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.

A graphite furnace‐atomic absorption spectrometry‐based rubidium efflux assay for screening activators of the Kv7.2/3 channel

“…Previous studies reported Z ’‐values of 0.73 and 0.81 for the Rb efflux assay using an automated F‐AAS procedure. [ 15,16 ]…”

“…Previous studies reported Z'-values of 0.73 and 0.81 for the Rb efflux assay using an automated F-AAS procedure. [15,16] The Z'-factor was also determined for the present GF-AAS-based Rb efflux assay based on the data from 10 experiments (Figure 4a) performed during assay development and validation. Figure 4b shows the calculated Z'-factors.…”

“…It is quite common to study the activity of potassium channels with a rubidium (Rb) efflux assay based on flame-AAS (F-AAS). [14][15][16] However, the use of F-AAS can pose hazards to laboratories due to the use of highly flammable gases. Since potassium ions are abundant in cells, the particular measurement of potassium flux by AAS is hardly feasible due to high background levels.…”

Can Graphene Bilayers Be the Membrane Mimetic Materials? Ion Channels in Graphene-Based Nanostructures

“…In literature one can find the description of all kinds of the ion channels implemented on the basis of the graphene nanopores/nanochannels [96,97]: for alkali metals [98], including biomimetic (bioinspired) analogs of the sodium and potassium channels [99] (as well as the other alkali metals, such as lithium, rubidium, and caesium [100][101][102], as follows from the thermodynamic calculations for the monovalent cations [103] and quantum chemical simulations for a number of the alkali metal ions [104], which can also operate in the channelome, but their clark is too low, and hence, does not allow to perform any significant functions at the macroscopic scale at the organism or the whole biosphere level), for the chloride ions [105] (the analogs of the known chloride ion channels [106,107]), etc. Consideration of the "exotic" pores and channels in graphene operating with the rubidium, gadolinium and other rare earth element ions [108,109] is beyond the scope of NANOSYSTEMS this paper for the purpose of maintenance of the biomimetic functional analogy.…”

Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Enhancers Using IonWorks HT

“…6B), potentially because of the nature of the non-GOhm seal of IonWorks and its resultant voltage error. When the IonWorks EC 50 s were compared with those generated with Rb + efflux using previously established methodology, 28 only a low correlation (R 2 = 0.4) was found (Table 3). It was also clear with the Rb + efflux assay that a poor correlation occurred between the maximum effects observed in Rb + and IonWorks (data not shown).…”