Recombinant Human Transmembrane protein 35 (TMEM35)

What is TMEM35 and what are its alternative designations?

TMEM35 (Transmembrane Protein 35) is a small neuronal-specific transmembrane protein that has been renamed NACHO (Novel Acetylcholine receptor Chaperone) due to its critical role in nicotinic acetylcholine receptor (nAChR) assembly and trafficking . The protein is encoded by the tmem35a gene and functions primarily in neurons, where it serves as an essential chaperone for specific nAChR subtypes . This protein has gained significant attention in pain research and neurological studies due to its specific expression pattern and functional significance in modulating nAChR activity.

What is the primary function of TMEM35/NACHO?

TMEM35/NACHO serves as a specialized chaperone protein with distinct roles in the processing of different nAChR subtypes. It is necessary and sufficient for the assembly and trafficking of homomeric α7 nAChRs, while also facilitating the assembly of heteromeric α3, α4, and α6-containing receptor subtypes . Studies have demonstrated that deletion of the tmem35a gene results in complete absence of α7 membrane expression and electrophysiological activity, highlighting its essential role in functional α7 nAChR expression . Additionally, tmem35a knockout mice show residual cell surface expression of α3, α4, and α6-containing receptors, suggesting that NACHO plays a more critical role in α7 nAChR assembly compared to other subtypes .

What physiological processes are affected by TMEM35/NACHO?

TMEM35/NACHO has been implicated in several important physiological processes, with pain modulation being particularly well-documented. Studies with tmem35a knockout mice have demonstrated that these animals exhibit thermal hyperalgesia and mechanical allodynia, indicating enhanced pain sensitivity . The absence of NACHO activity in neurons appears to contribute to heightened inflammatory responses, which may be responsible for the hyperalgesic phenotype observed in these mice . Additionally, the role of TMEM35 in regulating nAChR function suggests its involvement in other neurological processes where cholinergic signaling is important, including cognition, memory, and neuroinflammation.

How does TMEM35/NACHO regulate nicotinic acetylcholine receptor expression?

TMEM35/NACHO functions as a specialized chaperone protein that facilitates the proper assembly, folding, and trafficking of nicotinic acetylcholine receptors to the cell surface. For homomeric α7 nAChRs, NACHO is absolutely essential, as its absence results in complete loss of functional receptor expression . The mechanism involves direct interaction with the receptor subunits in the endoplasmic reticulum, facilitating proper folding and assembly before trafficking to the cell surface.

Research has shown that when NACHO is removed from cells (as in tmem35a knockout models), there is a complete absence of α7 membrane expression and electrophysiological activity . For heteromeric receptors containing α3, α4, or α6 subunits, NACHO plays a facilitatory but not essential role, as evidenced by residual expression of these receptors even after tmem35a deletion . This differential requirement suggests specialized mechanisms for different nAChR subtypes, making NACHO a potentially valuable target for selective modulation of specific cholinergic signaling pathways.

What are the implications of TMEM35/NACHO in pain research?

TMEM35/NACHO has emerged as a significant factor in pain processing and modulation. Studies with tmem35a knockout mice have revealed that these animals exhibit:

• Increased thermal hyperalgesia (heightened sensitivity to painful heat stimuli)

• Enhanced mechanical allodynia (pain response to normally non-painful touch stimuli)

• Altered responsiveness to cholinergic analgesics

Interestingly, intrathecal administration of nicotine and the α7-specific agonist PHA543613 still produced analgesic responses in tmem35a knockout mice, suggesting either residual expression of these receptors or off-target effects of these compounds . The specific involvement of neuronal α7 nAChRs in the spinal cord for heat nociception has been documented, with NACHO playing a critical role in this process through its chaperone function .

Transcriptomic analysis of spinal cord tissue from tmem35a knockout mice revealed 72 differentially expressed genes compared to wild-type controls, with functional gene network analysis suggesting increased neuroinflammation as a potential contributing factor to the hyperalgesic phenotype . This indicates that NACHO may modulate pain not only through direct effects on nAChR function but also through broader effects on inflammatory processes in the spinal cord.

How can recombinant TMEM35 be used in experimental studies?

Recombinant human TMEM35 protein can be utilized in various experimental settings to investigate its function and potential therapeutic applications:

Recombinant TMEM35 protein is available commercially, expressed in HEK293 cells with Myc-DYKDDDDK tags, with purity greater than 80% as determined by SDS-PAGE . This protein can be used for antibody production and as a standard in various biochemical and cellular assays .

What are the optimal experimental models for studying TMEM35/NACHO function?

Several experimental models have proven valuable for studying TMEM35/NACHO function:

In vitro models:

• HEK293 cells expressing recombinant TMEM35 and various nAChR subunits

• Primary neuronal cultures from wild-type or tmem35a knockout mice

• Neuroblastoma cell lines with endogenous or manipulated TMEM35 expression

In vivo models:

• Tmem35a knockout mice, which exhibit complete loss of α7 nAChR functional expression and specific pain phenotypes

• Conditional tmem35a knockout mice for tissue-specific deletion

• Transgenic mice overexpressing TMEM35 in specific neuronal populations

When selecting an experimental model, researchers should consider that TMEM35/NACHO is a neuronal-specific protein, so non-neuronal cell lines may require co-expression of additional factors to reconstitute physiologically relevant TMEM35 function .

How can researchers reliably assess nAChR function in relation to TMEM35/NACHO?

Assessment of nAChR function in relation to TMEM35/NACHO can be accomplished through multiple complementary approaches:

• Electrophysiological recordings:

  • Patch-clamp electrophysiology to measure nAChR currents in response to agonists like nicotine or PHA543613

  • Field potential recordings in tissue slices to assess network-level effects

• Calcium imaging:

  • Fluorescent calcium indicators to measure nAChR-mediated calcium influx

  • Real-time monitoring of receptor activation in live cells or tissues

• Radioligand binding assays:

  • Use of radiolabeled ligands (e.g., epibatidine) to quantify receptor density and binding affinity

  • Competition binding assays to assess pharmacological properties

• Behavioral assays (in vivo):

  • Thermal sensitivity tests (e.g., hot plate, Hargreaves test) with a 19-second cutoff to prevent tissue damage

  • Mechanical sensitivity tests using calibrated von Frey filaments (0.07-4.0g)

  • Intrathecal drug administration followed by sensory testing at 15, 30, 60, and 90 minutes post-injection

For valid results, researchers should include appropriate controls, such as wild-type littermates for tmem35a knockout mice, and consider blinding experimenters to genotype or treatment conditions .

What techniques are recommended for investigating TMEM35/NACHO in pain pathways?

Several specialized techniques have proven valuable for investigating TMEM35/NACHO in pain pathways:

• Intrathecal drug administration:

  • Direct delivery of compounds (e.g., nicotine at 0.5-1.5 nmol or PHA543613 at 10-50 nM) to the spinal cord

  • Insertion of a 30-gauge needle between the L5/L6 vertebrae at a 20° angle to the horizontal plane

  • Confirmation of correct placement by observing tail flick reflex

• Transcriptomic analysis:

  • RNA-seq of spinal cord tissue to identify differentially expressed genes

  • Functional gene network analysis using knowledge-based databases like Ingenuity Pathway Analysis

  • Validation of key genes by RT-qPCR or protein expression analysis

• Immunohistochemical analysis:

  • Assessment of microglial activation and neuroinflammatory markers in spinal cord sections

  • Quantification of nAChR subunit expression and localization

  • Co-localization studies of TMEM35/NACHO with nAChR subunits and other relevant proteins

• Behavioral testing protocols:

  • For heat sensitivity: Application of radiant heat to the plantar surface of each hind paw with 1-minute intervals between trials, using the mean of three trials per paw

  • For mechanical sensitivity: Up-down method with calibrated von Frey filaments, adapted for mouse paw sensitivity

These methodologies should be applied with appropriate controls and blinding procedures to ensure reliability and reproducibility of results.

How does TMEM35/NACHO compare to other nAChR regulatory proteins?

TMEM35/NACHO represents a unique class of nAChR regulatory proteins with several distinguishing features:

Unlike other regulatory proteins that modulate existing receptors, TMEM35/NACHO is absolutely essential for the functional expression of α7 nAChRs, making it a uniquely powerful target for selective manipulation of specific cholinergic pathways .

What is known about the role of TMEM35/NACHO in neuroinflammation?

Transcriptomic analysis of spinal cord tissue from tmem35a knockout mice has revealed dysregulation of genes associated with inflammatory responses, suggesting that TMEM35/NACHO may play a role in regulating neuroinflammation . The absence of NACHO activity in neurons appears to contribute to heightened inflammatory responses, which may explain the hyperalgesic phenotype observed in knockout mice .

This connection between TMEM35/NACHO and neuroinflammation could involve several mechanisms:

• Regulation of microglial activation through neuronal-microglial communication pathways

• Modulation of cytokine and chemokine release in the spinal cord

• Alteration of neuronal excitability that influences inflammatory processes

Previous studies have shown that activation of α7 or α4β2 nAChRs by selective agonists decreases neuropathic pain and reduces microglial activity and pro-inflammatory mediator release . Since NACHO is critical for α7 nAChR function, its absence would be expected to impair this anti-inflammatory cholinergic pathway, potentially explaining the increased neuroinflammation observed in knockout animals.

What are the emerging therapeutic applications of recombinant TMEM35/NACHO?

While recombinant TMEM35/NACHO is primarily used as a research tool, several potential therapeutic applications are being explored:

• Pain management: Given the role of TMEM35/NACHO in pain modulation through nAChRs, targeted modulation of this protein could offer novel approaches to pain management, particularly for conditions involving neuroinflammation .

• Neurological disorders: Since cholinergic signaling is implicated in various neurological conditions, including Alzheimer's disease and schizophrenia, TMEM35/NACHO-targeted approaches might provide new therapeutic avenues.

• Biomarker development: Recombinant TMEM35 could be used to develop antibodies and assays for measuring TMEM35 levels as potential biomarkers for neurological conditions or pain states.

• Drug screening platforms: Systems incorporating recombinant TMEM35 could be developed to screen for compounds that modulate nAChR assembly and function in a subtype-specific manner.

Research using recombinant TMEM35 is still in early stages, but the unique role of this protein in nAChR biology suggests significant untapped potential for therapeutic applications.

What are the key technical challenges in working with recombinant TMEM35?

Researchers working with recombinant TMEM35 face several technical challenges:

• Protein solubility and stability: As a transmembrane protein, TMEM35 can present challenges related to solubility and stability in aqueous solutions, potentially requiring specialized buffers or detergents.

• Maintaining native conformation: Ensuring that recombinant TMEM35 maintains its native functional conformation, especially when expressed in heterologous systems, is critical for meaningful experimental results.

• Functional assays: Developing reliable assays to assess the chaperone function of recombinant TMEM35 in reconstituted systems can be challenging, as this function involves complex multi-step processes of receptor assembly and trafficking.

• Specificity of effects: When using recombinant TMEM35 in cellular systems, distinguishing between specific effects on nAChR assembly versus non-specific effects on other cellular processes requires careful experimental design and appropriate controls.

Commercial recombinant human TMEM35 is available with Myc-DYKDDDDK tags, expressed in HEK293 cells with purity greater than 80% . While this preparation is suitable for many research applications, investigators should consider the potential impact of these tags on protein function when designing experiments.