Recombinant Bovine Short transient receptor potential channel 6 (TRPC6)

What is the basic structural organization of TRPC6?

TRPC6 tetramers are organized in a two-layered architecture, forming an inverted bell-shaped intracellular cytosolic domain (ICD) that caps the transmembrane domain (TMD). The ICD assembles through interactions between four ankyrin repeat domains (residues 96-243) in the N-terminus, linker helices (residues 256-393), and a coiled-coil domain in the C-terminus. This structure is critical for channel function, with ankyrin repeats (ARs) and linker helices (LHs) providing key inter-subunit interactions essential for tetramer assembly .

Methodologically, researchers investigating TRPC6 structure should consider:

• Conducting comparative analysis between bovine and human TRPC6 using alignment tools

• Employing cryo-electron microscopy for structural determination

• Using site-directed mutagenesis to explore structure-function relationships

• Applying molecular dynamics simulations to understand conformational changes

How does TRPC6 function in calcium signaling pathways?

TRPC6 primarily conducts calcium and represents a component of store-operated calcium entry (SOCE). In proximal tubular cells (PTC), calcium entry via TRPC6 has an inhibitory effect on H₂O₂-mediated autophagy through activating the PI3K/Akt/mTOR and Ras/Raf/ERK pathways . Studies have shown that oxidative stress triggers TRPC6-dependent Ca²⁺ influx, with significant consequences for cell survival mechanisms.

For methodological investigation of TRPC6 calcium signaling, researchers should:

• Use calcium imaging techniques with fluorescent indicators

• Compare calcium responses in wild-type versus TRPC6 knockout cells

• Apply specific channel blockers like SAR7334 to confirm TRPC6-specific responses

• Measure calcium transients under various stimulation conditions

What are the optimal methods for expressing recombinant bovine TRPC6?

For effective expression of recombinant bovine TRPC6, researchers typically use mammalian expression systems. Based on established protocols, HEK293T cells provide an excellent platform for heterologous expression, as demonstrated in studies with human TRPC6 .

Methodological recommendations include:

• Clone the bovine TRPC6 coding sequence into expression vectors with strong promoters (e.g., pcDNA3)

• Consider adding epitope tags (YFP, FLAG) to facilitate detection and purification

• Optimize transfection conditions using lipid-based transfection reagents

• Validate expression through Western blotting and immunofluorescence

• For stable expression, establish cell lines using antibiotic selection

How can post-translational modifications of TRPC6 be detected and analyzed?

TRPC6 undergoes several post-translational modifications, including O-GlcNAcylation and phosphorylation. For comprehensive analysis of these modifications, researchers should implement multiple complementary techniques .

Methodological approach for detecting O-GlcNAcylation:

• Immunoprecipitation with anti-TRPC6 antibodies followed by Western blotting with anti-O-GlcNAc antibodies

• Click-it™ assay using tetra-acetylated azide-modified N-acetylglucosamine (GlcNAz)

• Co-immunoprecipitation studies to detect interaction with O-GlcNAc transferase (OGT)

• Tandem mass spectrometry to identify specific modified residues

• Treatment with PNGaseF to remove N-glycosylation when necessary to distinguish from O-GlcNAcylation

Note that bovine TRPC6 may have species-specific patterns of post-translational modifications that should be compared with the human ortholog.

What techniques are most effective for studying TRPC6 localization in kidney cells?

TRPC6 localizes to the glomerular slit diaphragm in podocytes and is expressed in various kidney cell types. For accurate localization studies, a combination of techniques is recommended .

Methodological recommendations:

• Immunofluorescence microscopy using specific anti-TRPC6 antibodies (1:50-1:200 dilution)

• Confocal microscopy for co-localization with other slit diaphragm proteins (nephrin, podocin, CD2AP)

• Immunogold electron microscopy for ultrastructural localization:

  • Use perfusion-fixed kidney tissue

  • Apply rabbit anti-TRPC6 antibody followed by gold-labeled secondary antibody

  • Analyze using electron microscopy

• Expression of GFP-tagged TRPC6 in cultured cells for live imaging studies

How can protein-protein interactions of TRPC6 be investigated?

TRPC6 interactions with other proteins are crucial for its function and regulation. Based on established protocols, researchers should consider multiple complementary approaches .

Methodological guidelines:

• Co-immunoprecipitation studies:

  • Co-express recombinant TRPC6 with tagged potential interacting proteins

  • Immunoprecipitate using antibodies against the tag

  • Analyze eluates by immunoblotting for TRPC6

  • For endogenous interactions, use anti-TRPC6 antibody for immunoprecipitation from native tissues or cells

• Proximity ligation assays for detecting in situ interactions

• FRET or BiFC for analyzing interactions in living cells

• Pull-down assays using purified protein domains to map interaction sites

How does TRPC6 contribute to renal disease pathogenesis?

TRPC6 mutations have been associated with focal segmental glomerulosclerosis (FSGS), and the channel plays roles in renal oxidative stress injury .

Methodological approaches to study TRPC6 in disease contexts:

• Generation and characterization of TRPC6 knockout mice:

  • Confirm TRPC6 deletion by immunohistochemistry

  • Analyze SOCE in isolated proximal tubular cells (PTC)

  • Examine autophagic vacuoles by electron microscopy

• Analysis of TRPC6 disease-associated mutations:

  • Five families with autosomal dominant FSGS had mutations in the TRPC6 gene

  • Two mutations displayed increased current amplitudes (gain-of-function)

• Investigation of oxidative stress responses:

  • H₂O₂ treatment increases TRPC6 expression

  • TRPC6-triggered Ca²⁺ influx inhibits autophagy in response to oxidative stress

What are the functional consequences of TRPC6 mutations, and how can they be studied?

TRPC6 mutations linked to kidney disease show altered channel function, often with gain-of-function characteristics .

Methodological guidelines for mutation analysis:

• Site-directed mutagenesis to introduce disease-associated mutations

• Electrophysiological studies:

  • Patch-clamp analysis of TRPC6 current in transfected cells

  • Calcium imaging to measure channel-mediated calcium influx

• Structural analysis:

  • Many gain-of-function mutations reside in ankyrin repeat domains (e.g., G109S, P112Q, N125S, M132T)

  • Others are found in the C-terminus (Q899K, R895C, E897K)

  • These mutations likely destabilize electrostatic interactions at interfaces of AR domains and linker helices

• Immunofluorescence studies to assess altered protein localization

How can TRPC6 channel activity be accurately measured in different experimental systems?

Measuring TRPC6 activity requires specialized techniques to isolate the channel-specific responses from background activities.

Methodological recommendations:

• Electrophysiological approaches:

  • Whole-cell patch-clamp recordings in heterologous expression systems

  • Single-channel recordings to determine conductance properties

  • Use of specific inhibitors (SAR7334) to confirm TRPC6-mediated currents

• Calcium imaging:

  • Ratiometric calcium indicators (Fura-2) for quantitative measurements

  • Genetically encoded calcium indicators for long-term studies

  • Store-operated calcium entry (SOCE) protocol to isolate TRPC6 contribution

• TRPC6 knockout controls to verify channel-specific responses

What strategies can be employed to investigate the role of TRPC6 in autophagy regulation?

TRPC6 has been implicated in autophagy regulation, with evidence that TRPC6-mediated calcium influx inhibits cytoprotective autophagy in response to oxidative stress .

Methodological guidelines:

• Genetic approaches:

  • TRPC6 knockdown using shRNA lentivirus

  • TRPC6 overexpression using pcDNA3-TRPC6 plasmid

  • TRPC6 knockout mice for in vivo studies

• Autophagy assessment:

  • Western blot analysis of LC3-II expression

  • Electron microscopy to visualize autophagic vacuoles

  • Tandem fluorescent-tagged LC3 to distinguish autophagosome formation from autophagic flux

• Signaling pathway analysis:

  • Investigate PI3K/Akt/mTOR and ERK1/2 signaling pathways

  • Use specific pathway inhibitors to determine mechanism of action

What are common challenges in working with recombinant TRPC6, and how can they be addressed?

Researchers working with recombinant TRPC6 often encounter several technical challenges that require specific solutions.

Methodological recommendations for troubleshooting:

• Expression level variability:

  • Optimize codon usage for bovine sequence expression

  • Use inducible expression systems for tighter control

  • Screen multiple clones to identify high expressors

• Antibody specificity issues:

  • Validate antibodies using TRPC6 knockout tissues/cells as negative controls

  • Use multiple antibodies targeting different epitopes

  • For immunostaining controls, pre-incubate primary antibody with TRPC6 control peptide

• Channel functionality assessment:

  • Ensure proper membrane trafficking using surface biotinylation assays

  • Verify channel assembly using native PAGE or crosslinking approaches

  • Confirm calcium permeability using calcium-free external solutions

How can the impact of post-translational modifications on TRPC6 function be systematically investigated?

Post-translational modifications significantly affect TRPC6 function, with O-GlcNAcylation and phosphorylation being particularly important .

Methodological approach:

• Site-directed mutagenesis:

  • Generate non-modifiable mutants (e.g., T221A to prevent O-GlcNAcylation)

  • Create phosphomimetic mutants to simulate constitutive phosphorylation

• Modulation of cellular enzymes:

  • Inhibit OGT with specific inhibitors or siRNA

  • Use TMG to stimulate OGT activity

• Functional assessments:

  • Compare wild-type and mutant channel activity

  • Analyze calcium imaging data for altered responses

  • Assess protein-protein interactions with modified/unmodified channels

• Structural studies to understand how modifications affect channel conformation