A Soluble Ion Channel Binding Assay for Allosteric Modulator Screening

Abstract

Ion channels serve as potential therapeutic targets in diverse diseases including neuropathic pain and cardiovascular diseases. However, due to the hydrophobic nature of ion channels, current assays are largely medium-throughput screening, which limit the development of highly selective compounds for many channel subtypes. Here we developed a cell-free bioluminescence resonance energy transfer (BRET) assay to detect allosteric ligands based on the displacement of a fluorescent phosphatidylinositol 4,5-bisphosphate (FL-PIP2) probe as PIP2 binds to a wide range of ion channels in a conformation-dependent manner. We showed that the recently found allosteric agonist ML335 increased PIP2 binding to detergent-purified TREK-1 by 48% (p<0.001) while allosteric antagonist norfluoxetine completely blocked the fluorescent probe (p<0.0001). This assay also characterized the binding affinities of diverse signaling lipids to ion channels. We demonstrate a simple mix-and-read binding assay to screen for potent allosteric modulators of ion channels. Our assay could be further miniaturized to perform ultrahigh-throughput drug discovery. Cartoon representation showing a single subunit of detergent-purified HYPERLINK “https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/kcnk2” \o “Learn more about KCNK2” TREK-1 fused to the HYPERLINK “https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bioluminescence” \o “Learn more about Bioluminescence” bioluminescent protein, Nluc. Excitation of bound FL-PIP2 by Nluc produces a BRET-specific signal. A structure of FL-PIP2 (right) labeled with BODIPY-TMR (red circle) (542/574 nm). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.