TRPV1 Structure-Function study: Role of TRP domain in TRPV1 allosteric gating

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

The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating

Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention