Recombinant Mouse Transmembrane protein FAM155A (Fam155a)

What is mouse Fam155a and what is its relationship to human FAM155A?

Mouse Fam155a (Transmembrane protein FAM155A) belongs to the FAM155 family and is homologous to human FAM155A. In humans, this protein is also known as NALF1 (NALCN channel auxiliary factor 1) or NLF-1 . The protein plays a critical role as an auxiliary component of the NALCN channel complex. Sequence analysis shows conservation of key functional domains between mouse and human variants, particularly in regions involved in channel interaction. When designing experiments, researchers should consider these homologies when translating findings between species.

What are the primary protein interactions of mouse Fam155a?

Mouse Fam155a participates in several high-confidence protein interactions, with the strongest interactions observed with:

• Nalcn (Sodium leak channel non-selective protein) - 0.969 confidence score

• Unc79 (Protein unc-79 homolog) - 0.968 confidence score

• Unc80 (Protein unc-80 homolog) - 0.936 confidence score

Additional interactions with lower confidence scores include:

• Tafa2 (Chemokine-like protein TAFA-2) - 0.576 confidence score

• Nyap2 (Neuronal tyrosine-phosphorylated phosphoinositide-3-kinase adapter 2) - 0.531 confidence score

• Tmem179 (Transmembrane protein 179) - 0.522 confidence score

• Naaladl2 (N-acetylated alpha-linked acidic dipeptidase-like 2) - 0.514 confidence score

These interaction data highlight Fam155a's critical role in the NALCN channel complex formation.

How does Fam155a contribute to NALCN channel function?

Fam155a serves as an essential auxiliary component of the NALCN channel complex. The protein plays two key roles:

• Membrane localization: Fam155a is crucial for proper membrane localization of the NALCN channel. The NALCN-FAM155A subcomplex alone shows little surface localization, but when UNC79 and UNC80 are co-expressed, surface localization dramatically increases .

• Core complex formation: Fam155a and NALCN form the core complex of the channel. While this subcomplex can form without UNC79 and UNC80, the channel's activity essentially depends on the presence of both auxiliary proteins .

Without Fam155a, NALCN cannot properly localize to the cell membrane, severely limiting channel function. This relationship underscores Fam155a's importance in regulating neuronal excitability through its role in the NALCN complex.

What is the structural relationship between Fam155a and NALCN, and how does it influence channel function?

Recent cryo-electron microscopy (cryo-EM) studies have revealed the structural relationship between FAM155A and NALCN. Within the quaternary complex of NALCN-FAM155A-UNC79-UNC80, FAM155A and NALCN form the core channel complex . The structural studies show that:

• FAM155A interacts with specific domains of NALCN, influencing its membrane topology

• The interaction preserves NALCN's ability to form functional channels

• Recent high-resolution structures have identified a CTD-Interacting Helix (CIH) on the linker of NALCN domain II-III (D II-III)

The interaction between CTD and CIH appears unique to NALCN channels, as it has not been observed in related Na_V or Ca_V channels previously. Functional studies suggest this interaction may play a regulatory role, as mutations that disrupt the CIH-CTD interaction (such as the CIH-5A mutation combining I753A, L754A, R761A, R764A, and R765A) affect channel gating properties .

This structural arrangement suggests Fam155a doesn't just facilitate membrane localization but may also participate in regulating channel gating through its impacts on NALCN conformation.

What methodologies are most effective for expressing and purifying recombinant mouse Fam155a?

Based on research protocols used for NALCN complex studies, the following methodologies are recommended for recombinant mouse Fam155a expression and purification:

• Expression Systems:

  • HEK293T cells have been successfully used for co-expression of NALCN complex components

  • Xenopus oocytes provide an alternative system for functional expression

• Expression Tags:

  • 3×HA tag insertion at specific sites (such as after P1077 in NALCN) has been shown not to affect electrophysiological properties

  • This approach allows for tracking of surface localization using HA antibodies

• Co-expression Considerations:

  • For functional studies, co-expression of NALCN, FAM155A, UNC79, and UNC80 is necessary to reconstitute robust channel currents

  • For structural studies, co-purification of the entire complex may be necessary to maintain stability

• Functional Verification:

  • Surface expression can be quantified using antibody-based approaches targeting extracellular epitope tags

  • Electrophysiological recordings provide functional verification

When expressing Fam155a alone, protein stabilization may require optimization of buffer conditions and addition of stabilizing agents, as the protein normally exists in a complex with NALCN and other partners.

How do mutations in Fam155a affect NALCN channel complex assembly and function?

While the search results don't provide specific information about Fam155a mutations, we can infer their likely effects based on the protein's known functions:

• Impact on Complex Assembly:

  • Mutations in domains that interact with NALCN would likely disrupt the core complex formation

  • Alterations in regions that interact with UNC79 or UNC80 may affect the quaternary complex assembly

• Impact on Localization:

  • Since Fam155a is critical for membrane localization of NALCN, mutations that disrupt this function would lead to reduced surface expression of the channel complex

  • This would manifest as decreased channel currents in functional assays

• Disease Relevance:

  • The search results indicate that genetic mutations in NALCN channel components lead to neurodevelopmental diseases

  • By extension, pathogenic mutations in Fam155a would likely result in similar phenotypes

For researchers investigating Fam155a mutations, combining structural analysis with functional electrophysiology and cell biological approaches would provide comprehensive insights into mutation effects.

What experimental approaches can be used to study Fam155a-NALCN interactions?

Several complementary experimental approaches can be employed to study Fam155a-NALCN interactions:

• Biochemical Interaction Assays:

  • GST pull-down assays have been used successfully to demonstrate interactions between components of the NALCN complex

  • Co-immunoprecipitation can verify interactions in cell lysates

• Surface Expression Quantification:

  • Insertion of epitope tags (such as 3×HA) in extracellular loops followed by antibody labeling has been used to quantify surface localization

  • This approach allows assessment of how mutations or other perturbations affect trafficking

• Functional Electrophysiology:

  • Patch-clamp recording in heterologous expression systems (HEK293T cells, Xenopus oocytes)

  • G-V curve analysis to assess channel gating properties

• Structural Analysis:

  • Cryo-EM has been successfully used to determine the structure of the NALCN-FAM155A-UNC79-UNC80 quaternary complex

  • This approach provides atomic-level details of interaction interfaces

• Mutagenesis Studies:

  • Site-directed mutagenesis targeting specific residues (e.g., the CIH-5A mutation) can disrupt specific interactions to assess their functional importance

When designing these experiments, appropriate controls should include single component expressions and systematic omission of complex components to verify specificity of interactions.

What are the key considerations when designing knockout or knockdown studies of Fam155a?

When designing knockout or knockdown studies for Fam155a, researchers should consider:

• Model System Selection:

  • Cell lines: HEK293T cells have been used successfully for NALCN complex reconstitution

  • Animal models: Mouse models with inbred strains showing genetic diversity may be appropriate, similar to approaches used for other complex traits

• Knockout Verification:

  • Protein expression verification by western blot

  • Functional verification through electrophysiological assessment of NALCN currents

• Phenotypic Assessment:

  • Membrane localization of NALCN should be quantified, as this is a primary function of Fam155a

  • Electrophysiological properties, particularly sodium leak currents

  • Neuronal excitability parameters, given the role of NALCN in regulating resting membrane potential

• Compensatory Mechanisms:

  • Assess potential upregulation of related proteins

  • Consider conditional knockout approaches to avoid developmental compensation

• Rescue Experiments:

  • Include re-expression of wild-type Fam155a to confirm phenotype specificity

  • Consider expression of mutant forms to identify critical functional domains

Given that genetic complexity underlies many traits in inbred mouse strains, researchers should consider genetic background effects when interpreting knockout phenotypes .

How can structural data from cryo-EM studies of the NALCN-FAM155A complex be analyzed?

Analysis of cryo-EM structural data for the NALCN-FAM155A complex should follow these methodological steps:

• Map Quality Assessment:

  • Evaluate local resolution across different regions of the complex

  • Identify regions with poor map quality that might preclude confident assignment of molecular identities, as observed in some regions of the quaternary complex

• Structural Comparison:

  • Compare structures across different conformational states

  • Analyze similarities and differences with related channel complexes (Na_V or Ca_V channels)

  • Compare structures determined by different research groups to identify reliable features versus potential artifacts (as noted in the comparison that showed similarities in NALCN-FAM155A subcomplex and UNC79-UNC80 heterodimer, but differences in orientation)

• Interaction Interface Analysis:

  • Identify specific residues involved in protein-protein interactions

  • Correlate with functional data from mutagenesis studies

• Conformational Dynamics:

  • Analyze conformational heterogeneity in the dataset

  • Consider different orientations between NALCN-FAM155A subcomplex and UNC79-UNC80 heterodimer that might reflect functional states

• Integration with Functional Data:

  • Correlate structural features with electrophysiological properties

  • Use structure to generate hypotheses for mutagenesis studies

Researchers should be cautious when interpreting densities that might represent additional components, such as the reported calmodulin binding to NALCN's C-terminal helix, especially when local map quality is poor .

What approaches can reconcile contradictory results in Fam155a functional studies?

When faced with contradictory results in Fam155a functional studies, researchers should consider:

• Experimental System Differences:

  • Expression systems (HEK293T vs. Xenopus oocytes) may yield different results due to varying endogenous proteins

  • Primary cells versus heterologous systems may show different phenotypes due to cell-specific interacting partners

• Complex Component Variations:

  • Ensure all four components (NALCN, FAM155A, UNC79, UNC80) are correctly expressed when making functional assessments

  • Variations in expression levels of individual components may affect results

• Technical Considerations:

  • Surface expression quantification methods must be sensitive and specific

  • Electrophysiological recording conditions should be standardized

• Mutation Effects:

  • Different mutations may have distinct functional consequences

  • Consider the specific domains affected by mutations and their roles in the complex

• Statistical Analysis:

  • Ensure appropriate statistical tests are applied to electrophysiological data

  • Consider sample sizes necessary for detecting significant effects given biological variability

By systematically addressing these factors, researchers can identify the source of contradictions and develop a unified model of Fam155a function within the NALCN channel complex.

How does mouse Fam155a research inform understanding of human neurodevelopmental disorders?

Research on mouse Fam155a provides valuable insights into human neurodevelopmental disorders through several mechanisms:

• Disease Mechanism Insights:

  • Genetic mutations of NALCN channel components lead to neurodevelopmental diseases in humans

  • Understanding the structural and functional relationships between Fam155a and NALCN in mice helps elucidate how mutations might disrupt channel function in humans

• Functional Conservation:

  • The NALCN channel complex shows conserved composition and function across species

  • Mouse models can therefore provide relevant insights into human disease mechanisms

• Therapeutic Target Identification:

  • Detailed understanding of how Fam155a regulates NALCN activity can inform therapeutic approaches

  • The activation mechanism revealed by structural studies, such as the relief of self-inhibition by UNC79-UNC80 , suggests potential therapeutic strategies

• Model System Development:

  • Mouse genetic diversity models may capture aspects of human genetic variation relevant to disease susceptibility

  • Advanced intercross lines (AIL) of mice can provide fine mapping of genetic factors contributing to complex traits

When translating findings from mouse to human, researchers should consider species-specific differences while leveraging the substantial functional conservation of the NALCN channel complex.

What are the implications of targeting Fam155a in neurological disorder treatments?

Based on its role in the NALCN channel complex, targeting Fam155a in neurological disorders presents several implications:

• Therapeutic Potential:

  • Modulating Fam155a could affect NALCN channel surface expression and activity

  • This could normalize neuronal excitability in disorders where NALCN function is compromised

• Specificity Considerations:

  • Fam155a-targeted approaches might offer greater specificity than directly targeting the more conserved NALCN pore

  • The protein-protein interfaces between Fam155a and NALCN could provide selective targeting opportunities

• Delivery Challenges:

  • As a transmembrane protein involved in a complex with other large proteins, targeting Fam155a faces challenges in delivery and specificity

  • Small molecule approaches might focus on modulating specific interactions rather than the entire protein

• Potential Off-Target Effects:

  • Given Fam155a's interactions with multiple partners , intervention may affect pathways beyond NALCN

  • Comprehensive screening for additional biological effects would be essential

• Genetic Therapy Approaches:

  • Gene therapy to correct Fam155a mutations could restore proper NALCN function

  • For gain-of-function mutations, RNA interference approaches might be considered

These therapeutic considerations should be evaluated in the context of specific neurological disorders and their underlying pathophysiology related to NALCN channel dysfunction.