Construct design

Heteromer expression constructs were designed starting with genes for full-length rat GluK2 and GluK5. The genes were cloned into the pEZT-BM vector (Morales-Perez et al., 2016) and fused in frame via a thrombin recognition site to a Twin-Strep affinity tag (GluK2), and EGFP and a 1D4 tag (GluK5). The GluK2 gene underwent RNA editing at position 567 (I to V), and mutation of C576V and C595S to promote subunit expression (Schauder et al., 2013). The GluK5 gene was mutated at four cysteine positions (C559V, C578S, C619I, and C813A), truncated at position 827 after the M4 helix, and the GluA2 ‘tail’ (YKSRAEAKRMK) (Lee et al., 2014; Song et al., 2018) was added at the subunit C-terminus to improve heteromer monodispersity. The constructs are referred to as GluK2_em and GluK5_em and their heteromeric complex as GluK2/K5_em.