SUMMARY Type 2 Diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ~20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly-linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function, and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter, and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D.
Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2؉ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn 2؉ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on
Zinc and cadmium are similar metal ions, but though Zn 2þ is an essential nutrient, Cd 2þ is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn 2þ vs. Cd 2þ suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn 2þ transport, but reject Cd 2þ , thus constituting the first mammalian metal transporter with a refined selectivity against Cd 2þ . Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn 2þ and Cd 2þ . A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn 2þ transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd 2þ by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn 2þ and Cd 2þ , and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd 2þ binding.Cd transport | metal binding site | zinc | Zn transporter | Cd toxicity Z n 2þ and Cd 2þ are both d 10 closed shell metals with similar outer electronic structures. However, while Zn 2þ is an essential micro nutrient (1), Cd 2þ is a common environmental pollutant associated with severe toxicity and linked to many disorders, including hypertension, cancer, infertility, thyroid, renal, and bone diseases (2-4). Due to their chemical similarity, Cd 2þ can exploit Zn 2þ uptake routes to enter cells through, for example, Zn 2þ influx transporters such as ZIPs (5-7). Similarly, Metallothioneins, the major cellular metal buffering proteins, bind both Cd 2þ and Zn 2þ , and the divalent metal transporter 1 (DMT1) catalyzes H þ cotransport of Cd 2þ and Zn 2þ as well as other heavy metals (8-10). In plants, most of the Heavy metal ATPases (HMA) classified P-type ATPase share the same nonselective metal ions transport (11). Although bacterial metal pumps HMA1 and plant OsHMA3 are selective for Zn 2þ over Cd 2þ and their selectivity is associated with transmembrane domains (12, 13), the structural basis for their metal selectivity is not fully understood.Mammalian cells do not possess such a wide repertoire of heavy metal pumps and only express the ATP7A and ATP7B P-type cupper selective pumps (14). Yet once inside mammalian tissues, Cd 2þ is trapped with a retention half-time of more than 30 y (15) in a tissue restricted manner whereas Zn 2þ undergoes rapid bodily dissemination through a sequence of secretion and reabsorption processes (16). This difference in the extrusion rates of Zn 2þ and Cd 2þ suggests that in addition to Cd 2þ buffering by metallothioneins (MTs), a mammalian class of metal transporters may selective...
Edited by Roger J. ColbranManganese (Mn 2؉ ) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca 2؉ inward gradient for active cellular exchange of Mn 2؉ . In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn 2؉ and Ca 2؉ fluxes. We found that (a) application of Mn 2؉ -containing Ca 2؉ -free solution to ZnT10-expressing cells triggers an influx of Mn 2؉ , (b) reintroduction of Ca 2؉ leads to cellular Mn 2؉ extrusion against an inward Mn 2؉ gradient, and (c) the cellular transport of Mn 2؉ by ZnT10 is coupled to a reciprocal movement of Ca 2؉ . Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn 2؉ /Ca 2؉ exchange to an uncoupled channel mode, permeable to both Ca 2؉ and Mn 2؉ . The findings in our study identify the first ion transporter that uses the Ca 2؉ gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins.by guest on July 10, 2020 http://www.jbc.org/ Downloaded from Figure 2. ZnT10 is regulated by pH but does not conduct H ؉ /Mn 2؉ or Na ؉ /Mn 2؉ exchange. a, representative traces of Mn 2ϩ (5 M) uptake in ZnT10 transfected cells. Cells were superfused with Ringer's solution at the indicated pH values. Mn 2ϩ influx was monitored as described in Fig. 1c. b, mean rates of cellular Mn 2ϩ uptake derived from a (one-way ANOVA test; n ϭ 3; ***, p Ͻ 0.001; **, p Ͻ 0.01). c, Mn 2ϩ efflux in cells expressing ZnT10 is unaffected by extracellular pH changes. Representative traces of Mn 2ϩ (5 M) transport in ZnT10 (blue and red) and pcDNA (black) transfected cells that were loaded with Fura-2AM and monitored at the indicated pH values. Cells were first superfused as in Fig. 1c with pH 7.4 Ringer's solution containing Mn 2ϩ (5 M) as indicated by the left horizontal bar, then as indicated by the right horizontal bar with Mn 2ϩ -free Ringer at either pH 7.4 Ringer's solution (blue) or pH 6 (red and black) Ringer's solution. d, mean rates of cytoplasmic Mn 2ϩ fluxes of cells superfused with a Ringer's solution at pH 7.4 (n ϭ 11) or pH 6 (one-way ANOVA test, ZnT10 n ϭ 11; pcDNA n ϭ 4) taken from c. e, representative traces of cellular pH i values in ZnT10 (red and cream) or pcDNA (black) transfected cells preloaded with BCECF-AM. Ringer's solution with (red and black) or without (cream) Mn 2ϩ (5 M) that was added as indicated by the horizontal bar. f, mean rates of cytoplasmic pH changes taken from e of Mn 2ϩ -treated ZnT10 (n ϭ 21), pcDNA (n ϭ 13), and Mn 2ϩ untreated ZnT10 (n ϭ 5) transfected cells (one-way ANOVA test). g, effect of Na ϩ on Mn 2ϩ transport by ...
Cellular Zn2+ homeostasis is tightly regulated and primarily mediated by designated Zn2+ transport proteins, namely zinc transporters (ZnTs; SLC30) that shuttle Zn2+ efflux, and ZRT-IRT-like proteins (ZIPs; SLC39) that mediate Zn2+ influx. While the functional determinants of ZnT-mediated Zn2+ efflux are elucidated, those of ZIP transporters are lesser understood. Previous work has suggested three distinct molecular mechanisms: (I) HCO3− or (II) H+ coupled Zn2+ transport, or (III) a pH regulated electrodiffusional mode of transport. Here, using live-cell fluorescent imaging of Zn2+ and H+, in cells expressing ZIP4, we set out to interrogate its function. Intracellular pH changes or the presence of HCO3− failed to induce Zn2+ influx. In contrast, extracellular acidification stimulated ZIP4 dependent Zn2+ uptake. Furthermore, Zn2+ uptake was coupled to enhanced H+ influx in cells expressing ZIP4, thus indicating that ZIP4 is not acting as a pH regulated channel but rather as an H+ powered Zn2+ co-transporter. We further illustrate how this functional mechanism is affected by genetic variants in SLC39A4 that in turn lead to Acrodermatitis enteropathica, a rare condition of Zn2+ deficiency.
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