ABSTRACT
A high-throughput screening (HTS) assay was developed for the Na(+),K(+)-ATPase channel in order to study rubidium uptake as a measure of the functional activity and modulation of this exchanger. The assay uses elemental rubidium as a tracer for K(+) ions. Three cell lines were used to study the exchanger, and the assay was performed in a 96-well microtiter plate format. Rb(+) uptake was carried by the CHO-K1 cells at 37 degrees C; the maximum ion influx was at 80 min of incubation of the cell line in the medium containing 5.4 mM RbCl. The cells were incubated in Rb(+) uptake buffer (5.4 mM) and with the pump blocker ouabain for 1, 2, and 3 h, respectively. A complete block of the Rb(+) uptake was observed with a 5 mM concentration of ouabain for all the three time intervals. The ouabain 50% inhibitory concentration (IC(50)) value for CHO-K1 cell line ATPase was observed to be 298 microM after 3 h of incubation. In addition, IC(50) values of 94 and 89 microM were observed at 30 min of incubation, indicating that the protocol shows reproducible results. A Z' factor higher than 0.7 was observed in the assays. These studies extend the profile of Na(+),K(+)-ATPases and demonstrate the feasibility of this HTS assay system to screen for compounds that pharmacologically modulate the function of Na(+),K(+)-ATPase.
Subject(s)
Rubidium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Spectrophotometry, Atomic/methods , Animals , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Humans , Ouabain/pharmacology , Sensitivity and Specificity , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitorsABSTRACT
This article examines the utility of atomic absorption spectroscopy, in conjunction with cold flux assays, to ion channel screening. The multiplicity of ion channels that can be interrogated using cold flux assays and atomic absorption spectroscopy is summarized. The importance of atomic absorption spectroscopy as a screening tool is further elaborated upon by providing examples of the relevance of ion channels to various physiological processes and targeted diseases.
Subject(s)
Ion Channels/physiology , Spectrophotometry, Atomic/methods , Animals , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/trends , HumansABSTRACT
Ion channels are a key therapeutic target class that are relatively unexplored. New technologies and tools are enabling the interrogation of this target class for drug discovery and development. We present a summary here of the Aurora Biomed-sponsored Ion Channel Retreat held in June 2004 in Vancouver, British Columbia, Canada.
