Solute Carrier Family SLC41, what do we really know about it?

The 41st family of solute carriers (SLC41) comprises three members A1, A2, and A3, which are distantly homologous to bacterial Mg2+ channel MgtE. SLC41A1 was recently characterized as being an Na+/Mg2+ exchanger (NME; a predominant cellular Mg2+‐efflux system). Little is known about the exact function of SLC41A2 and SLC41A3, although, these proteins have also been linked to Mg2+ transport in human (animal) cells. The molecular biology (including membrane topology, cellular localization, transcriptomics, and proteomics) of SLC41A2 and SLC41A3 compared with SLC41A1 has only been poorly explored. Significantly more data with regard to function, functional regulation, involvement in cellular signaling, complex‐forming ability, spectrum of binding partners, and involvement in the pathophysiology of human diseases are available for SLC41A1. Three recent observations namely the identification of the null mutation, c.698G>T, in SLC41A1 underlying the nephronophthisis‐like phenotype, the recognition of a putative link between SLC41A1 and Parkinson's disease, and the observation that nearly 55% of preeclamptic placental samples overexpress SLC41A1, marks the protein as a possible therapeutic target of these diseases. A potential role of the SLC41 family of Mg2+ transporters in the pathophysiology of human diseases is further substantiated by the finding that SLC41A3 knockout mice develop abnormal locomotor coordination. WIREs Membr Transp Signal 2013. doi: 10.1002/wmts.9

Substitution p.A350V in Na⁺/Mg²⁺ exchanger SLC41A1, potentially associated with Parkinson's disease, is a gain-of-function mutation.

Parkinson's disease (PD) is a complex multifactorial ailment predetermined by the interplay of various environmental and genetic factors. Systemic and intracellular magnesium (Mg) deficiency has long been suspected to contribute to the development and progress of PD and other neurodegenerative diseases. However, the molecular background is unknown. Interestingly, gene SLC41A1 located in the novel PD locus PARK16 has recently been identified as being a Na⁺/Mg²⁺ exchanger (NME, Mg²⁺ efflux system), a key component of cellular magnesium homeostasis. Here, we demonstrate that the substitution p.A350V potentially associated with PD is a gain-of-function mutation that enhances a core function of SLC41A1, namely Na⁺-dependent Mg²⁺ efflux by 69±10% under our experimental conditions (10-minute incubation in high-Na⁺ (145 mM) and completely Mg²⁺-free medium). The increased efflux capacity is accompanied by an insensitivity of mutant NME to cAMP stimulation suggesting disturbed hormonal regulation and leads to a reduced proliferation rate in p.A350V compared with wt cells. We hypothesize that enhanced Mg²⁺-efflux conducted by SLC41A1 variant p.A350V might result, in the long-term, in chronic intracellular Mg²⁺-deficiency, a condition that is found in various brain regions of PD patients and that exacerbates processes triggering neuronal damage

Effect of imipramine and of cAMP-dependent PKA phosphorylation on SLC41A1-related [Mg²⁺]_i changes, cell adhesion, and cell proliferation in +tet p.A350V cells and wt cells.

<p>A: Summary of [Mg²⁺]_i changes after resuspension of Mg²⁺-loaded +tet p.A350V cells and wt cells in completely Mg²⁺-free Na⁺-containing solutions with or without (control) the Na⁺/Mg²⁺ exchanger inhibitor imipramine (250 µM). Values have been corrected for [Mg²⁺]_i changes in -tet cells and given as means ± SE; N_p.A350V  = 26 & N_wt  = 21 single experiments per condition; *P = 0.03; **P<0.005. B: Summary of [Mg²⁺]_i changes after resuspension of Mg²⁺-loaded +tet p.A350V cells and wt cells in completely Mg²⁺-free Na⁺-containing media with or without (control) the Na⁺/Mg²⁺ exchanger activator dB-cAMP (100 µM). Values have been corrected for [Mg²⁺]_i changes in -tet cells and are means ± SE; Np.A350V  = 15 & Nwt  = 15 single experiments per condition; **P = 0.01. C: Original growth curves of +tet p.A350V cells and wt cells under control conditions and after application of 250 µM imipramine and of 100 µM dB-cAMP. Cells were seeded at a density of 10×10⁵ per well, induced with tetracycline, and allowed to attach and proliferate for 24 h prior to treatment with the compounds (indicated by the arrow). The Cell Index, a dimensionless parameter reflecting cell adherence and number, was normalized (nCI) to the time just before modulator application. Values are means ± SD; N = 6 single experiments per condition; **P<0.001.</p

Confocal immunolocalization of HA-strep-SLC41A1

<p>(<b>wt</b>) <b>and HA-strep-SLC41A1-p.A350V in +tet</b> (<b>24 </b><b>h</b>) <b>cells.</b> Strep-tagged wt and p.A350V were immunolabeled with primary mouse anti-strep and secondary GAM Alexa-488 antibodies (green signal). Plasma membranes were fluorescently contrasted with wheat germ agglutinin (WGA) conjugated to Alexa-647 (red signal). Nuclei were stained with DAPI (blue signal). The merged images show that both Alexa-488 and Alexa-647 signals co-localize in +tet cells. Scale bar indicates 10 μm.</p

Immunodetection of phosphorylated recombinant flag-SLC41A1

<p>(<b>wt</b>)<b>, HA-strep-SLC41A1</b> (<b>wt</b>)<b>, and HA-strep-SLC41A1-p.A350V with the PhosphoProtein purification kit</b> (<b>Qiagen</b>)<b>.</b> Total (T), flow-through (U; containing unphosphorylated proteins) and elution (P; containing phosphorylated proteins) fractions were probed with antibodies against strep- or flag-tag. A signal specific for phosphorylated wt or p.A350V was detected in all three cell lines. The specificity of the fractionation was controlled with an antibody against phosphorylated Akt.</p

(A) Quantitative real time PCR analysis of <i>SLC41A1</i> (<i>wt</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are shown. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (B) Quantitative real time PCR analysis of <i>SLC41A1</i>-(<i>c.1049C>T</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are given. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (C) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in total protein isolate from -tet and +tet (24 h) cells. Strep-tagged wt and p.A350V were detected only in tet-induced cells. Positive control: flag-tagged SLC41A1 isolated from HEK293 cells, clone 17, which was extensively characterized in [20], [27]. Loading was controlled by immunodetection of RPL19 protein. (D) Immunodetection of recom

<p>(A) Quantitative real time PCR analysis of <i>SLC41A1</i> (<i>wt</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are shown. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (B) Quantitative real time PCR analysis of <i>SLC41A1</i>-(<i>c.1049C>T</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are given. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (C) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in total protein isolate from -tet and +tet (24 h) cells. Strep-tagged wt and p.A350V were detected only in tet-induced cells. Positive control: flag-tagged SLC41A1 isolated from HEK293 cells, clone 17, which was extensively characterized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071096#pone.0071096-Kolisek1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071096#pone.0071096-Kolisek2" target="_blank">[27]</a>. Loading was controlled by immunodetection of RPL19 protein. (D) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in soluble and membrane-protein-enriched fractions isolated from -tet and +tet (24 h) cells. Strep-tagged wt and p.A350V were detected almost exclusively in tet-induced cells and predominantly in membrane (M) protein fractions and in much lower quantities in soluble (S) protein fractions. Positive control: flag-tagged SLC41A1 isolated from HEK293 cells (clone 17). Soluble RPL19 was used to control the specificity of the separation between soluble and membrane proteins. (E) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in subcellular protein fractions isolated from +tet (24 h) cells. Wt and p.A350V were predominantly detected in membrane (M) protein fractions with much lower quantities in cytosolic (C) protein fractions and also for p.A350V in traces in the nuclear (N) protein fraction. Transgenic variants were not detected in cytoskeletal (S) fractions. Specificity of the fractionation was controlled on a parallel blot by immunodetection of PMCA4 (M).</p

[Mg²⁺]_i (mM) of uninduced (-tet) and induced (+tet) HEK293-(HA-strep-SLC41A1), and HEK293-(HA-strep-SLC41A1-p.A350V) cells.

<p>[Mg²⁺]_i values for cells successively incubated for 20 min in solutions containing 10 mM Mg²⁺ (loading conditions) and for 10 min in completely Mg-free media (efflux conditions) are given. Data are presented as means ± SE. N is being indicated. **P<0.001 vs. control (-tet cells); ^aP<0.001 vs. loaded +tet HEK293-(HA-strep-SLC41A1) or HEK293-(HA-strep-SLC41A1-p.A350V) cells.</p

SLC41A1-related Mg²⁺ efflux in p.A350V cells compared with wt cells.

<p>Before measurements, cells were pre-loaded with Mg²⁺ as described in Material and Methods ([Mg²⁺]_e  = 10 mM). The [Mg²⁺]_i change obtained after 10 min in media containing 145 mM Na⁺ is given for the following conditions: (1) completely Mg²⁺-free media (N_p.A350V  = 113 & N_wt  = 82); (2) media supplemented with 5 mM Mg²⁺ (N_p.A350V  = 13 & N_wt  = 14); and (3) media supplemented with 10 mM Mg²⁺ (N_p.A350V  = 13 & N_wt  = 14). Values have been corrected for [Mg²⁺]_i changes in -tet cells and are given as means ± SE; **P<0.001.</p

Methane emission, digestive characteristics and faecal archaeol in heifers fed diets based on silage from brown midrib maize as compared to conventional maize

The aim of the present experiment was to compare silage prepared from maize having a brown midrib (BMR) mutation with control (CTR) maize to identify their effects on enteric methane emission, digesta mean retention time (MRT), ruminal fermentation and digestibility. In addition, the utility of archaeol present in faecal samples was validated as a proxy for methane production. Seven German Holstein heifers were fed total mixed rations with a maize-silage proportion (either BMR or CTR) of 920 g/kg dry matter (DM) in a change-over design. Heifers were fed boluses with markers to measure MRT; faeces were collected for 7 days and rumen fluid was collected on the penultimate day. Methane emission was measured in respiration chambers on one day. Data were analysed by t-test and regression analysis. DM intake did not differ between the two diets. The apparent digestibility of DM and most nutrients was unaffected by diet type, but apparent digestibility of neutral and acid detergent-fibre was higher in those heifers fed BMR than in those fed CTR. Comparisons between diets revealed no difference in particle or solute MRT in the gastro-intestinal tract and the reticulorumen. Concentrations of short-chain fatty acid and ammonia in rumen fluid and its pH were not affected by silage type. Independent of the mode of expression [l/d, l/kg DM intake, l/kg digested organic matter], methane emissions were not affected by maize-silage type, but with BMR, there was a trend towards lower methane production per unit of digested neutral detergent fibre than there was with CTR silage. Results of the present study show that feeding heifers BMR silage does not increase methane emissions despite a higher fibre digestibility as compared to CTR silage. Therefore, it is assumed that improvements in animal productivity achieved by feeding BMR silage, as some studies have reported, can be obtained without extra environmental cost per unit of milk or meat. Neither faecal archaeol content [µg/g] nor daily amount excreted [mg/d] is suitable to predict methane production in absolute terms [l per day]. However, faecal archaeol content has a certain potential for predicting the methane yield [l per kg DM intake] of individual animals