Functional Analysis of Capsaicin Receptor (Vanilloid Receptor Subtype 1) Multimerization and Agonist Responsiveness Using a Dominant Negative Mutation

Kuzhikandathil, Eldo V.; Wang, Haibin; Szabó, Tamás; Morozova, Natasha; Blumberg, Peter M.; Oxford, Gerry S.

doi:10.1523/jneurosci.21-22-08697.2001

Cited by 114 publications

(100 citation statements)

References 19 publications

(49 reference statements)

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“…5C). In line with the assumption that the TRPV1-mediated acidification is due to proton entry through the channel pore, expression of a dominant negative TRPV1 NML676FAP mutation (25) failed to transmit a capsaicininduced intracellular acidification (data not shown). We conclude that the TRPV1-mediated acidification at acidic pH ext represents a true proton entry pathway rather than a buffer shuttle, and our data provide evidence for a direct H ϩ /H 3 O ϩ entry through the activated TRPV1 pore.…”

Section: Capsaicin-induced Changes Of the Ph I In Rat Dorsal Rootsupporting

confidence: 65%

TRPV1 Acts as Proton Channel to Induce Acidification in Nociceptive Neurons

Hellwig

Plant

Janson

et al. 2004

Journal of Biological Chemistry

139

101

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The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca 2؉ -dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na ؉ , Mg 2؉ , and Ca 2؉ . The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.During ischemia or inflammation, pain sensation is augmented by the acidic extracellular pH (pH ext ). 1 A␦-and C-fiber neurons sense extracellular acid by means of two different classes of cation channels, namely TRPV1, the founding member of the vanilloid receptor-like transient receptor potential channel family (1, 2), and the acid-sensing ion channels (ASIC) (3, 4). Both channel types are expressed in small diameter dorsal root ganglion (DRG) neurons, and their role in mediating inflammatory hyperalgesia has been proven by gene deletion techniques (5-7). TRPV1, a poorly selective cation channel, integrates multiple pain-inducing stimuli, including noxious heat, vanilloids, and acidic extracellular pH (1, 8 -10). Inflammatory mediators such as bradykinin, serotonin, histamine, or prostaglandins further stimulate TRPV1 activity either by protein kinase C-dependent signals (11-13), by a release from a phosphatidylinositol 4,5-bisphosphate-dependent inhibition (14,15), by formation of 12-lipoxygenase products (16), or by a protein kinase A-mediated recovery from inactivation (17). Furthermore, extracellular acidification shifts the activation of TRPV1 toward lower temperature or ligand thresholds by protonation of an amino acid located in the vicinity of the pore loop (18). Although the role of the external pH ext in modulating TRPV1 activity is well established, a possible impact of TRPV1 activation on the intracellular pH has not been studied.A Ca 2ϩ influx component through activated voltage-or ligand-gated cation channels has been recognized to lower the intracellular pH (pH i ) in neurons or in neuroendocrine cells (19,20). We therefore asked the question whether activation of the Ca 2ϩ -permeable TRPV1 may also mediate intracellular acidification in native rat dorsal root ganglion neurons or in a heterologous expression system. Our results provide evidence for two independent TRPV1-mediated acidification signals, including an as yet unrecognized direct proton conductance through the activated TRPV1 p...

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Section: Capsaicin-induced Changes Of the Ph I In Rat Dorsal Rootsupporting

confidence: 65%

TRPV1 Acts as Proton Channel to Induce Acidification in Nociceptive Neurons

Hellwig

Plant

Janson

et al. 2004

Journal of Biological Chemistry

139

101

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“…RTX binding clearly involves sites other than Met-547. In addition to sites of interaction in S2-S3 (29), sites in the cytosolic tails are essential for RTX binding (26), while mutations of sites in S6 of rat TRPV1 (30) have been shown to reduce [ 3 H]RTX binding affinity significantly.…”

Section: Discussionmentioning

confidence: 99%

Identification of Species-specific Determinants of the Action of the Antagonist Capsazepine and the Agonist PPAHV on TRPV1

Phillips

Reeve

Bevan

et al. 2004

Journal of Biological Chemistry

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The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC 50 values for capsazepine inhibition of the pH 5.5 response from >10,000 nM to 924 ؎ 241 nM in [Ca 2؉ ] i assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca 2؉ ] i assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by ϳ20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca 2؉ ] i assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca 2؉ ] i assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.The vanilloid compound capsaicin, the hot substance in chili peppers, activates a specific receptor on sensory nerves termed the vanilloid receptor 1 (VR1 or TRPV1), 1 which was cloned and initially characterized by Caterina et al. (2). TRPV1 is activated by a number of other compounds containing vanillyl groups (see Fig. 1) such as the capsaicin analogue olvanil (3), the phorbol compounds resiniferatoxin (RTX), and phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV) (4). A number of endogenous lipid molecules, such as anandamide (5) and lipoxygenase products, including some hydroxyeicosatetraenoic acids and leukotriene B 4 (6), which may be produced during inflammation also activate TRPV1. In addition to these agonists, TRPV1 is activated by low pH (Ͻ6.5) and by noxious heat (Ͼ42°C) (7-9)We have shown differences in the pharmacological properties of the mammalian orthologues of TRPV1 (1, 10). Human, rat and guinea pig TRPV1 expressed in mammalian cells can be similarly activated by capsaicin, low pH, and noxious heat (Ͼ42°C) (1, 2, 10). However, the competitive capsaicin antagonist capsazepine (11, 12) and the agonists PPAHV and RTX show species-specific differences (1, 10, 13). Capsazepine is an effective capsaicin antagonist at TRPV1 from rat, human and guinea pig. While capsazepine blocks the responses of human and guinea pig TRPV1 to low pH and heat, we observed that it only weakly inhibits these responses in cells expressing rat TRPV1 in [Ca 2ϩ ] i assays (1, 10). This pha...

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“…Such rare mutations have been recovered from screens in Escherichia coli for a mechanosensitive channel (24) or in yeast for native (25) and foreign K ϩ channel (26) after single-gene mutagenesis. Among TRP channels, disease or experimental point mutations reported to date all seem to cause reduction or loss of channel activities, including the dominant polycystic kidney disease (PKD) alleles (27) and a dominant-negative construct of TrpV1 (28). The Trp P365 mutation in the fly is an exception.…”

Section: Molecular Activities Of Y458h Y473hmentioning

confidence: 99%