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Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry, compatible with a trans-cyclooctene coupled fluorescent dye. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population. We identified T468 as a rapid labeling site (∼5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors.
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Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing
Abstract Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.
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- Award ID(s):
- 1918716
- PAR ID:
- 10185127
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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