Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter

Abstract

The calcium (Ca 2+ ) uniporter of mitochondria is a holocomplex consisting of the Ca 2+ -conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca 2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn 2+ ), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn 2+ specifically, whereas in the WT the two rings bind Mn 2+ cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca 2+ can specifically displace the bound Mn 2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn 2+ binding from the solventaccessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.) uptake by mitochondria is a long-known physiological phenomenon that is important for regulating various cellular activities such as aerobic metabolism and cell death (1-3). This uptake of Ca 2+ is achieved by a Ca 2+ uniporter whose activity can be blocked by ruthenium red (RuR) or ruthenium 360 (Ru360) (4). It was later shown by patch-clamping the inner mitochondrial membrane that this uniporter achieves remarkably high Ca 2+ conductance and selectivity (5). Only about 5 y ago was the molecular identity of the channel component of the uniporter identified using integrative genomics approaches (6, 7). This channel is commonly referred to as the mitochondrial calcium uniporter (MCU). In metazoans, however, the uniporter is much more complex than just the pore-forming subunit; it is a holocomplex (also called a uniplex) containing other regulatory components. Among them, the single-pass transmembrane (TM) protein EMRE (Essential MCU REgulator) is absolutely required for the MCU to conduct ions (8). Two soluble components in the intermembrane space, MICU1 and MICU2, are Ca 2+ -sensing proteins that gate the activity of MCU based on outside Ca 2+ concentrations through EMRE (9-12). The current consensus is that the minimum requirements for uniporter-mediated Ca 2+ flux in metazoans are MCU and EMRE, whereas in lower organisms such as Dictyostelium that lack EMRE, MCU i...

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening

“…MCU contains two trans-membrane helices, with amino and carboxyl termini facing into the matrix, with a short hydrophilic stretch of amino acids (YSWDI) linking them at the mouth of the channel facing the mitochondrial intermembrane space (Baughman et al, 2011; De Stefani et al, 2011). This region of the channel has highly conserved acidic residues, including an aspartate in position 261 that is involved in Ca 2+ permeation and selectivity (Cao et al, 2017; Chaudhuri and Clapham, 2014; Oxenoid et al, 2016). To predict a drug-target interaction and to identify key residues, we performed molecular docking using the YSWDI motif in the pentameric C. elegans MCU pore structure (Oxenoid et al, 2016) (Figure 7E).…”

Progress in understanding mitochondrial calcium uniporter complex‐mediated calcium signalling: A potential target for cancer treatment

“…Previously, the uniplex modulators were limited to lanthanides and ruthenium-related compounds, which had been shown to inhibit the (Anghileri, 1975;Cao et al, 2017;Kirichok et al, 2004;Moore, 1971). Ru360 and RuR ( (Cao et al, 2017).…”

“…The ubiquitously expressed MCU is a~35 kDa protein containing two transmembrane domains (TMDs) that are linked by a highly conserved sequence ( Figure 1b). The channel formed by MCU is sensitive to ruthenium red (RuR), RU360, and lanthanides (Cao, Wang, Cui, Su, & Chou, 2017;Crompton, Heid, Baschera, & Carafoli, 1979;De Stefani et al, 2011). The channel formed by MCU is sensitive to ruthenium red (RuR), RU360, and lanthanides (Cao, Wang, Cui, Su, & Chou, 2017;Crompton, Heid, Baschera, & Carafoli, 1979;De Stefani et al, 2011).…”

“…This conserved sequence faces the intermembrane space (IMS), while both the N-and C-terminal regions of MCU protrude into the mitochondria matrix (Baughman et al, 2011). The channel formed by MCU is sensitive to ruthenium red (RuR), RU360, and lanthanides (Cao, Wang, Cui, Su, & Chou, 2017;Crompton, Heid, Baschera, & Carafoli, 1979;De Stefani et al, 2011). The high affinity of MCU binding to Ca 2+ (K D < 2 nM) relies on its WDXXEP motif located between TM1 and TM2 (X denotes hydrophobic amino acids, WDIMEP in humans).…”

Pharmacological modulation of mitochondrial calcium homeostasis

“…Altogether, these observations confirmed that MCU is the Ca 2+ -conducting pore of the long-sought mitochondrial uniporter. Moreover, recent studies on the structural biology (Oxenoid et al 2016), coordination chemistry and molecular dynamics (Cao et al 2017) of MCU from Caenorhabditis elegans suggested that a functional channel results from the pentameric assembly of MCU multimers stabilized by a coiled-coil motif protruding into the mitochondrial matrix. In this structural model, the DIME motif forms the selectivity filter of the channel, whereby ion permeation and inhibitor binding occur.…”