title: Rb+ efflux assay for assessment of non-selective cation channel activities.
abstract: ABSTRACT Transient receptor potential (TRP) channels have been found to play important roles in cellular physiology and hold promise as therapeutic targets. These channels activate in response to a variety of chemical or physical stimuli and conduct non-selective cation currents (NSCC). Due to their unique activation properties, application of automated electrophysiology to measure the channel activity has been difficult. Using HEK293 cells stably expressing human TRP channels, hTRPC6 and hTRPA1, we developed and validated a high-throughput Rb(+) efflux assay for NSCC channels. The assay was performed in cell-based 96-well format. A significant increase in Rb(+) efflux can be detected upon channel activation by specific agonists, confirming that both TRPC6 and TRPA1 channels are permeable to Rb(+) ions. The agonists induced Rb(+) efflux can be blocked by known channel blockers and selected compounds from our high-throughput screening (HTS) hits. The assay is suitable for HTS with Z' factors of 0.53 and above. We also tested the Ca(2+) effect on channel activities in this assay. Both TRPC6 and TRPA1 channels were found to be inhibited by increasing the concentration of Ca(2+) in the assay buffer. However, Ca(2+) significantly reduced the potency of allyl isothiocyanate (AITC) on TRPA1 but did not affect the potency of carbochol on TRPC6. Using this assay for secondary confirmation screen, we successfully identified and confirmed the positive hits as TRPC6 inhibitors.
authors.affiliation: Screening Sciences, Wyeth Research , Collegeville, Pennsylvania, USA. liuk2@wyeth.com
authors.forename: Kun
authors.initials: K
authors.lastname: Liu
authors.forename: Manoj
authors.initials: M
authors.lastname: Samuel
authors.forename: Richard K
authors.initials: RK
authors.lastname: Harrison
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Paslay
mesh_terms: D002021:Buffers; D015220:Calcium Channels / Q000187:drug effects; D002412:Cations / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D007473:Ion Channels / Q000187:drug effects* / Q000378:metabolism*; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000187:drug effects; D018408:Patch-Clamp Techniques; D012413:Rubidium / Q000378:metabolism*; D000074025:TRPA1 Cation Channel; D050052:TRPC Cation Channels / Q000187:drug effects; D000074287:TRPC6 Cation Channel; D050051:Transient Receptor Potential Channels / Q000187:drug effects
chemical_list: D002021:Buffers; D015220:Calcium Channels; D002412:Cations; D007473:Ion Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D050051:Transient Receptor Potential Channels; D012413:Rubidium