Regulating Nociceptive Channel Trafficking by Site‐directed mutagenesis of a Calcineurin Interaction Motif

Transient receptor potential (TRP) channels are a family of receptor proteins regulating pain sensation. TRP receptors are activated by a variety of noxious stimuli, such as capsaicin (chili peppers), heat, and mechanical stress. Chronic TRPV1 stimulation leads to desensitization of the receptor, a process which is mediated by calcium/calmodulin dependent kinases. This attribute, however, has yet to been leveraged for treatment of chronic pain diseases. Here, we identify a C‐terminal phosphorylation site that is involved in regulating TRPV1 trafficking to the cell surface. Results and Conclusion In dorsal root ganglion cells, confocal microscopy and western blot show that de‐phosphorylation of threonine 705 is sufficient to induce significant trafficking to the plasma membrane. Mimicking constitutive phosphorylation at that site induces the receptor to be retained on intracellular membranes. TRPV4 ‐ another member of the TRPV family ‐ also has a similar homologous region to TRPV1 that contains two adjacent threonines T739 and T740, both potentially involved in regulating TRPV4 trafficking. Confocal and western blot data show that removing the T740 phosphorylation site is sufficient to significantly increase surface trafficking. However, the phosphomimetic mutation reduced but did not eliminate surface expression. These data lead us to believe that phosphorylation at both threonines is necessary for full internalization of TRPV4 to occur. Together, these data suggest that the highly conserved Calcineurin interaction motif is an important trafficking regulation site for multiple members of the TRP family, and that manipulating key phosphorylation sites is sufficient to significantly change protein trafficking patterns. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .