Integrative Binding Sites within Intracellular Termini of TRPV1 Receptor

Abstract

TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in E.coli in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C–terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our in silico predictions.

Role of TRPV channels in regulating various pancreatic β-cell functions: Lessons from <i>in vitro</i> studies

“…TRPV1 activity can be modulated by protein kinase A (23,24), protein kinase C (25) and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylation (26). Another regulatory molecule of TPRV1 is PIP2, which binds to the C-and N-terminal region of TRPV1 and thus regulates this channel activation (27,28). TRPV2 is approx.…”

Investigation of the transient receptor potential vanilloid 1 (TRPV1) ion channel

“…Grycova et al [206] found that the CaM-binding site overlapped with the PIP2-binding site in the C-terminal distal region (L777-S820) and that PIP2 interacted with the proximal region (I688-K718) of the TRPV1 Cterminal (Figure 1). Lishko et al [14] found that the TRPV1-ARD mutants K155A and K160A, which no longer bind ATP, did not interact with CaM in size exclusion chromatography, emphasizing that the binding surface on TRPV1-ARD is at least partially shared by both ligands.…”

“…Grycova et al [206] showed that two different PIP2-binding sites on the C-terminus L777-S820 and the Nterminus F189-V221 overlapped with the CaM-binding sites, and the third PIP2-binding site K688-K718 occupied the TRP domain on the C-terminus, a highly conserved sequence among the members of the TRP ion channel family. The presence of PIP2 was reported to prevent the interaction of the distal region of the Cterminus with CaM, which could play an important role in the regulation of TRPV1.…”

“…[206] K698 A The residue participates in PIP2 binding. [206] Q700 A The residue participates in PIP2 binding.…”

“…[206] K698 A The residue participates in PIP2 binding. [206] Q700 A The residue participates in PIP2 binding. [206] R701 A The residue participates in PIP2 binding.…”