Figure 5. ACCuRET measures short-range distances between the NTD and CTD of ASIC1a.

(A) Cartoon showing the ACCuRET approach (Created with Biorender.com). (B) Representative ACCuRET experiment. Fluorescence and bright field images at 60x of rASIC1a-Q14TAG at pH 6. In brief, the first image is taken in control buffer at pH 6. This is followed by washing on TETAC preloaded with Cu²⁺ and then washout of the excess Cu²⁺-TETAC. The second image is then taken which shows a decrease in L-ANAP fluorescence indicative of FRET. Finally, Cu²⁺-TETAC is removed by adding DTT and the final image is taken where the L-ANAP fluorescence is restored. mCitrine bleaches across all three images and the bright field is blank, indicating the cell is successfully unroofed. The scale bar in the top left is 10 μm and applies to all panels in B. (C) Normalized FRET efficiency for example experiment in B. Signals are normalized to fluorescence before addition of metal ions. The inset cartoon shows the position of L-ANAP denoted by a red star and the single cysteine denoted by a green circle. Grey symbols represent other positions that are mutated in this study in order to show the relative position mutated in this example. In this instance, L-ANAP is incorporated at Q14 and the single cysteine in the C-terminus is at position C469. (D) Cartoon illustrating the current hypothesis of the orientation between the NTD and CTD at rest (left) and after prolonged acidosis (right). The inset sequence shows the putative interaction between the intracellular domains.