title: 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(©)), 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.
authors.affiliation: 1 Department of Anesthesiology, Vanderbilt University School of Medicine , Nashville, Tennessee.
authors.forename: Rene
authors.initials: R
authors.lastname: Raphemot
authors.forename: Rishin J
authors.initials: RJ
authors.lastname: Kadakia
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Olsen
authors.forename: Sreedatta
authors.initials: S
authors.lastname: Banerjee
authors.forename: Emily
authors.initials: E
authors.lastname: Days
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Smith
authors.forename: C David
authors.initials: CD
authors.lastname: Weaver
authors.forename: Jerod S
authors.initials: JS
authors.lastname: Denton
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000098854:Kcnj10 Channel
chemical_list: D000098854:Kcnj10 Channel; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying