Ethosuximide: Subunit- and Gβγ-dependent blocker and reporter of allosteric changes in GIRK channels

The antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations in Gβ₁ protein, likely through the inhibition of G-protein gated K⁺ (GIRK) channels. Here, we investigated the mechanism of ETX inhibition (block) of different GIRKs.

We studied ETX inhibition of GIRK channels expressed in Xenopus oocytes with or without their physiological activator, the G protein subunit dimer Gβγ. ETX binding site and mode of action were analysed using molecular dynamic (MD) simulations and kinetic modelling, and the predictions were tested by mutagenesis and functional testing.

We show that ETX is a subunit-selective, allosteric blocker of GIRKs. The potency of ETX block is increased by Gβγ, in parallel with channel activation. MD simulations and mutagenesis locate the ETX binding site in GIRK2 to a region associated with phosphatidylinositol-4,5-bisphosphate (PIP₂) regulation, and suggest that ETX acts by closing the helix bundle crossing (HBC) gate and altering channel's interaction with PIP₂. The apparent affinity of ETX block is highly sensitive to changes in channel gating caused by mutations in Gβ₁ or GIRK subunits.

ETX block of GIRKs is allosteric, subunit-specific, and enhanced by Gβγ through an intricate network of allosteric interactions within the channel molecule. Our findings pose GIRK as a potential therapeutic target for ETX and ETX as a potent allosteric GIRK blocker and a tool for probing gating-related conformational changes in GIRK.