Functional analysis of large conductance Ca2(+)-activated K(+) channels: ion flux studies by atomic absorption spectrometry.
Although techniques such as (86)Rb(+) flux provide a sensitive measure of K(+) channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK(Ca) alpha subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BK(Ca) channel openers, including NS1608, NS1619, and NS-8, activated rubidium efflux with EC(50) values ranging from 1 to 4 microM in both radioactive and nonradioactive efflux formats. Compounds such as iberiotoxin, paxilline, and charybdotoxin inhibited rubidium efflux responses evoked by the BK(Ca) channel opener NS1608 in both radioactive and nonradioactive efflux formats. The IC(50) values of the inhibitors in AAS format were comparable to those derived from (86)Rb(+) efflux assays. The present studies show that the pharmacological profiles of BK(Ca) channels assessed by AAS compare well with those obtained using the (86)Rb(+) efflux assay, and support the utility of nonradioactive efflux format for higher throughput screening campaigns for novel K(+) channel modulators.
Parihar
Ashutosh S
AS
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6125, USA.
Groebe
Duncan R
DR
Scott
Victoria E
VE
Feng
Jianlin
J
Zhang
Xu-Feng
XF
Warrior
Usha
U
Gopalakrishnan
Murali
M
Shieh
Char-Chang
CC
D001562:Benzimidazoles / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects / Q000502:physiology*; D011865:Radioisotope Dilution Technique; D015203:Reproducibility of Results; D012413:Rubidium / Q000378:metabolism*; D012414:Rubidium Radioisotopes / Q000378:metabolism; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic / Q000379:methods*
D001562:Benzimidazoles; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; D012414:Rubidium Radioisotopes; C086491:NS 1619; D012413:Rubidium