Cell-Based hERG Channel Inhibition Assay in High-Throughput Format.

Human ether-a-go-go-related gene (hERG) channel plays an essential role in the repolarization of the cardiac action potential. Genetic mutations and some chemicals/drugs interfere with hERG channel activity, which may prolong the QT interval and potentially cause long QT syndrome. The FluxOR™ thallium flux assay performed in two cell lines, U2OS and HEK293, with stable hERG expression can be used to identify compounds that inhibit hERG channel activity. This chapter describes a cell-based hERG channel inhibition assay that has been optimized and performed in a 1536-well plate format. The homogeneous and robust assay can be used to identify compounds that inhibit hERG channel activity.

Discovery and Characterization of VU0529331, a Synthetic Small-Molecule Activator of Homomeric G Protein-Gated, Inwardly Rectifying, Potassium (GIRK) Channels.

G protein-gated, inwardly rectifying, potassium (GIRK) channels are important regulators of cellular excitability throughout the body. GIRK channels are heterotetrameric and homotetrameric combinations of the Kir3.1-4 (GIRK1-4) subunits. Different subunit combinations are expressed throughout the central nervous system (CNS) and the periphery, and most of these combinations contain a GIRK1 subunit. For example, the predominance of GIRK channels in the CNS are composed of GIRK1 and GIRK2 subunits, while the GIRK channels in cardiac atrial myocytes are made up mostly of GIRK1 and GIRK4 subunits. Although the vast majority of GIRK channels contain a GIRK1 subunit, discrete populations of cells that express non-GIRK1-containing GIRK (non-GIRK1/X) channels do exist. For instance, dopaminergic neurons in the ventral tegmental area of the brain, associated with addiction and reward, do not express the GIRK1 subunit. Targeting these non-GIRK1/X channels with subunit-selective pharmacological probes could lead to important insights into how GIRK channels are involved in reward and addiction. Such insights may, in turn, reveal therapeutic opportunities for the treatment or prevention of addiction. Previously, our laboratory discovered small molecules that can specifically modulate the activity of GIRK1-containing GIRK channels. However, efforts to generate compounds active on non-GIRK1/X channels from these scaffolds have been unsuccessful. Recently, ivermectin was shown to modulate non-GIRK1/X channels, and historically, ivermectin is known to modulate a wide variety of neuronal channels and receptors. Further, ivermectin is a complex natural product, which makes it a challenging starting point for development of more selective, effective, and potent compounds. Thus, while ivermectin provides proof-of-concept as a non-GIRK1/X channel activator, it is of limited utility. Therefore, we sought to discover a synthetic small molecule that would serve as a starting point for the development of non-GIRK1/X channel modulators. To accomplish this, we used a high-throughput thallium flux assay to screen a 100 000-compound library in search of activators of homomeric GIRK2 channels. Using this approach, we discovered VU0529331, the first synthetic small molecule reported to activate non-GIRK1/X channels, to our knowledge. This discovery represents the first step toward developing potent and selective non-GIRK1/X channel probes. Such molecules will help elucidate the role of GIRK channels in addiction, potentially establishing a foundation for future development of therapies utilizing targeted GIRK channel modulation.

Block of Kir channels by flonicamid disrupts salivary and renal excretion of insect pests.

Flonicamid is a selective insecticide for the control of sap-sucking insects; it exerts toxic effects by inhibiting insect feeding. However, its molecular target remains elusive. In this study, we functionally characterized NlKir1 channels of the brown planthopper (Nilaparvata lugens) in HEK293 cells. Homomeric NlKir1 channels generated inward-rectifying K+ currents. Flonicamid inhibited NlKir1 channels at nanomolar concentrations. Furthermore, flonicamid inhibited honeydew and salivary secretions of planthoppers, and reduced the renal excretion of female mosquitoes in a dose-dependent manner. The inhibitory effect of flonicamid on fluid secretion of isolated Malpighian tubules from Culex pipiens pullens was comparable to that of the selective Kir1 inhibitor. The observed physiological alterations by flonicamid are likely mediated by Kir1 channels and could lead to the disruption of feeding behaviors and eventually lethality. Our study establishes the Kir1 channel as the target of flonicamid and provided new insights into the mode of action of flonicamid.

Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay.

The family of inward rectifying potassium channels (Kir channels) plays crucial roles in the regulation of heart rhythms, renal excretion, insulin release, and neuronal activity. Their dysfunction has been attributed to numerous diseases such as cardiac arrhythmia, kidney failure and electrolyte imbalance, diabetes mellitus, epilepsy, retinal degeneration, and other neuronal disorders. We have recently demonstrated that the melanocortin-4 receptor (MC4R), a Gαs-coupled GPCR, regulates Kir7.1 activity through a mechanism independent of Gαs and cAMP. In contrast to the many other members of the Kir channel family, less is known about the biophysical properties, regulation, and physiological functions of Kir7.1. In addition to using conventional patch clamp techniques, we have employed a high-throughput Tl+ flux assay to further investigate the kinetics of MC4R-Kir7.1 signaling in vitro. Here, we discuss the employment of the Tl+ flux assay to study MC4R -mediated regulation of Kir7.1 activity and to screen compounds for drug discovery.