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. 2017 Jan 10;112(1):87–98. doi: 10.1016/j.bpj.2016.11.3197

Figure 4.

Figure 4

Effect of temperature on the pharmacological properties of TRPV1 and its activation, measured by BRET. (A and B) BRET ratios of HEK293T cells expressing either the TRPV1-Luc/YFP-CaM constructs (A) or the YFP-TRPV1-Luc BRET probe (B) were recorded in real time while the cell-culture medium was heated from 25 to 50°C using a Peltier plate. The effects of calcium-free extracellular medium (A) or preincubation of cells with 1 μM CPZ (A and B), were also tested. BRET ratios were corrected for the minor variation in Luc and YFP emissions due to the temperature increase (see Materials and Methods). In (A), the fact that the signal remained stable over the whole temperature range under Calcium-depleted conditions and in the presence of CPZ further excluded any bias in BRET measurements due to the denaturation of the Luc or YFP groups. Data represent one out of three independent experiments. (C and D) CAPS dose response curves measured by BRET using HEK293T cells, expressing either the TRPV1-Luc/YFP-CaM constructs (C) or the YFP-TRPV1-Luc BRET probe (D), incubated at 25, 31, 37, or 42°C. Results represent the mean ± SE of four independent experiments done in duplicate and are expressed as the difference between the net BRET measured in each condition and the basal BRET measured without any agonist at 37°C.