Dissecting Substance P-Mediated Signaling

The study was designed to examine the contribution of SP-induced endocytosis of the NK₁R to signal transduction in subcellular compartments, excitation of spinal neurons, and nociception. Endocytosis of the NK₁R was examined in HEK293 cells by using BRET to assess the proximity between the NK₁R and proteins resident in the plasma membrane and early endosomes and by localizing fluorescent SP by confocal microscopy. BRET was also used to examine the assembly of signaling complexes, which were localized in endosomes by immunofluorescence and super-resolution microscopy. Signaling in subcellular compartments of HEK293 cells was studied by expressing genetically encoded FRET biosensors, which allowed analysis of signaling with high spatial and temporal fidelity. NK₁R endocytosis was studied in spinal neurons in slice preparations and in vivo by immunofluorescence and confocal microscopy. To examine the excitation of pain-transmitting neurons, cell-attached patch clamp recordings were made from second-order neurons in slices of rat spinal cord. Nociceptive behavior was evaluated in conscious mice after intraplantar administration of capsaicin, formalin, or CFA. To examine the contribution of NK₁R endocytosis to signaling, neuronal excitation, and nociception, HEK293 cells, rat spinal cord slices, or mice were treated with pharmacological or genetic inhibitors of clathrin, dynamin, or βARRs, or with peptide inhibitors of NK₁R/βARR interactions. Peptidic and small-molecule antagonists of the NK₁R were conjugated to the lipid cholestanol, which facilitated endosomal targeting and retention of antagonists. Cholestanol-conjugated antagonists were used to directly evaluate the contribution of NK₁R signaling in endosomes to SP-induced compartmentalized signaling in HEK293 cells, excitation of spinal neurons, and nociception. Institutional Animal Care and Use Committees approved all studies.