# Mapping Molecular Pathways of Mitochondrial Iron Uptake: Implications for Iron Toxicity

> **NIH NIH R21** · MEDICAL UNIVERSITY OF SOUTH CAROLINA · 2020 · $224,250

## Abstract

Iron is an abundant metal in the environment providing challenges to human health by contributing to envi-
ronmental toxicity. Iron is a transition metal that exists in two pools within cells. Chelatable iron comprises free
iron and iron loosely bound to anionic metabolites like ATP and citrate, whereas non-chelatable iron is tightly
bound to ferritin, heme and iron-sulfur clusters. Chelatable iron promotes oxidative stress by catalyzing the Fen-
ton reaction, which produces highly reactive hydroxyl radicals that damage DNA, proteins and membranes. Sub-
stantial evidence implicates mitochondrial iron as an important contributor to toxicity, but the molecular pathways
of mitochondrial iron uptake are controversial. The prevailing view is that mitoferrins 1 and 2 (Mfrn1/2), proteins
localized in the mitochondrial inner membrane, are responsible for mitochondrial iron transport. However, previ-
ous studies from ~40 years ago show that the classical electrogenic mitochondrial calcium uniporter also cata-
lyzes uptake of Fe2+ but not Fe3+ driven by the mitochondrial membrane potential, a conclusion supported by our
own recent studies in intact hepatocytes. Our preliminary pull-down and Duolink studies indicate a physical as-
sociation between Mfrn2, the predominant isoform in non-erythropoietic cells, and MCU, the core protein of the
uniporter complex, which brings us to the fundamental questions to be addressed by this proposal: 1) Does
electrogenic mitochondrial Fe2+ and Ca2+ uptake occur via two independent pathways: one mediated by
Mfrn1/2 and another by MCU; or does electrogenic uptake of both cations occur exclusively by one or
the other carrier? 2) Alternatively, do Mfrn and the calcium uniporter act cooperatively in mediating up-
take of both iron and calcium? Therefore, the studies proposed here are intended to address these questions
utilizing Mfrn1/2 single knockout (KO) and double KO (DKO) hepatocytes and their wild type (WT) counterparts.
In Aim 1, mitochondrial Fe2+ uptake will be measured in permeabilized mouse hepatocytes by confocal micros-
copy using a newly developed mitochondria-specific Fe2+-indicating fluorophor, mitoferrofluor. In Aim 2, mito-
chondrial Ca2+ uptake will be measured using Ca2+-indicating Fluo5N. If there are two independent pathways
(#1 above), then Mfrn2 deficiency will prevent Fe2+ but not Ca2+ uptake, and MCU deficiency will prevent
Ca2+ but not Fe2+ uptake, whereas if Mfrn2 and MCU reside in a single complex (#2), then the kinetics of
both Fe2+ and Ca2 uptake should be altered by Mfrn2 deficiency, and both Fe2+ and Ca2 uptake will be
blocked by MCU deficiency. The concept that MCU and Mfrn are essential for both Fe2+ and Ca2 electrogenic
uptake is novel, innovative and paradigm-changing. The project will provide insights into an unexplored area
of biology and fill an important gap in our understanding of the pathways involved in mitochondrial iron uptake
and iron-dependent toxicities. Better understanding of ...

## Key facts

- **NIH application ID:** 9876797
- **Project number:** 1R21ES031335-01
- **Recipient organization:** MEDICAL UNIVERSITY OF SOUTH CAROLINA
- **Principal Investigator:** John J Lemasters
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $224,250
- **Award type:** 1
- **Project period:** 2020-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9876797

## Citation

> US National Institutes of Health, RePORTER application 9876797, Mapping Molecular Pathways of Mitochondrial Iron Uptake: Implications for Iron Toxicity (1R21ES031335-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9876797. Licensed CC0.

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