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

ABSTRACT: 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.

SUBMITTER: Levy M

PROVIDER: S-EPMC6463715 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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

Levy Moshe M Elkoshi Nadav N Barber-Zucker Shiran S Hoch Eitan E Zarivach Raz R Hershfinkel Michal M Sekler Israel I

The Journal of biological chemistry 20190212 15

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 ...[more]

PMID: 30755481

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Project description:ObjectiveHepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+/Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics.MethodsPrimary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knockout (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were used to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively.ResultsAnalysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations, underscoring NCLX's pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX's significant contribution to hepatic glucose metabolism.ConclusionsThe study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for regulating hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.

Dataset Information

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

Publications

Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn<sup>2+</sup> is driven by an active Ca<sup>2+</sup>-coupled exchange.

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