TMEM35A

TMEM35A (NACHO) is an endoplasmic reticulum-resident transmembrane chaperone that drives the assembly and surface delivery of nicotinic acetylcholine receptors (nAChRs) and, more broadly, pentameric ligand-gated ion channels (PMID:26875622, PMID:28445721). It is essential and selective for nAChRs: its loss in mice abolishes α7 receptor assembly entirely and profoundly reduces α4β2, α6-containing, and α3-containing subtypes, while leaving GABAA receptors unaffected, and it cooperates with RIC-3 to achieve surface expression (PMID:28445721, PMID:32204458). Mechanistically, NACHO engages unassembled subunits in the ER, recruits the oligosaccharyltransferase machinery and the lectin chaperone calnexin, and requires N-glycosylation and calnexin activity to promote correct folding and Golgi-dependent maturation (PMID:32783947, PMID:38967800). A cryo-EM structure of an assembly intermediate shows NACHO binding the principal (+) transmembrane interface of immature subunits with a second adjacent surface for the next subunit, accounting for stepwise oligomerization, and mutation of either surface impairs α7 pentamer formation (PMID:39553992). NACHO can also dictate receptor stoichiometry, favoring the (α4)2(β2)3 form of α4β2 (PMID:32676916). Loss of NACHO in mice impairs hippocampal long-term potentiation, fear and spatial memory, and reduces PSD95 and NMDA receptor levels, and produces thermal hyperalgesia and mechanical allodynia (PMID:27170659, PMID:33422618).

1. 2010 Medium

   Before its chaperone role was known, TMEM35/TUF1 was characterized as a specific binding partner of a neurotrophin receptor in adrenal tissue, the first functional handle on the protein.

   Evidence Competitive binding displacement and Co-IP in rat adrenal zona glomerulosa cells with mutant controls

   PMID:20685870

   Open questions at the time

   • Relationship between p75NTR binding and the later-established nAChR chaperone function unclear
   • Peripheral/adrenal context distinct from CNS role
   • No structural mapping of the binding interface
2. 2016 High

   Established that NACHO is an essential ER chaperone for α7 nAChR assembly, identifying the long-sought factor required for α7 folding, maturation, and surface expression.

   Evidence Genomic screening, siRNA knockdown in primary neurons, knockout mice, α-bungarotoxin binding, electrophysiology, and fractionation

   PMID:26875622

   Open questions at the time

   • Molecular interaction surface with α7 not defined
   • Whether function extends beyond α7 untested
   • Folding/glycosylation machinery involved unknown
3. 2016 Medium

   Linked NACHO loss to circuit- and behavior-level consequences, showing the chaperone is required for synaptic plasticity and memory.

   Evidence Knockout mice with fear conditioning, Morris water maze, CA1 LTP, and synaptosomal proteomics

   PMID:27170659

   Open questions at the time

   • Causal chain from nAChR loss to reduced PSD95/NMDA receptors not established
   • Elevated corticosterone vs. direct synaptic effects not separated
   • Single lab
4. 2017 High

   Broadened NACHO's role from α7 to all major nAChR classes and demonstrated subtype specificity, establishing it as a general nAChR assembly factor acting early and synergizing with RIC-3.

   Evidence Knockout mice with subtype-specific radioligand binding (α-bungarotoxin, epibatidine, conotoxin MII) and GABAA negative control

   PMID:28445721

   Open questions at the time

   • Mechanistic basis of subtype selectivity unresolved
   • Nature of synergy with RIC-3 undefined
   • Why GABAA is spared not explained at this stage
5. 2020 High

   Placed NACHO within the N-glycosylation ER chaperone pathway and identified its requirement for the α7 ectodomain and specific TM2 residues, defining how NACHO recognizes its substrate.

   Evidence α7 chimera/mutagenesis, surface trafficking assays, Co-IP proteomics identifying OST and calnexin, pharmacological N-glycosylation/calnexin inhibition, electrophysiology

   PMID:32783947

   Open questions at the time

   • Direct vs. indirect association with OST/calnexin not fully resolved
   • Order of calnexin vs. NACHO engagement unclear
   • Whether TM2 residues form the binding site or stabilize folding
6. 2020 Medium

   Showed NACHO controls receptor stoichiometry, not just assembly, distinguishing its output from a cytosolic chaperone.

   Evidence Heterologous expression with single-channel patch clamp and calcium imaging

   PMID:32676916

   Open questions at the time

   • Structural basis for stoichiometry selection unknown
   • Single heterologous system
   • In vivo relevance of stoichiometry control untested
7. 2020 Medium

   Demonstrated in vivo that NACHO, not RIC3, is the dominant determinant of brain α7 expression, refining the relative roles of the two cofactors beyond cell-culture inferences.

   Evidence Comparison of ric3 KO and tmem35a KO mice by α-bungarotoxin autoradiography and Western blot

   PMID:32204458

   Open questions at the time

   • Additional in vivo regulatory factors implied but unidentified
   • Reconciliation with in vitro RIC3 sufficiency incomplete
   • Preliminary analysis noted by authors
8. 2024 High

   Provided the first structural mechanism, showing NACHO shields the principal transmembrane interface of immature subunits and uses two distinct surfaces to template stepwise pentamer assembly.

   Evidence Cryo-EM of a GABAAR α1 assembly intermediate, crosslinking MS, AlphaFold prediction, and NACHO mutagenesis with functional α7 assembly assays (preprint)

   PMID:39553992

   Open questions at the time

   • Preprint; not peer-reviewed
   • Structure captured on GABAAR α1 while functional generality inferred
   • Dynamics of sequential subunit hand-off not directly observed
9. 2024 Medium

   Confirmed biochemically that NACHO is required for α7 N-glycosylation and Golgi maturation, separating its glycosylation-dependent role from RIC3.

   Evidence CHO cells expressing α7+RIC3 without NACHO, PNGase F digest, surface Western, voltage clamp

   PMID:38967800

   Open questions at the time

   • Whether NACHO directly recruits the glycosylation machinery to α7 not shown here
   • Single cell system
   • Step at which glycosylation fails not pinpointed
10. 2025 Medium

    Extended NACHO function to non-neuronal physiology, linking it to nicotine-induced ER stress, calcium handling, and airway smooth muscle proliferation.

    Evidence siRNA knockdown of TMEM35A and RIC-3 in human ASM cells with Ca2+ imaging, ER stress markers, and proliferation assays

    PMID:39236288

    Open questions at the time

    • Direct chaperone role vs. signaling effect in ASM not separated
    • Disease relevance to asthma correlative
    • Single lab
11. 2025 Medium

    Conservation studies in insect orthologs established that NACHO enhances channel expression/activity without altering ligand-binding pharmacology, clarifying that its action is on assembly rather than the binding site.

    Evidence Xenopus oocyte expression of insect nAChRs with two-electrode voltage clamp and binding-loop mutagenesis

    PMID:35082026 PMID:40082022

    Open questions at the time

    • Mechanism of activity enhancement vs. pure surface expression not distinguished
    • Heterologous systems only
    • Cofactor interplay in insects not mapped to mammalian context
12. 2025 Low

    Chimeric-receptor mapping challenged the TM4-binding model and pointed to a C-terminal SAP motif as functionally important for chaperone-dependent surface expression.

    Evidence α7-5HT3 chimera domain swaps in HEK/oocytes with α-bungarotoxin surface binding and electrophysiology

    PMID:41226195

    Open questions at the time

    • No direct binding assay performed; inference is indirect
    • Negative result on TM4 binding not independently confirmed
    • SAP motif role not structurally validated

• How NACHO's distinct binding surfaces are coordinated with calnexin/OST engagement to enforce subtype-specific assembly and stoichiometry in vivo, and the basis of its restriction to nAChRs versus other pLGICs, remain unresolved.
• No integrated structural model spanning glycosylation, calnexin, and sequential subunit binding
• Determinants of nAChR-versus-GABAAR selectivity unexplained
• Substrate hand-off timing to downstream trafficking factors unknown