Development of covalent chemogenetic K_2Pchannel activators

Abstract

K2Ppotassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2Pfunction remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (CovalentActivation ofTREK familyK+channels to cLAmpMembranePotential) that leverages the discovery of a site in the K2Pmodulator pocket that reacts with electrophile bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2Pfunction, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2Pchannel activity that should facilitate studies both molecular and systems level studies of K2Pfunction and enable the search for new K2Pmodulators.

Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation

“…Similar to that proposed for other TREK channel modulators (8, 10, 16), this also may also involve reorientation of the W306 and F164 side-chains, or their equivalents ( Fig 6 ). This model is also consistent with recent observations that covalent attachment of ML335 derivatives within the K2P modulator site via a conserved serine on PH1 can irreversibly activate TREK channels (34).…”

“…7 ). This model is also consistent with recent observations that covalent attachment of ML335 derivatives within the K2P modulator site via a conserved serine on PH1 can irreversibly activate TREK channels 34 .…”

“…ML335 is a small molecule TREK channel agonist that binds within the ‘K2P Modulator Pocket’, a cryptic binding site created by displacement of the top of M4 where its interactions with the P2-M4 loop and Pore-Helix-1 (PH1) are proposed to stabilise TREK-2 in a more conductive conformation 11, 16, 34 . It is therefore highly significant that interaction of Nb-Activator-76 with the P2-M4 loop also induces a displacement in the top of the M4 helix similar to that seen in the ML335-bound structures of TREK-2 ( Fig.…”

“…However, its robust activatory effect on recombinant homomeric TREK-2 channels, especially at lower concentrations, indicates that this nanobody may remain a useful tool in situations where the identity of its target is already known. ML335 is a small molecule TREK channel agonist that binds within the 'K2P Modulator Pocket', a cryptic binding site created by displacement of the top of M4 where its interactions with the P2-M4 loop and Pore-Helix-1 (PH1) are proposed to stabilise TREK-2 in a more conductive conformation (11,16,34). It is therefore highly significant that interaction of Nb-Activator-76 with the P2-M4 loop also induces a displacement in the top of the M4 helix similar to that seen in the ML335-bound structures of TREK-2 (Fig 4E).…”

“…A selective activatory nanobody. Unlike Nb-Activator-67, the second activatory nanobody (CA10776, hereafter referred to as Nb-Activator-76 or Nb76) exhibited highly selective activation of TREK-2 with an efficacy of 412 ± 14 nM and had no effect on TREK-1 or other K2P channels tested, even when used at high concentration (Fig 4A ML335 is a small molecule TREK channel agonist that binds within the 'K2P Modulator Pocket', a cryptic binding site created by displacement of the top of M4 where its interactions with the P2-M4 loop and Pore-Helix-1 (PH1) are proposed to stabilise TREK-2 in a more conductive conformation (11,16,34). It is therefore highly significant that interaction of Nb-Activator-76 with the P2-M4 loop also induces a displacement in the top of the M4 helix similar to that seen in the ML335-bound structures of TREK-2 (Fig 4E).…”