A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels

ACS Chem Biol. 2013 Aug 16;8(8):1841-51. doi: 10.1021/cb400289x. Epub 2013 Jun 17.

Abstract

K2P (KCNK) potassium channels generate "leak" potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure-activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Drug Evaluation, Preclinical*
  • Models, Biological
  • Molecular Structure
  • Potassium Channels, Tandem Pore Domain / chemistry
  • Potassium Channels, Tandem Pore Domain / metabolism*
  • Protein Binding / drug effects
  • Small Molecule Libraries / pharmacology*
  • Temperature*
  • Yeasts / enzymology
  • Yeasts / genetics

Substances

  • KCNK1 protein, human
  • Potassium Channels, Tandem Pore Domain
  • Small Molecule Libraries