Transient receptor potential (TRP) channels are thermosensitive and chemosensitive cation-permeable channels. TRPM8, a member of this family, facilitates calcium influx in sensory neurons in response to environmental cold and cooling agents such as menthol and icilin. However, the structural mechanisms by which TRPM8 responds to temperature and chemical stimuli remain elusive. Here, we performed a deep mutational scan (DMS) to investigate the phenotypes of 912 single-residue variants between amino acids 927–974, a region encompassing part of the S6 helix and the extracellular pore loop between the S5 and S6 helices, both implicated in channel gating. To assess the functional impact of these variants, we generated a library of cells co-expressing each TRPM8 variant and the calcium indicator GCaMP6s. Using fluorescence-activated cell sorting, we measured channel activity via GCaMP6s fluorescence and sorted cells into three bins: “low,” “medium,” and “high.” Each bin’s population was subjected to next-generation sequencing (NGS), enabling identification of genotypes represented in each sorted population. Sequence read-count distributions were used to assign a functional phenotype to each variant, classified as loss of function (LoF), wild type like (WT), or gain of function (GoF). To score the phenotype of each variant, we devised a system based on 3D vector projection. Bin read counts for each variant were normalized, and the variant with the highest “low” bin normalized frequency was selected as the reference vector, representing the strongest LoF phenotype. Each variant’s normalized frequency vector was projected onto the reference vector using the dot product, providing an objective measure of similarity or deviation from the most extreme LoF phenotype. We are comparing our scoring system with other methods and validating our scored data set by comparing DMS variant scores with data from 30 single-residue variants that we individually evaluated using our calcium reporter assay.
Le et al. (Sun,) studied this question.