Fig. 5: Photo-crosslinking of the photo-activatable non-canonical amino acid AzF leads to formation of multimers.

a, b Using the amber suppression methodology for incorporation of non-canonical amino acids (ncAAs), we introduced the photo-activatable non-canonical amino acid AzF (b) at M238 position into mutant K_v7.1-EGFP-M238AMBER-Stop. HEK cells were co-transfected with cDNA encoding K_v7.1-EGFP-M238amber-Stop, suppressor tRNA and AzF-tRNA synthetase. Addition of the ncAA p-azido-phenylalanine (AzF) at 0.5 mM to the cell culture medium allowed for incorporation of the ncAA. c I274 interacts with M238 from the adjacent subunit in activated state in silico. d Successful incorporation and full-length protein expression was assayed by confocal EGFP imaging. e K_v7.1-EGFP-M238AzF expressing HEK cells were incubated in high K⁺ solutions (137 mM KCl) leading to channels preferentially in depolarized (calculated V_m of about −8.9 mV) states. UV irradiation caused formation of multimers under high K⁺ / preferentially depolarizing conditions consistent with the predicted interaction. f Normalized currents of hK_v7.1 WT and hK_v7.1 M238C/I274C expressing oocytes in the absence and presence of (R)-L3 and Co²⁺ ions (number of oocytes for each condition see SI Table 2).