Figure 7.
CNP/NPR-C/PKC-induced sensitization of TRPV1 activity is dependent on two PKC phosphorylation sites in the channel protein. A, Representative traces of TRPV1 currents in HEK293T cells either coexpressing rTRPV1 and hNPR-C (top) or expressing rTRPV1 alone (bottom) in response to four successive applications of capsaicin (20 nm for 5 s; with 2 min interval), with the continuous extracellular perfusion of CNP (10 nm) after the second ICap. B, Quantification of ICap for experiments as shown in A. Data are presented as mean ± SEM of fold increase in peak ICap normalized to second ICap; n values are given for each expression group. *p < 0.05, significantly different compared with respective second ICap currents (one-way ANOVA with post hoc Dunnett's correction). C, Representative RT-PCR analysis of endogenous NPR expression in HEK293T cells with primers specific for NPR-A, NPR-B, and NPR-C, as well as for GAPDH as a housekeeping control. Amplicons of RT-PCR reactions from isolated mouse DRG neuron RNAs are loaded for comparison. D, Quantification of fold increase in ICap for experiments as shown in A, from HEK293T cells expressing rTRPV1 wild-type channel (TRPV1–WT), or single, double, and triple PKC phosphorylation site mutant rTRPV1 channels. Data are presented as mean ± SEM of fold increase in peak fourth ICap normalized to second ICap; n values are shown in parentheses for each expression group. *p < 0.05, significantly different compared with TRPV1–WT (one-way ANOVA with post hoc Dunnett's correction).