Abstract
TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast’s activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation.
Highlights
The KCNK gene family of two-pore domain potassium (K2P) channels contribute to background potassium current, helping to establish and maintain resting membrane potential
K2P channels have been shown to be expressed in sensory neurons, making them potential targets for the treatment of pain by using small molecules to increase channel activity and reduce neuronal excitability [3]
TREK2 and TREK1 share 79% sequence homology with each other, whereas TRAAK exhibits around 69% homology with the other TREK channels and exhibits different characteristics [4]
Summary
The KCNK gene family of two-pore domain potassium (K2P) channels contribute to background (or ‘leak’) potassium current, helping to establish and maintain resting membrane potential. TREK2 and TREK1 share 79% sequence homology with each other, whereas TRAAK exhibits around 69% homology with the other TREK channels and exhibits different characteristics [4] It has been shown in heterologous expressions systems that these channels can form heterodimers with each other [4]. All members of the TREK subfamily have been shown to be expressed in rodent and human dorsal root ganglion (DRG) cells [7,8] and, combined with their potential role modulating neuronal excitability, have been suggested as novel targets for the treatment of pain [3]. Screening of a bioactive lipid library identified 11-deoxy prostaglandin F2a as an activator of TREK2 but an inhibitor of TREK1 [20] This compound was shown to reduce Ca2þ influx in C-fiber primary DRG cells [20]. Pranlukast’s activity was confirmed in whole-cell patch clamp electrophysiology and a subset of Pranlukast analogues were screened for activity on K2P channels
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