title: Validation of an atomic absorption rubidium ion efflux assay for KCNQ/M-channels using the ion Channel Reader 8000.
abstract: M-channels (M-current), encoded by KCNQ2/3 K(+) channel genes, have emerged as novel drug targets for a number of neurological disorders. The lack of direct high throughput assays combined with the low throughput of conventional electrophysiology (EP) has impeded rapid screening and evaluation of K(+)-channel modulators. Development of a sensitive and efficient assay for the direct measurement of M-current activity is critical for identifying novel M-channel modulators and subsequent investigation of their therapeutic potential. Using a stable CHO cell line expressing rat KCNQ2/3 K(+) channels confirmed by EP, we have developed and validated a nonradioactive rubidium (Rb(+)) efflux assay in a 96-well plate format. The Rb(+) efflux assay directly measures the activity of functional channels by atomic absorption spectroscopy using the automated Ion Channel Reader (ICR) 8000. The stimulated Rb(+) efflux from KCNQ2/3-expressing cells was blocked by the channel blockers XE991 and linopirdine with IC(50) values of 0.15 microM and 1.3 microM, respectively. Twelve compounds identified as KCNQ2/3 openers were further assessed in this assay, and their EC(50) values were compared with those obtained with EP. A higher positive correlation coefficient between these two assays (r = 0.60) was observed than that between FlexStation membrane potential and EP assays (r = 0.23). To simplify the assay and increase the throughput, we demonstrate that EC(50) values obtained by measuring Rb(+) levels in the supernatant are as robust and consistent as those obtained from the ratio of Rb(+) in supernatant/lysate. By measuring the supernatant only, the throughput of ICR8000 in an eight-point titration is estimated to be 40 compounds per day, which is suitable for a secondary confirmation assay.
authors.affiliation: Discovery Neuroscience, Wyeth Research, CN-8000, Princeton, NJ 08543, USA. wangk@wyeth.com
authors.forename: Kewei
authors.initials: K
authors.lastname: Wang
authors.forename: Beal
authors.initials: B
authors.lastname: McIlvain
authors.forename: Eugene
authors.initials: E
authors.lastname: Tseng
authors.forename: Dianne
authors.initials: D
authors.lastname: Kowal
authors.forename: Flora
authors.initials: F
authors.lastname: Jow
authors.forename: Ru
authors.initials: R
authors.lastname: Shen
authors.forename: Howard
authors.initials: H
authors.lastname: Zhang
authors.forename: Qin Jennifer
authors.initials: QJ
authors.lastname: Shan
authors.forename: Lan
authors.initials: L
authors.lastname: He
authors.forename: Diana
authors.initials: D
authors.lastname: Chen
authors.forename: Qiang
authors.initials: Q
authors.lastname: Lu
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D051036:Large-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D012413:Rubidium / Q000378:metabolism*; D013054:Spectrophotometry, Atomic / Q000379:methods*
chemical_list: D007473:Ion Channels; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496911:Kcnq1 protein, rat; D051036:Large-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; D024642:Potassium Channels, Voltage-Gated; D012413:Rubidium