Agonist Recognition Sites in the Cytosolic Tails of Vanilloid Receptor 1

Abstract

Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N-and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [ 3 H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket.Capsaicin, the principal pungent ingredient of hot peppers, excites sensory neurons by opening an ion channel, the vanilloid receptor 1 (VR1), 1 thereby causing pain. VR1 is a ligand-gated, cationic channel that is present mainly in small nociceptive sensory neurons (1-3). The presence of VR1 in sensory neurons leads to questions concerning the existence of endogenous capsaicin-like substances, and various lipid metabolic products of lipoxygenases or anandamide have been suggested as candidates, because they activate VR1 and are structurally similar to capsaicin (4, 5). Accordingly, a role for lipoxygenase products in the activation of VR1 during inflammation was suggested (5), and in fact, bradykinin, a potent pain-causing inflammatory mediator, is now known to activate VR1 via the lipoxygenase/VR1 pathway (6). In addition, bradykinin also has a potential to sensitize VR1 via a phospholipase C or protein kinase C pathway (7-9).VR1 is also activated by acid or heat at over 43°C, a threshold temperature for pain (3, 10 -12). Moreover, because ischemic or inflamed tissues become acidic, the acid activation of VR1 is a pathologically relevant event (13). More direct evidence for the pathophysiological role of VR1 in the production of inflammatory pain came from knock-out experiments. In mice lacking VR1, thermal hyperalgesia evoked by inflammation is reduced (14, 15). Furthermore, hyperalgesia induced by the key inflammatory mediators, bradykinin and nerve growth factor, is reduced in mice lacking VR1 (8). Thus, VR1 is now considered a primary molecular transducer that mediates inflammatory hyperalgesia (13).The putative topology of VR1 indicates that it belongs to a class of transient receptor potential channels possessing six transmembrane domains and two cytosolic domains at each Nand C terminus (3, 16). VR1 appears to form a homotetramer when expressed heterologously (17). However, VR1 may form a heteromultimer with another temperature-sensitive channel, transient rec...

Role of the Transient Receptor Potential Vanilloid 5 (TRPV5) Protein N Terminus in Channel Activity, Tetramerization, and Trafficking

“…Mutation or removal of N-terminal regions abrogates function of many TRP channels studied (8,(33)(34)(35)(36); however, the underlying mechanism remains poorly understood. Within the TRPV subfamily, N-terminal deletion mutants or splice variants of TRPV1, TRPV4, and TRPV6 were investigated.…”

Phosphorylation of Vanilloid Receptor 1 by Ca2+/Calmodulin-dependent Kinase II Regulates Its Vanilloid Binding

“…Beads were washed with lysis buffer three times and subjected to SDS-PAGE. After transfer to a polyvinylidene difluoride membrane, Western blotting was performed using anti-NVR1 antiserum (diluted 1:1000) (13). Proteins were visualized using peroxidase-conjugated secondary antibody (Sigma) by enhanced chemiluminescence (ECL, Amersham Biosciences).…”

“…All mutant receptors were generated as previously described (13). Purified full-length DNAs were subcloned into pSDTF (a generous gift from T. Snutch of British Columbia University) or into pcDNA3 (Invitrogen) for expression in the oocytes of Xenopus laevis or in human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA) and were confirmed by DNA sequencing using Sequenase 2.0 (Amersham Biosciences).…”

“…2-5 days after injection, 2-electrode voltage clamp recordings were performed (E hold ϭ Ϫ60 mV) to record whole cell currents. The recording chamber was perfused at 2 ml/min with a bath solution containing 96 mM NaCl, 5 mM HEPES, 2 mM KCl, 1.8 mM CaCl 2 , and 1 mM MgCl 2 (pH 7.4) at room temperature, as previously described (13).…”

“…Using a chimeric construction with avian VR1, Jordt and Julius (12) find that transmembrane domain III and its vicinity are responsible for hydrophobic interactions with vanilloids. In addition, two individual sites in the N and C termini are known to be critical for the hydrophilic binding of vanilloids (13). Even though specific regions for ligand binding are now known, the gating mechanisms of VR1 facilitated by vanilloids are largely unknown.…”