC41A1 expression and its interaction with the Mg status in exercise requires further investigation.

"Inside-in" or "inside-out"? The membrane topology of SLC41A1.

Membrane topology is an important parameter for understanding the function and regulation of any integral protein. This aspect of the NME SLC41A1 is currently under debate. The most probable model, which has been computer-predicted, exhibits ten TMh with both termini being oriented intracellularly. However, other freely accessible online prediction programs predict that SLC41A1 possesses eleven ("outside-in" configuration), nine ("outside-in" configuration), or eight ("inside-in" configuration) TMh. The consensus based on published experimental data acquired by independent research teams is that the N-terminal flanking region is located intracellularly. However, controversy remains about the orientation of the C-terminus, which has lately been proposed to be extracellular in peer-reviewed bibliography. Here, we performed split-ubiquitin functional assays with transgenic SLC41A1 fused N- or C-terminally to a Cub-LexA-VP16 reporter cassette. The bait constructs were co-expressed in S. cerevisiae st. NMY51 with positive recombinant membrane markers (Ost1, Fur4, Alg5, Tom20) tagged with NubI (or NubG). Ubiquitin could only be reconstituted if the reporter moiety was exposed to the cytosol. Functional reconstitution of ubiquitin was observed when SLC41A1 C-terminally tagged with Cub was co-expressed with NubI-tagged membrane markers, thereby, indicating a cytosolic orientation of the C-terminus of SLC41A1. Thus, our experimental data are in favor of the - the in silico analyses being strongly preferred - ten TMh model of SLC41A1 topology, with both termini being oriented intracellularly.

Nature of SLC41A1 complexes: report on the split-ubiquitin yeast two hybrid assay.

The Na(+)/Mg(2+) exchanger SLC41A1 is involved in the pathophysiology of various disease conditions. It forms high-molecular-mass, possibly hetero-oligomeric protein complexes in transgenic HEK293 cells. Therefore, we attempted to identify binding partners of SLC41A1 by utilizing the split-ubiquitin modification of the yeast two-hybrid assay. As the most prominent binding partners in our experimental system, we identified 3-beta-hydroxysteroid-Δ(8),Δ(7)-isomerase and B-cell receptor associated-protein 31. Other polytons (interactors appearing in the screen more than once) included: IER3IP1, PPIB, UPF0480 protein C15orf24, SPINT2, C14orf1/PEBP28, NIFIE14, YIPF6, and KCP2. In total, 20 polytons and 38 singletons (interactors appearing in the screen only once) were identified. The polytons identified were mostly endoplasmic reticulum-located, integral proteins involved in protein maturation, N-glycosylation, protein folding, anterograde transport of proteins, protein secretion, and the regulation of apoptosis. Among the singletons, we identified SLC31A2, SLC35B1, SLC39A13, CRACM1, and MTCH2 as putative binding partners of SLC41A1. Interestingly, we did not identify interactions among SLC41A1 molecules. Most of the identified interactors are integral proteins localized in cellular compartments other than the cytoplasmic membrane, whereas SLC41A1 is targeted to the cytoplasmic membrane where it performs its core function. None of the interactors was confirmed by mass spectrometry. Instead, we identified among the proteins co-purified with strep-tagged SLC41A1: ACCA1, UBB, ATX2L, HSP7C and TBB. We therefore conclude that: (1) identified interactors form transient rather than stable complexes with SLC41A1, (2) the molecular interactors identified primarily among the polytons might contribute to the production, proper folding, and maturation of SLC41A1 in the endoplasmic reticulum and Golgi apparatus, (3) most of the interactors identified among singletons might undergo similar maturation steps (post-translational modification), anterograde transport, and protein sorting as SLC41A1.