# Molecular Architecture Of The Mitochondrial Calcium Uniporter

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2020 · $787,708

## Abstract

PROJECT SUMMARY
The goal of this proposal is to combine functional mutagenesis, biochemistry, and structural biology, to
understand the molecular architecture of the mitochondrial Ca2+ uniporter. Early biochemical studies
demonstrated that isolated mitochondria could transport and buffer huge amounts of Ca2+ across their inner
membrane via a highly selective, Ru360-sensitive channel called the “uniporter”. Uptake of Ca2+ via the
uniporter is known to activate the TCA cycle, while its overload leads to cell death. Although the uniporter has
been studied extensively for over 50 years, its molecular identity remained elusive until our group utilized
comparative genomics to discover its molecular components. In humans, the minimal genetic elements
required for uniporter current are MCU (the pore forming protein) and EMRE (a single-pass membrane
protein). We have recently shown that Dictyostelium discoideum harbors a simpler uniporter, requiring only
one component (DdMCU, the homolog of MCU), which is necessary and sufficient for uniporter activity and
complements the requirement for MCU or EMRE in human knockout cells. At present, the molecular basis for
the uniporter’s selectivity, mechanistic basis for inhibition by Ru360, and why the animal uniporter requires
EMRE are unclear. Recently, we have solved the NMR structure of the channel-forming region of MCU using
a N-Terminal Domain (NTD) truncated but fully functional construct from C. elegans, named cMCU-NTD.
The structure, which represents a new architecture for Ca2+ channel, provides the much-needed framework
for addressing the above questions. Aims 1 and 2 will use DdMCU, which is active by itself, to address the
open state of MCU, whereas Aims 3 and 4 will focus on animal MCU and EMRE, to address the mechanism
of how EMRE activates the MCU channel. Specifically, in Aim 1, we will perform systematic but hypothesis-
driven mutagenesis of DdMCU to identify key residues important for the function and pharmacologic
properties of this channel. In Aim 2, we will determine the structure of DdMCU using independent structural
approaches including crystallography, EM, and NMR. In Aim 3, we will investigate the biochemistry and
topology of EMRE and perform systematic mutagenesis to identify protein regions critical for MCU-EMRE
interaction. Finally, in Aim 4, we will investigate Ca2+ and Ru360 binding to MCU using the cMCU-NTD NMR
system, and perform biochemical and structural characterization of MCU-EMRE interaction, including solving
the high resolution structure of EMRE alone in lipid bilayer. The proposed studies will yield deep insights into
the molecular architecture and mechanisms of the uniporter that promise to have implications for a range of
human diseases.

## Key facts

- **NIH application ID:** 9847983
- **Project number:** 5R01HL130143-04
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** JAMES Jeiwen CHOU
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $787,708
- **Award type:** 5
- **Project period:** 2017-02-01 → 2021-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9847983

## Citation

> US National Institutes of Health, RePORTER application 9847983, Molecular Architecture Of The Mitochondrial Calcium Uniporter (5R01HL130143-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9847983. Licensed CC0.

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