Development of an HTS Assay for Na +,K + -ATPase Using Nonradioactive Rubidium Ion Uptake

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°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 (IC₅₀) value for CHO-K1 cell line ATPase was observed to be 298 µM after 3 h of incubation. In addition, IC₅₀ values of 94 and 89 µM 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.