SLC25A23 Antibody, HRP conjugated

What is SLC25A23 protein and what cellular functions does it perform?

SLC25A23, also known as SCaMC-3, APC2, MCSC2, or MGC2615, is a 467-468 amino acid mitochondrial inner membrane protein with a calculated molecular weight of 52 kDa and observed molecular weight of 49-54 kDa . The protein has a distinctive bipartite structure characteristic of calcium-binding mitochondrial solute carriers (CaMSC), featuring three canonical EF-hand calcium-binding domains in the amino-terminal portion . SLC25A23 functions primarily as an ATP-Mg/P(i) exchanger, regulating the transport of magnesium-bound ATP across the mitochondrial inner membrane in exchange for phosphate . This exchange mechanism facilitates the net uptake or efflux of adenine nucleotides into or from mitochondria, making it essential for maintaining mitochondrial energy metabolism and cellular homeostasis .

Recent research has revealed that SLC25A23 augments mitochondrial Ca²⁺ uptake through interaction with the mitochondrial Ca²⁺ uniporter (MCU) and MICU1, which are key components of the mitochondrial calcium uptake machinery . Knockdown studies demonstrate that SLC25A23 reduction decreases mitochondrial Ca²⁺ uptake while also attenuating basal mitochondrial reactive oxygen species (mROS) accumulation and reducing oxidant-induced ATP decline and cell death .

What is the tissue distribution profile of SLC25A23?

SLC25A23 displays a distinctive tissue expression pattern with predominant expression in:

Western blot analyses have specifically detected SLC25A23 in mouse pancreas tissue, mouse skeletal muscle tissue, mouse testis tissue, and SH-SY5Y cells . Additionally, immunohistochemistry has successfully detected this protein in human pancreas tissue . This tissue distribution pattern suggests specialized roles in tissues with high energy demands and calcium signaling requirements.

What are the optimal applications and dilutions for SLC25A23 antibodies?

SLC25A23 antibodies have been validated in multiple experimental applications with specific recommended dilutions:

For optimal results in IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may be used as an alternative . It is strongly recommended that researchers titrate the antibody in each specific testing system to obtain optimal results, as sensitivity can be sample-dependent . HRP-conjugated formats of these antibodies are particularly useful for direct detection in Western blot applications without the need for secondary antibodies.

How does SLC25A23 mechanistically interact with the mitochondrial calcium uptake machinery?

SLC25A23 physically and functionally interacts with the core components of the mitochondrial calcium uniporter complex. Co-immunoprecipitation experiments have demonstrated that Flag-tagged SLC25A23 interacts with GFP-tagged MCU and HA-tagged MICU1 . When GFP-tagged MCU was immunoprecipitated, SLC25A23 was pulled down, confirming their physical association . Similarly, HA-tagged MICU1 successfully pulled down SLC25A23, establishing that SLC25A23 forms protein complexes with both MCU and MICU1 .

Functionally, SLC25A23 appears to increase the activity of MCU (IMCU), suggesting it may serve as a positive regulator of the uniporter complex . The calcium-binding EF-hand domains of SLC25A23 are critical for this function, as expression of SLC25A23 EF-hand domain mutants exhibits a dominant-negative phenotype characterized by reduced mitochondrial Ca²⁺ uptake . This indicates that the calcium-sensing ability of SLC25A23 directly influences the calcium uptake capacity of mitochondria.

Methodologically, researchers investigating these interactions should consider combining co-immunoprecipitation with calcium imaging techniques using indicators such as rhod-2 AM or genetically encoded calcium sensors like GCaMP2-mt to comprehensively assess both physical interactions and functional outcomes .

What experimental approaches can researchers use to study the effects of SLC25A23 manipulation on mitochondrial function?

Researchers can employ several complementary approaches to investigate SLC25A23's role in mitochondrial function:

• Gene Silencing Techniques:

  • shRNA-mediated knockdown with validated constructs (e.g., clone #864 for maximum knockdown, clone #863 for moderate effects)

  • siRNA targeting specific regions of SLC25A23 mRNA

  • Rescue experiments with shRNA-insensitive SLC25A23 cDNA to confirm specificity

• Calcium Flux Measurements:

  • Chemical calcium indicators (e.g., rhod-2 AM for mitochondrial calcium, Fluo-4 for cytosolic calcium)

  • Genetically encoded calcium sensors targeted to mitochondria (e.g., GCaMP2-mt)

  • Extended time-course measurements (>1000 seconds) to evaluate cytosolic calcium clearance

• Mitochondrial Membrane Potential (ΔΨm) Assessment:

  • Fluorescent indicators such as TMRE and rhodamine 123

  • Real-time monitoring during calcium challenge experiments

  • Correlation of membrane potential changes with calcium uptake capacity

• Reactive Oxygen Species Measurements:

  • Mitochondrial-specific ROS indicators

  • Assessment of basal and stimulus-induced ROS production

  • Correlation with calcium uptake parameters

• ATP Production Assays:

  • Measurement of ATP levels before and after oxidative challenge

  • Assessment of ATP-Mg/Pi exchange rates in isolated mitochondria or reconstituted liposomes

When analyzing experimental data, researchers should note that SLC25A23 knockdown preserves ΔΨm during calcium challenge compared to control cells, suggesting a protective role against calcium overload-induced mitochondrial dysfunction .

How do calcium-binding EF-hand domains influence SLC25A23 function and experimental outcomes?

The calcium-binding EF-hand domains of SLC25A23 are crucial determinants of its functional activity. Similar to its paralog SLC25A25, SLC25A23 likely undergoes significant conformational changes upon calcium binding . These conformational changes directly impact its transport activity, as demonstrated by increased uptake rates of radio-labeled [¹⁴C]-Mg-ATP into proteoliposomes containing reconstituted SLC25A25 after calcium addition .

Experimental evidence supporting the critical nature of these domains includes:

• Dominant-negative effects: Expression of SLC25A23 EF-hand domain mutants results in reduced mitochondrial Ca²⁺ uptake, even in the presence of endogenous wild-type protein .

• Thermal shift assays: Similar studies with the paralog SLC25A25 show that calcium binding changes the protein's thermal stability profile from a heterogeneous population (multiple apparent melting temperatures) to a homogeneous one (single apparent melting temperature), indicating calcium-induced conformational uniformity .

• Functional consequences: The calcium-dependent activation of transport activity suggests that SLC25A23 functions as a calcium-responsive metabolite carrier, linking calcium signaling to mitochondrial bioenergetics .

For researchers designing experiments involving SLC25A23, it is advisable to consider:

• Creating point mutations in individual EF-hand domains to assess their relative contributions

• Performing experiments under varying calcium concentrations to determine dose-dependent effects

• Conducting parallel measurements of transport activity and calcium binding to establish direct correlations

• Using purified protein in reconstituted systems to directly measure calcium-dependent conformational changes

What is the relationship between SLC25A23 and reactive oxygen species (ROS) production in mitochondria?

SLC25A23 plays a significant role in regulating mitochondrial reactive oxygen species (mROS) production, with important implications for cellular stress responses and survival. Experimental evidence from knockdown studies has revealed that SLC25A23 depletion lowers basal mROS accumulation . This reduction in basal oxidative stress appears to be protective, as SLC25A23 knockdown cells also show attenuated oxidant-induced ATP decline and reduced cell death when challenged with oxidative stressors .

The mechanism linking SLC25A23 to ROS production appears to involve its role in mitochondrial calcium homeostasis. Since mitochondrial calcium overload is a known trigger for ROS production, SLC25A23's function in augmenting calcium uptake likely contributes to baseline ROS levels. Supporting this connection, reconstitution with shRNA-insensitive SLC25A23 cDNA restores both mitochondrial Ca²⁺ uptake and superoxide production in knockdown cells .

For researchers investigating this relationship, recommended experimental approaches include:

• Simultaneous measurement of mitochondrial calcium uptake and ROS production in the same experimental system

• Time-course analysis to determine whether calcium changes precede ROS alterations

• Pharmacological interventions with calcium chelators or antioxidants to dissect causality

• Site-directed mutagenesis of calcium-binding domains to determine their specific roles in ROS regulation

• Metabolic flux analysis to connect changes in ATP-Mg/Pi exchange with ROS production

Importantly, when designing experiments to investigate SLC25A23's role in oxidative stress, researchers should control for potential confounding factors such as changes in mitochondrial membrane potential, which can independently affect both calcium uptake and ROS production.

What are the best practices for using HRP-conjugated SLC25A23 antibodies in advanced research applications?

HRP-conjugated SLC25A23 antibodies offer significant advantages for research applications requiring direct detection without secondary antibodies. To maximize the utility of these reagents in advanced research applications, consider the following methodological recommendations:

When working specifically with SLC25A23 detection in mitochondria, researchers should be aware that fixation methods can significantly impact antibody accessibility to this inner membrane protein. For optimal detection in immunofluorescence and immunohistochemistry applications, mild permeabilization steps and appropriate fixation protocols should be empirically determined for each cell type or tissue.

Additionally, when detecting SLC25A23 in tissues with high expression (brain, skeletal muscle, and pancreas) , lower antibody concentrations may be sufficient, while tissues with lower expression levels may require more sensitive detection methods or signal amplification strategies.

How can researchers distinguish between SLC25A23 and related family members in functional studies?

Distinguishing between SLC25A23 and closely related family members (SLC25A24, SLC25A25) requires careful experimental design and validation approaches:

• Antibody Specificity Validation:

  • Verify antibody specificity through Western blot analysis of tissues with differential expression patterns

  • Use knockdown/knockout controls to confirm specificity, similar to the validation showing that SLC25A23 #864 shRNA did not affect SLC25A24 or SLC25A25 mRNA levels

  • Consider using multiple antibodies targeting different epitopes

• Functional Differentiation:

  • Design experiments exploiting known functional differences, such as the observation that SLC25A23 knockdown, but not SLC25A24 or SLC25A25 knockdown, reduces mitochondrial Ca²⁺ uptake

  • Use GCaMP2-mt fluorescence assays after histamine stimulation to distinguish functional roles in calcium handling

  • Measure cytosolic Ca²⁺ clearance rates using Fluo-4 over extended time intervals (>1000s)

• Gene-Specific Manipulations:

  • Employ highly specific shRNA or siRNA constructs validated for target selectivity

  • Conduct parallel knockdown experiments of each family member to compare phenotypes

  • Perform rescue experiments with shRNA-insensitive constructs to confirm specificity of observed effects

• Molecular Characterization:

  • Use RT-qPCR to quantify relative expression levels of all three family members in the experimental system

  • Consider the use of isoform-specific primers to detect alternative splice variants

It is worth noting that functional studies have demonstrated clear differences among these family members, with SLC25A23 knockdown specifically affecting mitochondrial calcium uptake while SLC25A24 and SLC25A25 knockdowns showed no significant alterations in this parameter .

What experimental controls are essential when studying SLC25A23's role in mitochondrial calcium regulation?

When investigating SLC25A23's role in mitochondrial calcium regulation, several critical controls must be included to ensure reliable and interpretable results:

• Knockdown/Expression Validation Controls:

  • Quantitative RT-PCR to confirm specific reduction of SLC25A23 mRNA without affecting related family members (SLC25A24, SLC25A25)

  • Western blot confirmation of protein level changes

  • Rescue experiments with shRNA-insensitive SLC25A23 cDNA to confirm phenotype specificity

• Mitochondrial Integrity Controls:

  • Measurements of basal mitochondrial membrane potential (ΔΨm) using indicators such as TMRE or rhodamine 123 to ensure knockdown does not alter the driving force for calcium uptake

  • Assessment of mitochondrial morphology to rule out structural changes affecting calcium handling

  • Confirmation of other mitochondrial functions (e.g., respiration) to isolate calcium-specific effects

• Calcium Signaling Controls:

  • Parallel measurements of cytosolic and mitochondrial calcium using specific indicators (e.g., Fluo-4 for cytosolic, rhod-2 AM for mitochondrial)

  • Extended time-course measurements to assess both immediate responses and delayed effects on calcium homeostasis

  • Use of calcium chelators and ionophores as positive and negative controls

• Specificity Controls for Protein Interactions:

  • Reciprocal co-immunoprecipitation experiments when studying interactions with MCU and MICU1

  • Use of known interaction partners (e.g., MCU-MICU1) as positive controls

  • Inclusion of irrelevant proteins as negative controls

• Functional Consequence Controls:

  • Assessment of ΔΨm during calcium challenge to connect calcium uptake to potential downstream effects

  • Measurement of ROS production and ATP levels to link calcium regulation to bioenergetic outcomes

  • Cell viability assays to determine physiological relevance of observed molecular changes

These controls collectively ensure that observed effects on calcium regulation can be specifically attributed to SLC25A23 function rather than to indirect effects or experimental artifacts.

How can researchers leverage SLC25A23 antibodies to study mitochondrial dynamics and bioenergetics?

SLC25A23 antibodies, particularly HRP-conjugated versions, offer powerful tools for investigating the intersection of calcium signaling, metabolite transport, and mitochondrial bioenergetics:

• Live-Cell Imaging Applications:

  • Combining immunofluorescence detection of SLC25A23 with live-cell calcium imaging can reveal spatial relationships between protein localization and functional calcium microdomains

  • Co-localization studies with markers of mitochondrial fission/fusion machinery can explore whether SLC25A23 distribution changes during dynamic mitochondrial rearrangements

• Bioenergetic Analysis:

  • Correlation of SLC25A23 expression levels with mitochondrial respiration parameters measured by Seahorse extracellular flux analysis

  • Investigation of how calcium-dependent activation of SLC25A23 influences ATP production under different metabolic conditions

  • Assessment of how SLC25A23-mediated ATP-Mg/Pi exchange affects mitochondrial ATP content and cytosolic ATP availability

• Stress Response Studies:

  • Examination of how oxidative stress modifies SLC25A23 expression, localization, and function

  • Investigation of whether post-translational modifications of SLC25A23 occur during cellular stress responses

  • Assessment of SLC25A23's role in calcium-dependent mitochondrial adaptation to changing metabolic demands

• Disease Model Applications:

  • Analysis of SLC25A23 expression and function in neurodegenerative disease models, given its high expression in brain tissue

  • Investigation of SLC25A23's contribution to pancreatic β-cell function and potential role in diabetes, based on its high pancreatic expression

  • Exploration of whether alterations in SLC25A23 contribute to skeletal muscle pathologies

• Therapeutic Target Assessment:

  • Screening for compounds that modulate SLC25A23 activity as potential tools for manipulating mitochondrial calcium and energy metabolism

  • Evaluation of whether existing drugs with mitochondrial effects interact with or regulate SLC25A23 function

These advanced applications will benefit from combining antibody-based detection methods with functional assays of mitochondrial calcium uptake, ATP transport, and metabolic activity to provide comprehensive insights into SLC25A23's integrative role in cellular energetics.

What are the implications of SLC25A23's interaction with the MCU complex for experimental design?

The discovery that SLC25A23 interacts with the mitochondrial calcium uniporter (MCU) complex components has significant implications for experimental design and interpretation:

• Complex Composition Analysis:

  • When studying MCU complex composition, researchers should consider SLC25A23 as a potential component or regulator

  • Co-immunoprecipitation experiments should test for SLC25A23 alongside established components (MCU, MICU1/2/3, EMRE, etc.)

  • Blue native PAGE followed by Western blotting can help determine whether SLC25A23 is part of the core complex or acts as an accessory protein

• Functional Studies of MCU:

  • Interpretation of MCU functional data should consider potential regulation by SLC25A23

  • When measuring MCU current (IMCU), researchers should account for potential modulation by SLC25A23 expression levels

  • Experiments manipulating MCU expression should monitor potential compensatory changes in SLC25A23

• Calcium Signaling Pathway Integration:

  • Experimental designs should incorporate analysis of both cytosolic and mitochondrial calcium dynamics to capture the full impact of SLC25A23-MCU interactions

  • The influence of SLC25A23 on cytosolic calcium clearance kinetics should be considered when interpreting calcium signaling data

  • Calcium concentration-response studies can help determine whether SLC25A23 alters the calcium sensitivity of the MCU complex

• Mitochondrial Membrane Potential Considerations:

  • Since SLC25A23 knockdown preserves ΔΨm during calcium challenge, experimental designs should include membrane potential measurements alongside calcium uptake assays

  • Time-resolved measurements can help establish causal relationships between calcium influx, membrane potential changes, and metabolic outcomes

• Tissue-Specific Variations:

  • Given SLC25A23's differential expression across tissues, experimental designs should account for potential tissue-specific functions of the MCU-SLC25A23 interaction

  • Comparisons between high-expressing tissues (brain, skeletal muscle, pancreas) and low-expressing tissues may reveal context-dependent roles

Understanding these implications will allow researchers to design more comprehensive experiments that capture the full complexity of mitochondrial calcium regulation and its downstream effects on cellular physiology.

What are the emerging research questions regarding SLC25A23 that remain to be addressed?

Despite significant advances in understanding SLC25A23's structure and function, several important questions remain unresolved:

• Structural Biology:

  • What is the precise atomic structure of SLC25A23, particularly in calcium-bound and calcium-free states?

  • How does calcium binding to the EF-hand domains trigger conformational changes that affect transport activity?

  • What are the specific interaction interfaces between SLC25A23 and MCU complex components?

• Regulatory Mechanisms:

  • How is SLC25A23 expression regulated at the transcriptional and post-transcriptional levels?

  • Are there post-translational modifications that modulate SLC25A23 function?

  • Do specific signaling pathways target SLC25A23 to regulate its activity?

• Physiological Functions:

  • What is the physiological significance of SLC25A23's tissue-specific expression pattern?

  • How does SLC25A23-mediated ATP-Mg/Pi exchange contribute to tissue-specific energy metabolism?

  • What is the evolutionary conservation of SLC25A23 function across species?

• Pathological Implications:

  • Is SLC25A23 dysfunction involved in mitochondrial diseases or metabolic disorders?

  • Could targeting SLC25A23 provide therapeutic benefits in conditions characterized by calcium dysregulation or bioenergetic deficits?

  • Are there disease-associated variants of SLC25A23 with functional consequences?

• Technical Developments:

  • What new tools and approaches can be developed to study SLC25A23 dynamics in living cells with greater spatial and temporal resolution?

  • How can HRP-conjugated antibodies be optimized for advanced imaging applications?

  • What high-throughput approaches might identify modulators of SLC25A23 function?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, cell biology, and physiology, ultimately advancing our understanding of mitochondrial calcium regulation and bioenergetics in health and disease.

How can researchers integrate SLC25A23 studies with broader mitochondrial research?

Integrating SLC25A23 research into the broader context of mitochondrial biology offers opportunities for synergistic discoveries:

• Systems Biology Approaches:

  • Incorporate SLC25A23 into computational models of mitochondrial calcium handling and metabolism

  • Use network analysis to identify functional relationships between SLC25A23 and other mitochondrial proteins

  • Develop multi-omics approaches to correlate SLC25A23 expression with global metabolic profiles

• Cross-Disciplinary Integration:

  • Connect SLC25A23 function to mitochondrial dynamics (fission/fusion) research

  • Explore links between SLC25A23-mediated calcium regulation and mitochondrial quality control mechanisms

  • Investigate the relationship between SLC25A23 activity and mitochondrial-nuclear communication

• Translational Applications:

  • Examine SLC25A23 expression and function in patient-derived samples across various diseases

  • Develop SLC25A23-targeted interventions for conditions involving mitochondrial dysfunction

  • Explore SLC25A23 as a biomarker for mitochondrial health in clinical settings

• Methodological Innovations:

  • Develop CRISPR/Cas9-based approaches for precise genome editing of SLC25A23

  • Create improved fluorescent reporters for real-time monitoring of SLC25A23 activity

  • Design engineered variants of SLC25A23 with altered calcium sensitivity or transport selectivity

• Collaborative Research Frameworks:

  • Establish repositories of validated reagents and protocols for SLC25A23 research

  • Develop standardized assays for comparing results across different experimental systems

  • Create interdisciplinary research consortia focused on mitochondrial calcium transporters