A thallium transport FLIPR-based assay for the identification of KCC2-positive modulators

KCC2, potassium chloride cotransporter 2, is expressed exclusively in the CNS (on inhibitory neurons) and plays a major role in maintaining appropriately low intracellular chloride levels that ensure inhibitory actions of GABA(A) and glycine receptors. As such, it plays a pivotal role in inhibitory mechanisms that control neuronal excitation in the CNS. KCC2 downregulation has been implicated in various excitatory disorders, such as epilepsy and neuropathic pain. Positive modulators of KCC2 expression or activity may thus provide effective therapy for these disorders. However, the identification of such agents is hindered by the lack of a high-throughput screening method. Here the authors report the development of a fluorescence-based thallium (Tl(+)) transport assay using a Fluorometric Imaging Plate Reader (FLIPR), in which KCC2 activity is assessed by measuring the initial rate of KCC2-mediated Tl(+) transport/influx. The authors demonstrate Tl(+)/Cl(-) cotransport by KCC2, which exhibits a high apparent affinity for Tl(+) and dependency on the presence of the Cl(-) ion. Pharmacological studies revealed anticipated effects and potencies of known KCC-positive (NEM, staurosporine) and KCC-negative (DIOA, furosemide) modulators. The authors demonstrate that the assay is robust and reproducible and can be employed in high-throughput screening for positive modulators of KCC2 as potential therapeutic agents.