Recombinant Macaca fascicularis Magnesium transporter MRS2 homolog, mitochondrial (MRS2)

What is MRS2 and how does it function in mitochondrial magnesium transport?

MRS2 (Mitochondrial RNA Splicing 2) forms a magnesium (Mg²⁺) entry protein channel in the inner mitochondrial membrane. The protein contains two transmembrane domains that constitute a pore, while most of the protein resides within the mitochondrial matrix as an amino terminal domain (NTD) . Unlike its bacterial ortholog CorA which forms a pentameric assembly, the human MRS2 NTD self-associates into a homodimer . The channel functions through a mechanism that involves regulation by its NTD, which appears to provide negative feedback when bound to magnesium ions .

Structurally, MRS2 features a narrow, electropositive R332 ring that creates an energy barrier controlling cation permeation. Research has demonstrated that replacing R332 with a smaller polar amino acid (serine) significantly facilitates cation conduction by reducing this energy barrier .

How does the amino terminal domain (NTD) regulate MRS2 function?

The NTD of MRS2 plays a crucial regulatory role through a negative feedback mechanism. When Mg²⁺ binds to the NTD, it disrupts homomeric interactions and inhibits mitochondrial Mg²⁺ uptake . This autoregulation mechanism has been demonstrated through experiments where:

• Mg²⁺ and calcium suppress lower and higher order oligomerization of the MRS2 NTD

• Mutating residues that mediate Mg²⁺ binding to the NTD selectively decreases Mg²⁺-binding affinity approximately sevenfold

• Disruption of NTD Mg²⁺ binding potentiates mitochondrial Mg²⁺ uptake in both wild-type and Mrs2 knockout cells

These findings reveal an important self-regulatory mechanism where MRS2 responds to changing Mg²⁺ levels to maintain appropriate mitochondrial magnesium homeostasis.

How does MRS2 differ from its bacterial ortholog CorA?

Unlike CorA, MRS2 shows much slower kinetics, which may be advantageous for maintaining mitochondrial membrane potential while still facilitating necessary ion transport .

What vectors are effective for studying MRS2 in non-human primate models?

Canine adenovirus type 2 (CAV-2) vectors have proven to be highly effective gene transfer tools in the Macaca fascicularis brain. Research has demonstrated their:

• Strong neuronal tropism

• Effective retrograde transport capabilities

• Reliable biodistribution throughout neural networks

• High transduction efficiency

In one study, CAV-2 vectors expressing GFP were injected into the left putamen of M. fascicularis, resulting in GFP expression within cell bodies and projections with neuron-like morphology. The GFP signal was detected in the putamen and caudate, corresponding to a volume of 540 mm³ (approximately 47% of total striatum of 1,140 mm³), with an approximate ratio between injected volume and transduced volume of 1:9 .

The vector efficacy is likely related to the neuronal expression of the coxsackievirus and adenovirus receptor (CAR) in the primate brain, although more research is needed on CAR expression patterns specifically in M. fascicularis .

What techniques are most effective for measuring MRS2-mediated magnesium transport?

Several complementary techniques have proven valuable for assessing MRS2-mediated magnesium transport:

• Patch-clamp electrophysiology: This technique allows direct measurement of ion currents through MRS2. When studying the R332S mutant of MRS2, researchers observed μA levels of Mg²⁺ currents that were reduced by cobalt hexammine (a known CorA inhibitor) with an IC₅₀ of 0.3 mM .

• Radioisotope flux assays: Using ²⁸Mg to track influx into mitochondria provides quantitative data on transport rates. Studies have noted slow ²⁸Mg influx into mitochondria, consistent with the limited MRS2 ion conduction rate .

• Fluorescent magnesium indicators: These allow real-time monitoring of magnesium transport in intact cells or isolated mitochondria.

• Dynamic light scattering (DLS): This technique has been used to study the oligomerization state of MRS2 and how it changes in response to different divalent cations. For example, researchers observed that MRS2 58-443 samples showed autocorrelation functions with decay times corresponding to distributions centered at ~4 and ~20 nm at 37°C .

How can protein-protein interactions of MRS2 be assessed experimentally?

To study MRS2 protein-protein interactions, researchers have employed several approaches:

• Dynamic light scattering (DLS): This technique revealed that MRS2 NTD self-associates into homodimers. DLS also demonstrated that Mg²⁺ and Ca²⁺ suppress lower and higher order oligomerization of MRS2 NTD, while cobalt has no effect on the NTD but disassembles full-length MRS2 .

• Size-exclusion chromatography: This complements DLS by separating protein complexes based on their molecular size.

• Co-immunoprecipitation: This allows investigation of MRS2 interactions with other mitochondrial proteins.

• Structural analysis using cryo-electron microscopy: This has helped reveal key structural features of MRS2, such as the narrow, electropositive R332 ring that creates an energy barrier controlling cation permeation .

When conducting these studies, it's important to note that experimental buffer conditions can significantly impact results. For example, studies with full-length MRS2 (excluding the mitochondrial targeting sequence) used buffers containing CHAPS, which showed autocorrelation functions consistent with ~1-1.5 nm micelles at 37°C .

How might MRS2 function relate to neurological diseases such as Parkinson's disease?

The relationship between MRS2 function and neurological diseases, particularly Parkinson's disease (PD), represents an emerging area of research. Although the direct link between MRS2 and PD has not been fully established, several lines of evidence suggest potential connections:

• Parkinson's disease is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction is a well-established factor in PD pathogenesis .

• MRS2, as a critical regulator of mitochondrial Mg²⁺ homeostasis, may influence mitochondrial function in neurons. Mg²⁺ is an important protein-stabilizing cofactor within mitochondria, forms biologically functional Mg²⁺-ATP complexes, and regulates crucial enzymatic activities .

• Research has demonstrated that CAV-2 vectors can be used to express leucine-rich repeat kinase 2 (LRRK2 G2019S), one of the most common genetic mutations causing Parkinson's disease, in the Macaca fascicularis brain. This approach could help model the neurodegenerative processes of this genetic subtype of Parkinson's disease in monkeys .

Studies have shown that following unilateral HD-LRRK2 G2019S injection into the substantia nigra, LRRK2 G2019S increased phosphorylated Tau (pTau Ser395/Ser404) levels, which may be relevant to neurodegeneration processes .

What is the interplay between MRS2-mediated magnesium transport and calcium signaling?

The relationship between MRS2-mediated magnesium transport and calcium signaling is complex and bidirectional:

• Regulatory effects of calcium on MRS2: Unlike CorA (which is inhibited by cytoplasmic Mg²⁺), MRS2 is not regulated by matrix Mg²⁺ but is instead inactivated by Ca²⁺ . This suggests that mitochondrial calcium signaling directly modulates magnesium influx.

• MRS2 as a nonselective cation channel: Recent research has revealed that MRS2 functions as a Ca²⁺-regulated nonselective cation channel, permeable not only to Mg²⁺ but also to Ca²⁺, K⁺, and Na⁺. This has profound implications for mitochondrial ion homeostasis beyond just magnesium transport .

• Competition between Mg²⁺ and Ca²⁺: Mg²⁺ has the capability to compete with Ca²⁺ for binding sites on proteins and membranes. This competition significantly influences intracellular Ca²⁺ dynamics and affects Ca²⁺-dependent processes within mitochondria .

• Experimental observations: Studies have shown that MRS2RS-mediated Na⁺ currents (100 mM Na⁺) are not affected by submicromolar levels of Mg²⁺ or Ca²⁺, suggesting that MRS2 does not selectively transport Mg²⁺ or Ca²⁺ under these conditions .

How do mutations in the MRS2 magnesium-binding domain affect channel function?

Mutations in the MRS2 magnesium-binding domain can dramatically alter channel function, as demonstrated by several key studies:

• Mutating specific residues that mediate Mg²⁺ binding to the NTD selectively decreases Mg²⁺-binding affinity approximately sevenfold and abrogates Mg²⁺ binding-induced changes in secondary, tertiary, and quaternary structure .

• Disruption of NTD Mg²⁺ binding significantly potentiates mitochondrial Mg²⁺ uptake in both wild-type and Mrs2 knockout cells, demonstrating that these mutations can create gain-of-function effects .

• Replacement of R332 with a smaller polar amino acid, serine (R332S), produced μA levels of Mg²⁺ currents, demonstrating that charge neutralization and reduced side chain size at this constriction site greatly facilitated cation conduction .

• Alanine substitution at N314 (part of the conserved GMN motif that generates a divalent cation binding site) affects the channel's ion selectivity properties .

These findings reveal that the magnesium-binding domain plays a critical regulatory role in MRS2 function, and targeted mutations can create channels with altered conductance, selectivity, and regulatory properties.

What controls should be included when studying MRS2 in expression systems?

When studying MRS2 in expression systems, the following controls are essential for valid data interpretation:

• Empty vector controls: Essential for distinguishing between effects of MRS2 expression versus effects of the expression system itself. For instance, in electrophysiology studies, it was shown that Mg²⁺ currents were not observed in oocytes without MRS2 expression or in oocytes expressing the mitochondrial Ca²⁺ uniporter .

• Known channel inhibitor controls: Cobalt hexammine, a known CorA inhibitor, reduced MRS2-mediated currents with an IC₅₀ of 0.3 mM . Including such inhibitors helps confirm that observed effects are specifically due to MRS2 activity.

• Cross-species orthologs: Comparing the function of MRS2 with TmCorA provides important context for interpreting results, as both showed different levels of surface expression and functional properties .

• Buffer composition controls: The experimental buffer for full-length MRS2 (excluding the mitochondrial targeting sequence) should include appropriate detergents like CHAPS, as this affects protein stability and function .

• Temperature controls: Studies have shown that temperature affects MRS2 function, with experiments typically conducted at physiologically relevant temperatures (37°C) .

How can researchers address the challenge of studying MRS2 in its native mitochondrial environment?

Studying MRS2 in its native mitochondrial environment presents several challenges that can be addressed through these methodological approaches:

How should researchers interpret the seemingly contradictory data about MRS2 ion selectivity?

The apparent contradictions in data regarding MRS2 ion selectivity can be reconciled by considering:

• Experimental conditions: Different studies may use varying ionic conditions, which can significantly affect channel behavior. For instance, MRS2 exhibits the anomalous mole fraction effect (AMFE) where, in the presence of millimolar levels of Mg²⁺, additional Mg²⁺ entering the pore creates electrical repulsion between ions, leading to rapid Mg²⁺ permeation .

• Regulatory state of the channel: Ca²⁺ regulation of MRS2 means that the presence or absence of Ca²⁺ will dramatically alter the channel's behavior. MRS2 functions as a Ca²⁺-regulated nonselective cation channel .

• Structural considerations: The narrow, electropositive R332 ring creates an energy barrier that suppresses cation permeation in wild-type MRS2, whereas mutations at this position (e.g., R332S) dramatically increase conductance .

• Comparison with bacterial orthologs: Unlike CorA, which is more selective for Mg²⁺, MRS2 appears to have broader cation permeability. This difference may relate to the different physiological roles of these channels in bacteria versus mitochondria .

• Physiological context: MRS2's slow kinetics could be advantageous for mitochondrial function, as it could "mitigate the problem of mitochondrial inner-membrane potential dissipated by typical channels with fast kinetics" .

What are the promising approaches for studying MRS2 function in neurodegenerative disease models?

Several promising approaches could advance our understanding of MRS2 function in neurodegenerative disease models:

• CAV-2 vector-mediated gene transfer: These vectors have demonstrated efficacy in the Macaca fascicularis brain and could be used to express both wild-type and mutant forms of MRS2 alongside disease-related genes such as LRRK2 G2019S .

• Combined PET imaging and histological analysis: This approach has been used to evaluate the effects of LRRK2 G2019S expression in primates and could be adapted to study the impact of MRS2 manipulation .

• Analysis of phosphorylated protein biomarkers: Following unilateral HD-LRRK2 G2019S injection into the substantia nigra, researchers observed increased pTau Ser395/Ser404 levels. Similar approaches could be used to assess how MRS2 manipulation affects neurodegenerative processes .

• Gain-of-function MRS2 mutants: The identification of mutations that disrupt NTD Mg²⁺ binding and potentiate mitochondrial Mg²⁺ uptake provides novel tools for investigating the role of enhanced mitochondrial Mg²⁺ in neurodegeneration .

• Integration with mitochondrial dynamics studies: Investigating how MRS2-mediated ion fluxes affect mitochondrial morphology, fission/fusion, and quality control mechanisms that are known to be dysregulated in neurodegenerative diseases.

How might MRS2's role in regulating mitochondrial cation homeostasis beyond magnesium be further explored?

The discovery that MRS2 functions as a Ca²⁺-regulated nonselective cation channel opens several new avenues for research:

• Investigation of Na⁺ and K⁺ cycles: MRS2 may contribute to mitochondrial Na⁺ and K⁺ uniport. These ions are imported into the matrix via slow, electrophoretic uniport mechanisms and are extruded via Na⁺/H⁺ and K⁺/H⁺ exchangers. MRS2's slow kinetics could make it well-suited for this role .

• Relationship to mitochondrial volume regulation: K⁺ plays a prominent role in regulating mitochondrial volume and inner membrane potential. Studies could investigate how MRS2-mediated K⁺ transport contributes to these processes .

• Impact on mitochondrial Ca²⁺-dependent processes: Na⁺ is known to regulate mitochondrial Ca²⁺-dependent processes, such as oxidative phosphorylation and cell death, by participating in Na⁺/Ca²⁺ antiport. Research could explore how MRS2-mediated Na⁺ transport affects these pathways .

• Development of MRS2-specific modulators: Creating compounds that specifically target MRS2 to modify its ion selectivity or regulation could provide valuable tools for understanding its physiological roles.

• Integration with whole-cell ionic homeostasis: Studies should explore how MRS2-mediated mitochondrial ion transport integrates with cytosolic ion homeostasis and cellular signaling pathways.

What technological advances could facilitate deeper understanding of MRS2 structure-function relationships?

Several emerging technologies could provide new insights into MRS2 structure-function relationships:

• Cryo-electron microscopy (cryo-EM): This technique has already revealed key structural features of MRS2, such as the narrow, electropositive R332 ring . Further advances in cryo-EM resolution could provide more detailed insights into conformational changes associated with ion permeation and regulatory binding events.

• Single-molecule fluorescence resonance energy transfer (smFRET): This could allow real-time observation of MRS2 conformational changes during ion permeation and in response to regulatory signals.

• Molecular dynamics simulations: As computational power increases, more sophisticated simulations of MRS2 in a lipid bilayer environment could predict ion permeation pathways and regulatory mechanisms.

• Optogenetic control of MRS2: Development of light-sensitive MRS2 variants could allow precise temporal control of channel function in living cells.

• CRISPR-mediated genome editing: This could facilitate the creation of animal models with specific MRS2 mutations, allowing for in vivo assessment of structure-function relationships in a physiological context.