Ongoing Research

Extracellular labeling of ion channels

Two strategies for extracellular labeling of TRPV1. (A) TRPV1 expression in DRG neurons is distributed across the plasma membrane and intracellular compartments (top). If the extracellular side of TRPV1 can be labeled at a click-chemistry site, labeling the plasma membrane population of TRPV1 is possible (bottom). (B) Amber codon suppression techniques permit substitution of a non-canonical amino acid (ncAA) at a single residue in the TRPV1 sequence. Addition of an ncAA with click-chemistry properties in an extracellular loop of TRPV1 allows surface labeling of the ion channel (PDB 7lqy; B,i). By introducing the HaloTag domain (blue PDB 6u32) to an extracellular loop of TRPV1, cell impermeable HaloLigands can be specifically targeted to the population of channels that are expressed on the plasma membrane (B, ii).

Variant-structure analysis

Variant analysis of TRPV1 surface residues exposes N-terminal/C-terminal interface. (A) Ribbon representation of rat TRPV1 residues 111-320 from structure 3J5P. Variant frequency color scheme adopted from Figure 1. (B) Surface representation of TRPV1 fragment shown in panel (A). (C) Panel (B) rotated 90 degrees as indicated. (D) TRPV1 C-terminal region (7LQY) residues 746-775 shown with variant coloring scheme (left) and aligned with structure 3J5P overlaid onto N-terminal ARD (right, cyan). (E) Inset of box indicated by dashed outline in panel (D) with highlighted residues from N-terminal ARD shown in green (K155, K160, R211) and blue (R212) in accordance with variant coloring scheme and highlighted residues in C-terminal region shown in cyan (W752, E761, D762). (Adapted from Mott et al., 2023)

Unroofed cells

Cell unroofing is the process by which we remove the body of an adherent cell grown on a coverslip so that the only remaining cellular material on the coverslip is a plasma membrane sheet. Our recent structural studies of TRPV1 with an unnatural amino acid (UAA) inserted at different sites along its polypeptide sequence relied on sonication as the unroofing method (Zagotta et al., JGP 2016). To the left are examples of unroofed HEK293T/17 cells. (A) A plasma membrane sheet loaded with fluorescent Rhodamine B C-18. Brighter regions on periphery indicate partial unroofing where two bilayers are still present. Clicking here shows an unroofing movie of a cell expressing the PIP2 marker PLCd-PH-GFP. The fluorescence intensity decreases after unroofing as the PLCd-PH-GFP dissociates from the membrane. (B) A cell (outline in white) expressing TRPV1-GFP has a region of diffuse background fluorescence (yellow dashed line). The plasma membrane with resident TRPV1-GFP single channels remains after unroofing while intracellular TRPV1-GFP (presumably in endoplasmic reticulum and other intracellular membranes) is lost (Sponholtz and Senning, 2021).

Figure 3.

Single molecule lipid imaging is conducted in unroofed cells with genetically encoded lipid headgroup recognition motifs known as PH domains. The image shows two unroofed cells with PLCdelta1-PH domain labeled GFP bound to various positions on the inner leaflet. The image links to a movie (PLCdelta1-PH-GFP single molecule), showing the highly dynamic character of the labaled PH domains as these bind PI(4,5)P2 lipids and traverse the plasma membrane sheet before dissociating or photobleaching (Sponholtz and Senning, 2021).