Figure 2. Characterizing murine cold sensor(s) in keratinocytes.

(A).) In order to characterize the proteins involved in cold transduction, cold-induced calcium transients were measured in keratinocytes exposed to extracellular buffer containing different ionic concentrations or pharmacological agents, or in keratinocytes from transgenic mice. (B).) Extracellular calcium chelation (EGTA, 0 µM Ca²⁺) decreases the proportion of keratinocytes that respond to cold; substituting NMDG for extracellular sodium did not further decrease the percentage of cold-responsive cells. Cold responses were only abolished when endoplasmic reticulum Ca²⁺ stores were depleted and unable to be refilled with extracellular sources (EGTA, 0 µM Ca²⁺, thapsigargin). CRAC channel inhibition (5J4) did not decrease the percentage of cold-responsive cells, and similar proportions of keratinocytes from C57BL/6, global TRPA1, and global TRPC5 knockout mice responded to decreasing buffer temperature (Chi square p<0.0001; Fisher’s Exact tests ***p<0.001 vs. vehicle; n ≥ 4). (C).) Peak calcium responses to cold were lower in the absence of extracellular calcium; responses were unaltered in other conditions (1-way ANOVA p<0.0001; Bonferroni’s multiple comparisons: ****p<0.0001 vs. vehicle, #### p<0.0001 vs. EGTA, 0 µM Ca²⁺, &&&& p<0.0001 vs. EGTA, 0 µM Ca²⁺, NMDG). (D).) Altering extracellular buffer contents increased or decreased the temperature at which keratinocytes responded to cold by ≤0.6°C. Mean temperature thresholds (°C) for vehicle: 20.9, EGTA, 0 µM Ca²⁺,±NMDG: 20.3, 20 µM 5J4: 20.5, TRPA1 KO: 21.5, TRPC5 KO: 20.7 (Kruskal-Wallis test p<0.0001; Dunn’s multiple comparisons: *p<0.05, ****p<0.0001 vs. vehicle).