The transient receptor potential vanilloid-1 (TRPV1) ion channel is essential for sensation of thermal and chemical pain. TRPV1 activation is accompanied by Ca 2+ -dependent desensitization; acute desensitization reflects rapid reduction in channel activity during stimulation, whereas tachyphylaxis denotes the diminution in TRPV1 responses to repetitive stimulation. Acute desensitization has been attributed to conformational changes of the TRPV1 channel; however, the mechanisms underlying the establishment of tachyphylaxis remain to be defined. Here, we report that the degree of whole-cell TRPV1 tachyphylaxis is regulated by the strength of inducing stimulation. Using light-sheet microscopy and pH-sensitive sensor pHluorin to follow TRPV1 endocytosis and exocytosis trafficking, we provide real-time information that tachyphylaxis of different degrees concurs with TRPV1 recycling to the plasma membrane in a proportional manner. This process controls TRPV1 surface expression level thereby the whole-cell nociceptive response. We further show that activity-gated TRPV1 trafficking associates with intracellular Ca 2+ signals of distinct kinetics, and recruits recycling routes mediated by synaptotagmin 1 and 7, respectively. These results suggest that activity-dependent TRPV1 recycling contributes to the establishment of tachyphylaxis. desensitization | calcium | synaptotagmin | pain | TRP channel T ransient receptor potential vanilloid-1 (TRPV1) is a Ca 2+permeable cation channel expressed in sensory nerves specialized for pain detection (1, 2). Ca 2+ influx upon TRPV1 activation induces channel desensitization, with acute desensitization referring to the rapid reduction in the evoked inward current, and tachyphylaxis denoting current diminutions over repetitive stimulation (1,3,4). Desensitization can be leveraged to treat clinical pain. For instance, the TRPV1 agonist capsaicin has been used as a therapeutic analgesic (5, 6).The mechanism underlying TRPV1 acute desensitization has been widely explored and converged on agonist-induced conformational changes at the channel level (1, 2). In this regard, TRPV1 interacts in a Ca 2+ -dependent manner with either or both calcinerin (7), calmodulin (8), and phosphatidylinositol 4,5-bisphosphate (9), to regulate channel gating and inactivate response. Relatively, much remains to be understood on the mechanisms underlying the establishment of tachyphylaxis. Early studies show that phosphorylation of TRPV1 by protein kinase A and mutation at the corresponding phosphorylation site affect the induction of tachyphylaxis, which yet display little effect on acute desensitization (1, 7, 10). Likely, the establishment of tachyphylaxis recruits other mechanisms besides those assigned for acute desensitization.Here, we observed by whole-cell patch clamp that the extent of tachyphylaxis is regulated by the strength of inducing stimulation. Imaging TRPV1 exocytosis and endocytosis trafficking in real time by light-sheet microscopy and a pH-sensitive sensor, we show that TRPV1 channels unde...