Transient receptor potential vanilloid receptor subtype I (TRPV1) is a polymodal sensory receptor gated by chemical and physical stimuli. Akin to other TRP channels, TRPV1 contains in its C-terminus, adjacent to the pore, a highly conserved domain referred to as TRP domain. This region is necessary for both channel multimerization and correct coupling of activating sensors and channel gate. Centered in this region it appears a 6-mer sequence called TRP box which is also preserved in other TRP channels members. Previous studies have identified that changes in this area, and specifically in TRP box residues, drastically affect the channel response to all activating stimuli, thus implying a pivotal role of these residues in channel gating. We have further interrogated the role of this domain in TRPV1 function by using two complementary approaches based on site-directed mutagenesis. On one hand, we performed sequential mutations to recover the original sequence of TRP domain in TRPV1 from a non-functional chimera containing the cognate sequence from TRPV2 (TRPV1-AD2). Minor changes in this region severely affected channel response to voltage, capsaicin and heating temperatures. In turn, protein structure was also impaired by these mutations since we detected a dramatic decrease in protein expression level. Furthermore, we studied the involvement of TRP box residues I696 and W697 in TRPV1 by incorporating 18 natural L-amino acids and evaluating their impact on voltage and capsaicin gating. Analysis of the experimental data from both approaches with an allosteric model of activation indicates that mutations in this region primarily affected the equilibrium constant of gate opening and the allosteric coupling constants of ligand, voltage and temperature sensors to the channel pore. Taken together, our findings substantiate the notion that inter- and/or intra-subunit interactions at the level of the TRP box, and TRP domain, are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions has a drastic impact on the efficacy and potency of the activating stimuli. Furthermore, our results signal to these interactions as potential sites for pharmacological intervention.