The small molecule CLP257 does not modify activity of the K+–Cl− co-transporter KCC2 but does potentiate GABAA receptor activity
Abstract
To the Editor: The neuronal K+–Cl– co-transporter KCC2 (also known as solute carrier family 12 member 5, or SLC12A5) critically maintains the neuronal Cl– gradient to establish hyperpolarizing signaling by GABAA receptors, the primary mediators of fast synaptic inhibition in the brain. These receptors are also the principal targets of benzodiazepines, neurosteroids, and intravenous general anesthetics1. The downregulation of KCC2 in neuropathic pain models and seizurerelated disorders indentifies it as a putative therapeutic target. Through a small-molecule screen in neuroblastoma/glioma-derivative NG108-15 cells, Gagnon et al.2 identified CLP257 as a ‘KCC2 activator’. We therefore sought to confirm and further determine its mechanism of action. We began our investigation by using gramicidin perforated-patch recordings to measure the levels of intracellular Cl– ([Cl]i) in NG108-15 cells that were cultured as detailed in the original report. We observed no change in intracellular Cl– values after a 5-h exposure to CLP257, which according to the original paper would produce an easily detectable ~23 mM reduction in Cl– (Fig. 1a). Furthermore, acute administration of the selective KCC2 inhibitor VU0463271 did not affect Cl– levels in either vehicleor CLP257-treated cells (Supplementary Fig. 1)3. In parallel experiments, we analyzed the actions of CLP257 on KCC2 function using fluorescence resonance energy transfer (FRET)-based Cl– imaging that reproduced the conditions of the…
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