Synthesis of a positional scanning library of pentamers of N-alkylglycines assisted by microwave activation and validation via the identification of trypsin inhibitors.

A positional scanning library of 625 N-alkylglycine pentamers has been synthesized on solid-phase, employing a set of 10 commercially available primary amines as a source of chemical diversity. The iterative synthetic steps were carried out in tea bags and accelerated by using microwave assisted organic synthesis (MAOS). The reactivity study of the primary amines used as diversity sources led to determine their relative reactivity values and equireactivity factors, which were applied to the library synthesis to ensure comparable concentrations of all final oligomers in the mixtures. This library was validated by the screening, deconvolution, and identification of trypsin inhibitors. These compounds are of potential interest for controlling the intracellular transport of TRPV1 channel.

Identification of molecular determinants of channel gating in the transient receptor potential box of vanilloid receptor I.

Transient receptor potential vanilloid receptor subtype I (TRPV1) is an ion channel gated by physical and chemical stimuli that belongs to the TRPV protein family. TRPV receptors contain a highly conserved, 6-mer segment near the channel gate, known as the TRP box, whose function remains unknown. Here, we performed an alanine scanning mutagenesis of the TRP box of TRPV1 (IWKLQR) and found that mutation of this motif affected channel gating by raising the free energy of channel activation. Functional characterization of TRPV1 mutants showed that substitution of I696, W697, and R701 by alanine severely affected voltage- and heat-dependent activation and notably reduced the capsaicin responsiveness and tachyphylaxia, while mutation of K698, L699, and Q700 had minor effects. In addition, mutation of I696 to alanine promoted a strong outward rectification at negative membrane potentials, and slowed the kinetics of channel activation. Taken together, our findings suggest that modification of I696, W697, and R701 to alanine altered channel function by affecting events downstream of the initial stimuli-sensing step and imply that intersubunit interactions within the TRP box play an important role in TRPV1 gating.

A role of the transient receptor potential domain of vanilloid receptor I in channel gating.

Transient receptor potential vanilloid receptor subtype 1 (TRPV1) is an ionotropic receptor activated by temperature and chemical stimuli. The C-terminal region that is adjacent to the channel gate, recognized as the TRP domain, is a molecular determinant of receptor assembly. However, the role of this intracellular domain in channel function remains elusive. Here, we show that replacement of the TRP domain of TRPV1 with the cognate region of TRPV channels (TRPV2-TRPV6) did not affect receptor assembly and trafficking to the cell surface, although those receptors containing the TRP domain of the distantly related TRPV5 and TRPV6 did not display ion channel activity. Notably, functional chimeras exhibited an impaired sensitivity to the activating stimuli, consistent with a significant contribution of this protein domain to channel function. At variance with TRPV1, voltage-dependent gating of chimeric channels could not be detected in the absence of capsaicin and/or heat. Biophysical analysis of functional chimeras revealed that the TRP domain appears to act as a molecular determinant of the activation energy of channel gating. Together, these findings uncover a role of the TRP domain in intersubunit interactions near the channel gate that contribute to the coupling of stimulus sensing to channel opening.

Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia.

Neurogenic inflammation is produced by overstimulation of peripheral nociceptor terminals by injury or inflammation of tissues. Excessive activity of sensory neurons produces vasodilation, plasma extravasation and hypersensitivity. Mechanistically, neurogenic inflammation is due to the release of substances from primary sensory nerve terminals that act directly or indirectly at the peripheral terminals, either activating or sensitizing nociceptors, endothelial cells and immunocytes. Notably, small-diameter sensory neurons that are sensitive to capsaicin play a key role in the generation of neurogenic inflammation. The cloning of the vanilloid receptor 1 (TRPV1) has been a breakthrough that has propelled our understanding of the molecular mechanisms involved in neurogenic inflammation. TRPV1 pivotally contributes to the integration of various stimuli and modulates nociceptor excitability, thus making it a true gateway for pain transduction. In addition, TRPV1 is the endpoint target of intracellular signalling pathways triggered by inflammatory mediators. Phosphorylation-induced potentiation of TRPV1 channel activity, along with an incremented TRPV1 surface expression are major events underlying the nociceptor activation and sensitization that leads to thermal hyperalgesia. The important contribution of TRPV1 receptor to the onset and maintenance of neurogenic inflammation has validated it as a therapeutic target for inflammatory pain management. As a result, the development of specific TRPV1 antagonists is a central focus of current drug discovery programs.

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

Molecular architecture of the vanilloid receptor. Insights for drug design.

The transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a molecular integrator of physical and chemical stimuli in the peripheral nociceptor terminals. TRPV1 is an ionotropic channel that plays a critical role in both thermal nociception and inflammatory hyperalgesia. Structure-function relationships are providing fundamental insights of the modular architecture of this neuronal receptor. As a result, the molecular determinants that endow TRPV1 with its physiological properties, namely activation by heat, potentiation by extracellular acidic pH, and interaction with vanilloid-like compounds, as well as its permeation properties are being unveiled. This information can now be used to build up molecular models for the protein which, upon experimental validation, could be used as tools to thrust the target-oriented design of druggable TRPV1 ligands.

Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity.

The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activity-dependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.