Basal tearing is crucial to maintaining ocular surface wetness. Corneal cold thermoreceptors sense small oscillations in ambient temperature and change their discharge accordingly. Deletion of the cold-transducing ion channel Transient receptor potential cation channel subfamily M member 8 (TRPM8) in mice abrogates cold responsiveness and reduces basal tearing without affecting nociceptor-mediated irritative tearing. Warming of the cornea in humans also decreases tearing rate. These findings indicate that TRPM8-dependent impulse activity in corneal cold receptors contributes to regulating basal tear flow.

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 activitydependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.TRPV1 1 is a capsaicin-, proton-and heat-sensitive, cationselective ion channel expressed in nociceptors that participates in the transduction of noxious chemical and thermal stimuli by sensory nerve endings in peripheral tissues (1-3). Heterologous expression of TRPV1 cDNA results in ionic currents that recapitulate most of the functional properties displayed by native capsaicin-and heat-activated currents in sensory neurons (1, 2). For instance, TRPV1 exhibits a time-and Ca 2ϩ -dependent desensitization, a long lasting refractory state during which the receptor does not respond to vanilloids or other stimuli (2). In addition, the channel activity of TRPV1 is remarkably upregulated by inflammatory mediators through the activation of phospholipase C and protein kinases A and C (PKA and PKC) signaling pathways (4 -13). Recent evidence shows that an increase in TRPV1 expression in peripheral nociceptors is critical for the maintenance of inflammatory hyperalgesia (14, 15). The involvement of TRPV1 in heat hypersensitivity is further underscored by the reduced sensitivity of mice lacking TRPV1 (16, 17) and by mice treated with receptor-specific antagonists (18).TRPV1 belongs to the family of transient receptor potential channels, which structurally resembles the family of voltagegated potassium or cyclic nucleotide-gated channels (19). Accordingly, these channels are presumed to be tetrameric assemblies of identical subunits (Fig. 1A), although heteromeric assemblies have be...

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu 648 and Glu 651 rendered minor effects on VR1 pore attributes, while Glu 636 slightly modulated pore blockade. In contrast, replacement of Asp 646 by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg 2؉ with respect to Na ؉ without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp 646 is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.The molecular mechanism underlying chemical and thermal nociception is starting to be understood, thanks to the cloning of a capsaicin-operated neuronal receptor referred to as the vanilloid receptor subunit 1 (VR1) 1 (1). VR1 is a nonselective cation channel with high Ca 2ϩ permeability that integrates both types of pain-producing stimuli (1-5). These channels are activated by vanilloids such as capsaicin, the pungent ingredient of hot red peppers, and by temperatures higher than 40°C (1, 2, 4). Recently, the lipid-based anandanamide was shown to be a potential endogenous VR1 agonist (6). Activation of the VR1 channel raises intracellular Ca 2ϩ and excites a subset of dorsal root and trigeminal ganglion primary neurons (5). These neurons transmit noxious information to the central nervous system and release proinflammatory neuropeptides at peripheral terminals (5, 7). In addition to playing a role in nociception, the high Ca 2ϩ permeability exhibited by VR1 strongly desensitizes capsaicin-operated responses (5, 7). This property partially accounts for the antinociceptive activity exhibited by vanilloids (5,8,9).VR1 subunits are membrane proteins with a predicted relative molecular mass of 95 kDa that show similarity to the family of putative store-operated calcium channels (1,3,10). Although the molecular composition and stoichiometry of neuronal VR1 channels is undetermined, heterologous expression of VR1 subunits gives rise to homomeric receptors that recapitulate most of the reported physiological properties (1, 2, 4, 5, 7). Nonetheles...

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