Development and validation of fluorescence-based and automated patch clamp-based functional assays for the inward rectifier potassium channel Kir4.1.

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(©)), 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'=0.75±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 IC50 of ∼6 μM. 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 (IC50=10 μM), VU717 (IC50=6 μM), and structurally related calcium channel blocker prenylamine (IC50=6 μM). 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.