The forebrain specific
AMPA receptor antagonist, LY3130481/CERC-611,
which selectively antagonizes the AMPA receptors associated with TARP
γ-8, an auxiliary subunit enriched in the forebrain, has potent
antiepileptic activities without motor side effects. We designated
the compounds with such activities as γ-8 TARP dependent AMPA
receptor antagonists (γ-8 TDAAs). In this work, we further investigated
the mechanisms of action using a radiolabeled γ-8 TDAA and ternary
structural modeling with mutational validations to characterize the
LY3130481 binding to γ-8. The radioligand binding to the cells
heterologously expressing GluA1 and/or γ-8 revealed that γ-8
TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling
of γ-8, based on the crystal structures of a distant TARP homologue,
murine claudin 19, in conjunction with knowledge of two γ-8
residues previously identified as critical for the LY3130481 TARP-dependent
selectivity provided the basis for a binding mode prediction. This
allowed further rational mutational studies for characterization of
the structural determinants in TARP γ-8 for LY3130481 activities,
both thermodynamically as well as kinetically
