Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl– channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on α1, α2, α1β and α2β GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the β subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by β subunit expression. However, ginkgolide A sensitivity was increased in the α2β GlyR relative to the α2 GlyR but not in the α1β GlyR relative to the α1 GlyR. We hypothesised that the subunit‐specific differences were mediated by residue differences at the second transmembrane domain 2′ and 6′ pore‐lining positions. The increased ginkgolide A sensitivity of the α2β GlyR was transferred to the α1β GlyR by the G2′A (α1 to α2 subunit) substitution. In addition, the α1 subunit T6′F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore‐lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2′ residues of both α1 and β subunits. These findings provide strong evidence for ginkgolides binding at the 2′ pore‐lining position.