Recombinant Human Transmembrane protein FAM155A (FAM155A)

What is the molecular structure of FAM155A and how does it interact with NALCN?

FAM155A contains an extracellular cysteine-rich domain (CRD) consisting of three consecutive α-helices and connecting loops with a high content of cysteine residues that form stabilizing disulfide bonds. The CRD interacts extensively with the extracellular loops of NALCN domains I, III, and IV (L5 I, L5 III, L5 IV, and L6 IV), which create a platform for FAM155A binding. Specifically:

• K287 and E290 on α1 of FAM155A make cation-π and electrostatic interactions with W1085 and R1062 on L5 III of NALCN

• K307 on the α1-α2 loop interacts with D1408 on L6 IV

• R1094 on L5 III interacts with the carbonyl group of G364 of FAM155A, D1397 on P2 IV, and E1364 on L5 IV

The post-α3 loop of FAM155A extends over the extracellular ion entrance of the NALCN pore, with L5 III of NALCN covering this loop and locking it in a sandwiched conformation. This extensive interaction network likely helps stabilize NALCN in the membrane .

High-resolution cryo-EM studies have revealed that the C-lobe of FAM155A contains a fold similar to the frizzled-like cysteine-rich domain of receptor tyrosine kinase MuSK (PDB: 3HKL), with 84 Cα atoms aligned with an r.m.s.d. of 2.8 Å. Three disulfide bonds are structurally conserved between these domains .

What are the key structural features and post-translational modifications of FAM155A?

FAM155A has several important structural features and modifications:

• Glycosylation: One glycosylation site (Asn217) has been identified on FAM155A through cryo-EM structural studies .

• Disulfide bonds: Six disulfide bonds have been recognized in FAM155A, which are critical for maintaining its tertiary structure . These disulfide bonds are highly conserved from C. elegans NLF-1 to human FAM155A/B, suggesting their functional importance in stabilizing the CRD structure .

• Structural domains: FAM155A contains a transmembrane region and an extracellular cysteine-rich domain (CRD). The CRD forms a specific interaction interface with NALCN that is electrostatically complementary, ensuring favorable interaction stability .

• Conservation: The key residues mediating complex formation in FAM155A are highly conserved among orthologs from different species, indicating evolutionary conservation of the interaction mode between NALCN and FAM155A .

How do mutations in FAM155A affect NALCN channel function and neuronal excitability?

Mutations in the FAM155A-NALCN interaction interface can significantly disrupt channel function. For example, the R1094Q mutation in NALCN, which is located at the interface between NALCN and FAM155A, leads to disordered respiratory rhythm with central apnea, highlighting the critical nature of these interactions for proper channel function .

FAM155A's primary role appears to be in the proper membrane localization and stabilization of NALCN. When co-expressed with NALCN, FAM155A enables measurable sodium leak currents even without the presence of UNC79 and UNC80 auxiliary subunits. Electrophysiological recordings show that NALCN co-expressed with FAM155A alone exhibits nonlinear current-voltage relationships similar to those seen when all three auxiliary subunits are present .

Since NALCN is responsible for the resting Na⁺ permeability that controls neuronal excitability, disruptions in FAM155A function can alter neuronal resting membrane potential and consequently affect neuronal excitability. This potentially explains why dysfunctions of the NALCN channelosome are associated with various neurological disorders .

What is the role of FAM155A in the complete NALCN-FAM155A-UNC79-UNC80 quaternary complex?

Within the complete quaternary complex:

• FAM155A and NALCN form the core complex, with FAM155A playing a crucial role in membrane localization of NALCN .

• UNC79 and UNC80 form a large piler-shaped heterodimer that attaches to the intracellular side of the NALCN channel through tripartite interactions with the cytoplasmic loops of NALCN .

• While UNC79 and UNC80 are essential for robust channel function in vivo, FAM155A alone can facilitate some NALCN currents in heterologous expression systems .

• One specific interaction in the quaternary complex relieves the self-inhibition of NALCN by pulling the auto-inhibitory CTD Interacting Helix (CIH) out of its binding site, suggesting a regulatory role for the complete complex .

Cryo-EM structures of the quaternary complex reveal that UNC79-UNC80 interactions with NALCN are essential for proper cell surface localization and channel function. There is significant conformational heterogeneity within the UNC79-UNC80 heterodimer as shown by 3D variability analysis, suggesting dynamic regulation of channel function by these auxiliary proteins .

What is the genetic association between FAM155A variants and human diseases?

Genome-wide association studies (GWAS) have identified a significant association between variants in the FAM155A gene and diverticulitis, but interestingly not with diverticulosis. Specifically:

• The variant rs67153654 in FAM155A shows a markedly reduced minor allele frequency (MAF) in patients with prior diverticulitis compared to controls (odds ratio 0.66; 95% CI 0.47–0.92) .

• This association remains significant after adjusting for environmental cofactors such as age, BMI, alcohol consumption, and smoking status (adjusted OR 0.49 [95% CI 0.27–0.89], p = 0.002) .

• The specific variant is located in an intron, suggesting a molecular mechanism at the level of RNA expression or linkage disequilibrium with another, yet unidentified causal variant .

Additionally, since FAM155A is a critical component of the NALCN channelosome, mutations affecting its function may contribute to neurological disorders associated with NALCN dysfunction. The NALCN channel and its auxiliary proteins have been implicated in a variety of human diseases, particularly neurodevelopmental disorders .

What experimental approaches are optimal for expressing and purifying recombinant human FAM155A?

Based on successful structural studies, the following methodological approaches have proven effective:

Expression System:

• Co-expression of NALCN and FAM155A in HEK293F cells using baculovirus-mediated gene transduction has been successful .

• The addition of protein expression enhancers like sodium butyrate can improve yields.

Purification Protocol:

• Cell Lysis: Use detergent-based lysis buffers containing protease inhibitors to extract membrane proteins.

• Affinity Purification: Tag-based purification using FLAG or His tags has been effective. For the NALCN-FAM155A complex, researchers have successfully used FLAG-tagged NALCN to pull down the entire complex .

• Size Exclusion Chromatography: Further purification using size exclusion chromatography in buffers containing sodium chloride and mild detergents like GDN (glyco-diosgenin) .

Buffer Conditions:

• For structural studies, symmetric sodium solution buffers have been used to mimic electrophysiological recording conditions .

• Detergent selection is critical, with GDN showing good results for maintaining protein stability while preserving the native conformation of the complex .

Complex Stabilization:
For cryo-EM studies, ensuring stable complex formation is essential, which may require optimization of pH, salt concentration, and detergent composition .

What are the challenges in detecting FAM155A expression and localization in experimental models?

Researchers face several challenges when detecting FAM155A:

• Antibody Specificity: Commercial antibodies like Proteintech's Rabbit Polyclonal FAM155A antibody (24929-1-AP) have been validated in Western blot, immunohistochemistry, and ELISA techniques, but optimization is required for different sample types .

• Expression Levels: FAM155A expression may vary across tissues, with higher expression reported in certain cell lines like K-562 and COLO 320 cells .

• Co-localization Studies: Since FAM155A functions in complex with NALCN, co-localization studies are often necessary to understand its physiological role. This requires simultaneous labeling of multiple proteins.

• Recommended Approaches:

  • For Western blot: dilutions of 1:200-1:1000 have been effective

  • For immunohistochemistry: dilutions of 1:20-1:200 with antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0)

  • The observed molecular weight is typically 49-55 kDa, compared to the calculated 51 kDa

• Tissue-Specific Protocols: Protocols may need optimization for different tissue types, with successful detection reported in human colon cancer tissue and human liver cancer tissue .

How can researchers effectively study the electrophysiological properties of the NALCN-FAM155A complex?

To study the electrophysiological properties of NALCN-FAM155A:

• Expression Systems:

  • Heterologous expression in HEK293 cells or Xenopus oocytes has been successful .

  • Co-expression of NALCN with FAM155A alone produces measurable currents, though the full quaternary complex with UNC79 and UNC80 yields more robust currents .

• Recording Techniques:

  • Whole-cell patch-clamp recordings using voltage steps protocols can effectively measure NALCN currents.

  • Current-voltage relationships for NALCN-FAM155A show nonlinear patterns that can be analyzed for voltage sensitivity .

• Ion Permeability Studies:

  • Studies of the NALCN selectivity filter (EEKE) have revealed a Na⁺ ion-binding site reminiscent of Ca²⁺ binding sites in Ca_v channels .

  • Experimental designs should account for the channel's "leaky" nature and its physiological role in maintaining resting membrane potential .

• Mutational Analysis:

  • Point mutations at key interaction residues (e.g., K287, E290 on FAM155A or R1062, W1085, R1094 on NALCN) can be used to study the functional importance of specific protein-protein interactions .

  • The CIH-5A mutation (I753A, L754A, R761A, R764A, R765A) has been shown to disrupt interaction between the CTD Interacting Helix and CTD, potentially releasing self-inhibition of NALCN .

• Control Experiments:

  • Expression of NALCN alone (without auxiliary subunits) typically yields minimal currents, serving as an important negative control .

  • Comparison of NALCN-FAM155A currents with the full quaternary complex can reveal the functional contributions of UNC79 and UNC80 .

What are the differences between FAM155A and FAM155B, and how can researchers distinguish between them?

FAM155A and FAM155B share significant homology but have distinct properties:

Structural Similarities:

• Key residues mediating complex formation with NALCN are highly conserved between FAM155A and FAM155B, suggesting that both can form stable complexes with NALCN .

Functional Redundancy:

• Studies have shown that FAM155A can be functionally substituted by human FAM155B in experimental settings, indicating overlapping functions .

Research Approaches to Distinguish Them:

• Sequence-Specific Detection: Design primers or antibodies that target non-conserved regions to specifically identify each isoform.

• Tissue Distribution Analysis: Though not fully characterized in the provided search results, the two proteins may have different tissue expression patterns that can be used for differentiation.

• Functional Assays: While functionally similar, subtle differences in channel modulation might exist and could be detected through detailed electrophysiological studies.

• Genetic Manipulation: Selective knockdown or knockout of each gene can help determine their specific contributions to NALCN function in different cellular contexts.

• Protein-Protein Interaction Studies: While both interact with NALCN, they might have different affinities or slightly different binding interfaces that could be characterized through detailed interaction studies.

Researchers should note that although FAM155B can substitute for FAM155A, the genetic association with diverticulitis has been specifically linked to variants in FAM155A , suggesting some non-redundant functions that warrant further investigation.

What are the most promising avenues for therapeutic targeting of the NALCN-FAM155A complex?

Several promising therapeutic approaches could target the NALCN-FAM155A complex:

• Structure-Guided Drug Design: The high-resolution structures of the NALCN-FAM155A complex (3.1 Å) provide templates for rational drug design targeting specific interaction interfaces or channel functions.

• Targeting the Na⁺ Binding Site: The identified Na⁺ ion-binding site in the unique EEKE selectivity filter of NALCN could be targeted to modulate channel function in conditions where abnormal sodium leak affects neuronal excitability .

• Modulating FAM155A-NALCN Interactions: Small molecules or peptide mimetics that enhance or disrupt specific aspects of FAM155A-NALCN interactions could be developed to modulate channel function in neurological disorders associated with NALCN dysfunction.

• Gene Therapy Approaches: For NALCN channelosome-related disorders with identified mutations, gene therapy approaches could be developed to restore proper channel function.

• Targeting Disease-Specific Variants: The association between FAM155A variants and diverticulitis suggests potential for targeted therapeutics in this condition, although the molecular mechanisms require further elucidation.

Future research should focus on understanding the tissue-specific regulation of the NALCN-FAM155A complex and how its dysfunction contributes to various human diseases, including both neurological disorders and potentially intestinal conditions like diverticulitis.

How might single-cell techniques advance our understanding of FAM155A function?

Single-cell approaches offer powerful tools for dissecting FAM155A function:

• Single-Cell RNA Sequencing: Can reveal cell type-specific expression patterns of FAM155A, FAM155B, and other NALCN channelosome components, particularly in the nervous system where NALCN function is critical.

• Single-Cell Electrophysiology: Patch-clamp recordings from individual neurons with manipulated FAM155A expression can provide direct insights into how this protein influences neuronal excitability and resting membrane potential.

• Single-Molecule Imaging: Techniques like single-molecule FRET could reveal dynamic interactions between FAM155A and NALCN in living cells, potentially uncovering regulatory mechanisms not apparent in static structural studies.

• CRISPR Screening at Single-Cell Resolution: Could identify genetic modifiers of FAM155A function and reveal unexpected pathways connected to the NALCN channelosome.

• Single-Cell Proteomics: May uncover cell type-specific interactomes of FAM155A beyond the known NALCN channelosome, potentially identifying new regulatory mechanisms.