A graphite furnace‐atomic absorption spectrometry‐based rubidium efflux assay for screening activators of the K_v7.2/3 channel

Abstract

For the characterization of K v 7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flameatomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for K v 7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known K v 7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of K v 7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z'-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.

Development and Application of an Atomic Absorption Spectrometry-Based Method to Quantify Magnesium in Leaves of Dioscorea polystachya

“…Unfortunately, the latter is often inadvertently dismissed as an old-fashioned technique in crisis [ 8 ]. Nevertheless, AAS has recently been used in modern pharmaceutical research, for example, in the screening of potential activators of potassium channels [ 9 ] or bioanalytical investigations of anticancer and antibacterial drug candidates [ 10 , 11 ]. This documents the usefulness of AAS.…”

“…Electrothermal atomization (heating with electricity) of a graphite furnace is used for GF AAS. While F AAS certainly harbors risks given the use of highly flammable gases, GF AAS requires a smaller sample volume and is even more sensitive [ 9 ]. The lower sensitivity of F AAS can be related to the processes taking place in the burner flame.…”