Elucidating the H+ Coupled Zn2+ Transport Mechanism of ZIP4; Implications in Acrodermatitis Enteropathica.

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.

Gain-of-Function Claims for Type-2-Diabetes-Associated Coding Variants in SLC16A11 Are Not Supported by the Experimental Data.

Human genetic variants in SLC16A11 are associated with increased risk of type 2 diabetes (T2D). We previously identified two distinct mechanisms through which co-inherited T2D-risk coding and non-coding variants disrupt SLC16A11 expression and activity, thus implicating reduced SLC16A11 function as the disease-relevant direction of effect. In a recent publication, Zhao et al. (2019a) argue that human SLC16A11 coding variants confer gain of function, basing their conclusions on phenotypic changes observed following overexpression of mutant murine Slc16a11. However, data necessary to demonstrate gain-of-function activity are not reported. Furthermore, several fundamental flaws in their experimental system-including inaccurate modeling of the human variant haplotype and expression conditions that are not physiologically relevant-prevent conclusions about T2D-risk variant effects on human physiology. This Matters Arising paper is in response to Zhao et al. (2019a), published in Cell Reports. See also the response by Zhao et al. (2019b) in this issue of Cell Reports.

Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn2+ is driven by an active Ca2+-coupled exchange.

Manganese (Mn2+) 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 Ca2+ inward gradient for active cellular exchange of Mn2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn2+ and Ca2+ fluxes. We found that (a) application of Mn2+-containing Ca2+-free solution to ZnT10-expressing cells triggers an influx of Mn2+, (b) reintroduction of Ca2+ leads to cellular Mn2+ extrusion against an inward Mn2+ gradient, and (c) the cellular transport of Mn2+ by ZnT10 is coupled to a reciprocal movement of Ca2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn2+/Ca2+ exchange to an uncoupled channel mode, permeable to both Ca2+ and Mn2+ The findings in our study identify the first ion transporter that uses the Ca2+ 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.

Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms.

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. VIDEO ABSTRACT.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Zinc and cadmium are similar metal ions, but though Zn(2+) is an essential nutrient, Cd(2+) is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn(2+) vs. Cd(2+) suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn(2+) transport, but reject Cd(2+), thus constituting the first mammalian metal transporter with a refined selectivity against Cd(2+). Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn(2+) and Cd(2+). 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(2+) transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd(2+) by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn(2+) and Cd(2+), and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd(2+) binding.

Identification of the Zn2+ binding site and mode of operation of a mammalian Zn2+ transporter.

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 Zn2+ 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 ZnT5. Consistent with this model, replacement of these residues, Asp599 and His451, with alanine was sufficient to block Zn2+ transport. These findings indicate, for the first time, that Zn2+ transport mediated by a mammalian ZnT is catalyzed by H+/Zn2+ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.