A Cell-Based Rb⁺-Flux Assay of the Kv1.3 Potassium Channel

Abstract

The Kv1.3 channels expressed by human T lymphocytes are emerging as important therapeutic targets. Peptides like agitoxin and margatoxin in scorpion venom and some non-peptide small molecules are known to inhibit this channel. Since such blockers cannot be used as drugs, pharma has a need to discover effective blockers. The major limiting factor for such development has been the lack of a reliable high-throughput screening (HTS) technology. A cell-based HTS assay for this target was developed in 96-well format to facilitate screening of many candidates. The assay incorporates rubidium ion as a tracer for potassium ion, which can be analyzed by the atomic absorption spectroscopy. The assay provided a Z' factor of 0.813 with more than a 4.5-fold window of detection. The two known blockers agitoxin and margatoxin gave a 50% inhibitory concentration (IC(50)) of 1.52 and 2 nM, respectively. These values are about five- and 2.8-fold higher than their IC(50) values obtained from patch clamp. Some non-peptide compounds like tamoxifen, nifedipine, and fluoxetine also inhibited the efflux through these channels, whereas astemizole and pimozide (potent human ether-a-go-go-related gene blockers) did not block Kv1.3 activity.

The Antipsychotic Drug Loxapine Is an Opener of the Sodium-Activated Potassium Channel Slack (Slo2.2)

“…More recently, AAS methods have been developed for measurement of nonradioactive Rb ϩ flux through several K ϩ channels, including Kv1.3 (Gill et al, 2007), KCNQ2/3 , MaxiK (Parihar et al, 2003), and hERG (Chaudhary et al, 2006) channels. These results confirm that Rb ϩ efflux measurement with AAS represents a reliable, safe, low-cost alternative for K ϩ channelscreening purposes (Parihar et al, 2003;Terstappen, 2004;Gill et al, 2007). EC 50 values determined by using AAS and patch-clamp methods were in good keeping for loxapine (3.5 and 4.4 M, respectively) but showed some discrepancies for niclosamide (0.7 and 2.9 M, respectively).…”

The Design and Application of Target-Focused Compound Libraries

“…BioFocus screened a small set of SFI01 compounds against two potassium VGICs (KCNQ2/3 and IK), one calcium VGIC (Cav) and one sodium VGIC (Nav1.5), using ion flux as a measure of activity in non voltage-clamped cells [32]. Two micromolar hits exhibited clear signs of selectivity for the calcium channel (compound A for Cav) and for one of the potassium channels (compound B for KCNQ2/3), respectively (Table 3 ), demonstrating that exquisite selectivity can be obtained with a single library.…”

Impact of Novel Screening Technologies on Ion Channel Drug Discovery

“…Commercialization of non-isotopic detection methods, such as atomic absorption detection, for various ions makes this assay more suitable for HTS [28]. For example, atomic absorption detection of Rb + (as a surrogate ion for K + ) in a multi-well plate format has been used to produce very robust and reliable assays for a variety of potassium channels [29][30][31]. In this case, cells are pre-loaded with Rb + .…”