TRPA1 agonist activity of probenecid desensitizes channel responses: consequences for screening.
The transient receptor potential channel subtype A member 1 (TRPA1) is a nonselective cation channel widely viewed as having therapeutic potential, particularly for pain-related indications. Realization of this potential will require potent, selective modulators; however, currently the pharmacology of TRPA1 is poorly defined. As TRPA1 is calcium permeable, calcium indicators offer a simple assay format for high-throughput screening. In this report, we show that probenecid, a uricosuric agent used experimentally in screening to increase loading of calcium-sensitive dyes, activates TRPA1. Prolonged probenecid incubation during the dye-loading process reduces agonist potency upon subsequent challenge. When Chinese Hamster Ovary (CHO)-hTRPA1 or STC-1 cells, which endogenously express TRPA1, were dye loaded in the presence of 2 mM probenecid TRPA1, agonists appeared less potent; EC(50) for allyl isothiocyante agonists in CHO-hTRPA1 was increased from 1.5±0.19 to 7.32±1.20 μM (P<0.01). No significant effect on antagonist potency was observed when using the agonist EC(80) concentration determined under the appropriate dye-loading conditions. We suggest an alternative protocol for calcium imaging using another blocker of anion transport, sulfinpyrazone. This blocker significantly augments indicator dye loading and the screening window, but is not a TRPA1 agonist and has no effect on agonist potency.
Novartis Institutes for BioMedical Research, Horsham Research Centre, Horsham, United Kingdom.
Conor
C
McClenaghan
Fanning
F
Zeng
Jan Martin
JM
Verkuyl
D000818:Animals; D016466:CHO Cells; D015220:Calcium Channels; D004396:Coloring Agents; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D007201:Indicator Dilution Techniques; D007473:Ion Channels / Q000187:drug effects*; D009419:Nerve Tissue Proteins / Q000819:agonists*; D018408:Patch-Clamp Techniques; D011339:Probenecid / Q000494:pharmacology*; D012076:Renal Agents / Q000494:pharmacology*; D013442:Sulfinpyrazone / Q000494:pharmacology; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000819:agonists*
D015220:Calcium Channels; D004396:Coloring Agents; D007473:Ion Channels; D009419:Nerve Tissue Proteins; D012076:Renal Agents; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; D011339:Probenecid; D013442:Sulfinpyrazone