Development and Validation of Fluorescence-Based and Automated Patch Clamp–Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1

Abstract

The inward rectifier potassium (Kir) channel Kir4.1 plays essential roles in modulation of neurotransmission and renal sodium transport and may represent a novel drug target for temporal lobe epilepsy and hypertension. The molecular pharmacology of Kir4.1 is limited to neurological drugs, such as fluoxetine (Prozac ª ), exhibiting weak and nonspecific activity toward the channel. The development of potent and selective small-molecule probes would provide critically needed tools for exploring the integrative physiology and therapeutic potential of Kir4.1. A fluorescence-based thallium (Tl + ) flux assay that utilizes a tetracycline-inducible T-Rex-HEK293-Kir4.1 cell line to enable high-throughput screening (HTS) of small-molecule libraries was developed. The assay is dimethyl sulfoxide tolerant and exhibits robust screening statistics (Z 0 = 0.75 -0.06). A pilot screen of 3,655 small molecules and lipids revealed 16 Kir4.1 inhibitors (0.4% hit rate). 3,3-Diphenyl-N-(1-phenylethyl)propan-1-amine, termed VU717, inhibits Kir4.1-mediated thallium flux with an IC 50 of *6 lM. An automated patch clamp assay using the IonFlux HT workbench was developed to facilitate compound characterization. Leak-subtracted ensemble ''loose patch'' recordings revealed robust tetracycline-inducible and Kir4.1 currents that were inhibited by fluoxetine (IC 50 = 10 lM), VU717 (IC 50 = 6 lM), and structurally related calcium channel blocker prenylamine (IC 50 = 6 lM). Finally, we demonstrate that VU717 inhibits Kir4.1 channel activity in cultured rat astrocytes, providing proof-of-concept that the Tl + flux and IonFlux HT assays can enable the discovery of antagonists that are active against native Kir4.1 channels.

An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

“…gambiae Malpighian tubules (Genbank Accession # KJ596497)17 was sub-cloned into the pcDNA5/TO expression vector (Life Technologies) and used for stable cell line generation, as described previously4445. Expression constructs for mammalian Kir channels were developed and validated in previous studies by our group464748.…”

ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels

“…The 3.7-fold shift in IC 50 determined with patch clamp electrophysiology, as compared with Tl + flux, is consistent with previous observations of other Kir channel inhibitors 18–20 . Quantitative Tl + flux assays were utilized to evaluate the selectivity of VU714 for Kir7.1 over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, Kir4.1, and Kir6.2/SUR1, as reported previously 16, 21, 22 . The concentration-response curves (CRCs) shown in Fig.…”

“…HTS for Kir7.1-M125R modulators was performed using a Tl + -flux reporter assay essentially as described previously 16, 20, 21 . The Kir7.1-M125R mutation increases the channel unitary conductance and enables robust Tl + flux measurement that cannot be achieved with the WT channel 18 .…”

Application of chemical biology in target identification and drug discovery

“…Metabolite-based screening assay which detects major changes in cell metabolism triggered by ligand binding to a specific receptor showed its promise (Chauret et al 2001). Fluorescence-based screenings also demonstrated suitability to discover chemical blockers to P/Q-type calcium channel (Mezler et al 2012) and Kir4.1 channel (Raphemot et al 2013). Last but not least is the wide application of second messengers (Fig.…”