Ethosuximide is an allosteric blocker of girk channels with Gβγ and is subunit dependent.

Abstract

Ethosuximide (ETX) is a common drug in the treatment of absence epilepsy. ETX is known to act via inhibition of T-type voltage gated Ca2+ channels. Later, it was shown that ETX blocks GIRK channels as well. Recently, ETX was shown to reduce absence-like epileptiform activity in mice carrying disease-causing mutations in GNB1 gene, which encodes for Gβ1 subunit. Gβγ subunits are the main gating factor of GIRK channels. Here, we study the mechanism by which ETX blocks GIRK channels. We confirmed that CaV3.2, as well as CaV1.2 channels are weakly blocked by ETX at therapeutic doses (0.25-0.75 mM), as was shown previously. Homotetrameric GIRK2 channels co-expressed with Gβγ subunits were almost completely inhibited at therapeutic doses of ETX, while heterotetrameric GIRK1/2 channels were mildly inhibited, and GIRK1/3 were non-sensitive. We found that the open channel mutant GIRK2N94H or ivermectin activated GIRK2WT channel were not sensitive to ETX, suggesting that it is not an open channel blocker. The affinity of ETX to GIRK2 homotetramers was increased with increasing doses of Gβγ, suggesting an allosteric inhibition. Exchange of the GIRK2 transmembrane domain (TM) with GIRK3's (GIRK2G3TM) turned the channel non-sensitive to ETX block, the opposite exchange made GIRK3G2TM more sensitive to ETX block. This data suggests that ETX blocks GIRK channels in subunit-dependent manner, it is an allosteric- rather than open channel blocker, and its binding site may be located within the TM or extracellular domain of the channel.