Figure 2.

Screening of the serine mutants within the peptide LHSSNKSSLYSGR (817–829) for TRPM8 activity in intracellular Ca²⁺-measurements.

Fluorescence measurements of intracellular Ca²⁺ concentration were performed on HEK-293 cells transiently transfected with the wild type or mutants TRPM8 (0.7 μg) and GFP (0.2 μg) constructs.

Panels A–I are representatives from single cover slips, the total number of measurements (n) and total number of cells for each variety are indicated in parentheses A: TRPM8 wild type (n = 5, n_cells = 71); B: double serine mutant S823G/S824G (n = 8, n_cells = 99); C: quadruple serine mutant S819G/S820G/S823G/S824G or GGNKGG (n = 4, n_cells = 76); D: quintuple serine mutant 5S-G: S819G/S820G/S823G/S824G/S827G or GGNKGGLYG (n = 8, n_cells = 61); E: double serine mutant S819P/S820P (n = 4, n_cells = 53); F: single serine mutant S827G (n = 7, n_cells = 171); G: triple serine mutant S823G/S824G/S827P or GGLYP (n = 5, n_cells = 67); H: quintuple serine mutant S823G/S824G/L825G/Y826G/S827P or GGGGP (n = 7, n_cells = 88); I: single serine mutant S827P (n = 6, n_cells = 83); J: the summary of cold, menthol, icilin, and second cold applications (error bars stand for ±s.e.m.). Alternatively, these measurements were carried with a single application of icilin, which resulted in the similar observations (Figure S6). We have also performed measurements with subsequent ligand washout, but those resulted in a slow Ca²⁺ run down and prolonged experiment time that might have an effect on cells (data not shown). K: Cartoon of the putative PHB-modification sites on the TRPM8 protein with a sequence indication for the extracellular PHBylated peptides targeted for the mutagenesis.