title: Kinetics of penetration influence the apparent potency of vanilloids on TRPV1.
abstract: Evidence that the ligand binding site of TRPV1 lies on the inner face of the plasma membrane and that much of the TRPV1 itself is localized to internal membranes suggests that the rate of ligand entry into the cell may be an important determinant of the kinetics of ligand action. In this study, we synthesized a BODIPY TR-labeled fluorescent capsaicin analog (CHK-884) so that we could directly measure ligand entry. We report that CHK-884 penetrated only slowly into Chinese hamster ovary (CHO) cells expressing rat TRPV1, with a t1/2 of 30 +/- 4 min, and localized in the endoplasmic reticulum and Golgi. Although CHK-884 was only weakly potent for TRPV1 binding (Ki = 6400 +/- 230 nM), it was appreciably more potent when assayed by intracellular calcium imaging and was 3.2-fold more potent with a 1-h incubation time (37 nM) than with a 5-min incubation time. Olvanil, a highly lipophilic vanilloid, yielded an EC50 of 4.3 nM upon intracellular calcium imaging with an incubation time of 1 h, compared with an EC50 value of 29.5 nM for calcium imaging assayed at 5 min. Likewise, the antagonist 5-iodo-resiniferatoxin (5-iodo-RTX) displayed a Ki of 4.2 pM if incubated with CHO-TRPV1 cells for 2 h before addition of capsaicin compared with 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may affect assessment of structure activity relations and may represent a significant factor for vanilloid drug design.
authors.affiliation: Molecular Mechanism of Tumor Promotion, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bldg. 37, Room 4048, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255, USA.
authors.forename: Jozsef
authors.initials: J
authors.lastname: Lazar
authors.forename: Derek C
authors.initials: DC
authors.lastname: Braun
authors.forename: Attila
authors.initials: A
authors.lastname: Tóth
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Larry V
authors.initials: LV
authors.lastname: Pearce
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Pavlyukovets
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Susan H
authors.initials: SH
authors.lastname: Garfield
authors.forename: Stephen
authors.initials: S
authors.lastname: Wincovitch
authors.forename: Hyun-Kyung
authors.initials: HK
authors.lastname: Choi
authors.forename: Jeewoo
authors.initials: J
authors.lastname: Lee
mesh_terms: D000818:Animals; D001547:Benzaldehydes / Q000737:chemistry / Q000493:pharmacokinetics*; D016466:CHO Cells; D006224:Cricetinae; D018613:Microscopy, Confocal; D015394:Molecular Structure; D050916:TRPV Cation Channels / Q000187:drug effects*
chemical_list: D001547:Benzaldehydes; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C100058:vanillin