Recombinant Mouse Protein tweety homolog 2 (Ttyh2)

What is Ttyh2 and what is known about its structure?

Ttyh2 is a member of the tweety family of proteins that has traditionally been described as a Ca²⁺-activated large conductance chloride (Cl⁻) channel containing five transmembrane regions. Recent cryo-EM studies have determined that Ttyh2 adopts a previously unobserved protein fold. The protein consists of 532 amino acids with a calculated molecular weight of 58,772 Da, though it typically appears at approximately 68 kDa in experimental conditions .

Structurally, Ttyh2 can exist in different oligomeric states depending on calcium concentration:

• In the presence of Ca²⁺: Forms cis-dimers (protomers in the same membrane)

• In Ca²⁺-free conditions: Forms monomers or trans-dimers (protomers in separate membranes associate head-to-head)

The cis-dimer interface buries approximately 1,556 Ų and involves residues in both transmembrane and extracellular domains. The trans-dimer interface is smaller, burying about 908 Ų, and partially overlaps with the cis-dimer interface, making these two dimerization states mutually exclusive .

What are the current contradictions in Ttyh2 functional characterization?

A significant scientific contradiction exists regarding Ttyh2's function:

These contradictions highlight the evolving understanding of this protein and suggest that Ttyh2 may have multiple functions or indirect effects on ion channel activity .

What expression systems are effective for producing recombinant mouse Ttyh2?

For structural studies, full-length M. musculus TTYH2 has been successfully expressed in HEK293T cells with a cleavable C-terminal fusion to EGFP. The recommended workflow includes:

• Expression in HEK293T cells

• Extraction and purification in detergent

• Reconstitution into lipid nanodiscs formed by the scaffold protein MSP1E3D1

This approach has yielded protein suitable for high-resolution cryo-EM structural determination to 3.3 Å resolution in the presence of Ca²⁺ and 4.0 Å resolution in Ca²⁺-free conditions .

What antibodies and detection methods are available for Ttyh2 research?

Validated antibodies are available for multiple applications as detailed below:

When using antibodies for detection, avoid repeated freeze-thaw cycles and exposure to high temperatures to maintain antibody integrity .

How does calcium affect Ttyh2 oligomerization and potential function?

Calcium plays a critical role in determining Ttyh2's oligomeric state, which may regulate its biological functions:

The cis-dimer interface features juxtaposed conserved negatively charged residues from each protomer, including those from extracellular domain 1 (Y109, S112, E113, E116) and extracellular domain 4b (D384, E387). This electronegative surface appears to be bridged by Ca²⁺, explaining the calcium dependence of dimerization .

What is the significance of the RGD motif in Ttyh2?

Ttyh2 contains an RGD (Arg-Gly-Asp) motif that forms part of the trans-dimerization interface. This motif is particularly significant because:

• RGD motifs are known to interact with integrins, suggesting a potential role in cell adhesion

• In cis-dimers (Ca²⁺ present), the RGD is exposed with approximately 250 Ų of solvent-accessible area

• In trans-dimers (Ca²⁺ absent), the RGD is sequestered at the dimerization interface

This suggests that extracellular Ca²⁺ levels could regulate Ttyh2-integrin interactions: high Ca²⁺ favors cis-dimers with exposed RGD available for integrin binding, while low Ca²⁺ promotes trans-dimerization that sequesters the RGD surface .

How do human and mouse Ttyh2 compare for translational research?

The apparent difference in Ca²⁺-free oligomerization may reflect intrinsic protein properties or methodological differences (e.g., residual Ca²⁺ in supposedly Ca²⁺-free samples) .

What approaches should be used to resolve the contradictory findings about Ttyh2's channel function?

To address the contradiction between Ttyh2's reported role as an ion channel and structural evidence suggesting otherwise, researchers should consider a multi-faceted approach:

• Functional characterization: Compare volume-regulated anion currents in wild-type versus TTYH2 knockout cells using patch-clamp electrophysiology

• Interaction studies: Investigate whether Ttyh2 associates with known VRAC components, potentially modulating their function indirectly

• Structure-function analysis: Create targeted mutations in key regions of Ttyh2 to test their impact on:

  • Ca²⁺-dependent oligomerization

  • RGD-mediated interactions

  • Effects on chloride conductance

• Reconstitution experiments: Test whether purified Ttyh2 alone can form functional channels in artificial membrane systems or requires additional components

What methods are recommended for studying potential Ttyh2-integrin interactions?

Given the presence of the RGD motif and its Ca²⁺-dependent accessibility, the following approaches are recommended for investigating Ttyh2-integrin interactions:

• Co-immunoprecipitation experiments under different Ca²⁺ conditions to identify Ca²⁺-dependent protein interactions

• Cell adhesion assays using:

  • Recombinant Ttyh2 extracellular domains (wild-type and RGD mutants)

  • Varying Ca²⁺ concentrations

  • Specific integrin blocking antibodies

• Förster resonance energy transfer (FRET) to detect Ttyh2-integrin proximity in living cells

• Site-directed mutagenesis of the RGD motif (R164) and assessment of functional consequences - previous research has shown that mutation of R164 affects function

What are the optimal experimental conditions for studying Ca²⁺-dependent structural changes?

When investigating Ca²⁺-dependent changes in Ttyh2 structure and function, researchers should consider these methodological details:

• For Ca²⁺-free conditions:

  • Use EGTA to chelate Ca²⁺ in protein samples

  • Pre-treat filter paper used for EM grid blotting with EGTA to prevent Ca²⁺ transfer to samples

  • Control for residual Ca²⁺ that may affect results

• For Ca²⁺-containing conditions:

  • 1 mM Ca²⁺ has been used successfully for structural studies

  • Monitor Ca²⁺ concentration throughout experimental procedures

• For structural investigations:

  • Cryo-EM has proven effective for determining Ttyh2 structures in different oligomeric states

  • Lipid nanodisc reconstitution using MSP1E3D1 scaffold protein provides a membrane-like environment

What are the most pressing unresolved questions about Ttyh2?

Several critical questions remain unanswered about Ttyh2:

• Does Ttyh2 function directly as an ion channel, or does it modulate ion channel activity indirectly?

• What is the physiological significance of Ca²⁺-dependent changes in oligomeric state?

• Do Ttyh2-integrin interactions occur in vivo, and what cellular processes do they regulate?

• What is the function of the trans-dimer configuration that bridges separate membranes?

• How do the four different Ttyh2 isoforms differ in structure and function?

• What is the relationship between Ttyh2 and other TTYH family members (TTYH1, TTYH3)?

Addressing these questions will require interdisciplinary approaches combining structural biology, electrophysiology, cell biology, and in vivo studies.