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First published on January 17, 2006; DOI: 10.1124/mol.105.019158

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Received for publication September 23, 2005.
Revised December 16, 2005.
Accepted for publication January 13, 2006.

Kinetics of penetration influence the apparent potency of vanilloids on TRPV1

Jozsef Lazar 1, Derek C. Braun 1, Attila Toth 1, Yun Wang 1, Larry V. Pearce 1, Vladimir A. Pavlyukovets 1, Peter M. Blumberg 1*, Susan H. Garfield 2, Stephen Wincovitch 2, Hyun-Kyung Choi 3, Jeewoo Lee 3

1 LCCTP, National Cancer Instute, National Institutes of Health 2 LEC, National Cancer Instute, National Institutes of Health 3 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University

* Address correspondence to: E-mail: blumberp{at}dc37a.nci.nih.gov

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 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 hour 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 hr, compared with an EC50 value of 29.5 nM for calcium imaging assayed at 5 min. Similarly, the antagonist 5-Iodo-RTX displayed a Ki of 4.2 pM if incubated with CHO-TRPV1 cells for 2 hours before addition of capsaicin compared to 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may impact assessment of structure activity relations and may represent a significant factor for vanilloid drug design.


Key words: Capsaicin/vanilloid, Ca imaging, Receptor binding studies


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