Modular control of AMPA and Kainate receptor gating revealed by systematic domain swapping

Ionotropic glutamate receptors (iGluRs) are modular proteins whose functions arise from complex inter-domain allostery. However, the principles governing functional compatibility between domains from different iGluR subfamilies remain unclear. To address this, we created a comprehensive library of 14 reciprocal chimeric receptors between the AMPA receptor GluA2 and the kainate receptor GluK2, systematically swapping the amino-terminal (ATD), ligand-binding (LBD), transmembrane (TMD), and C-terminal (CTD) domains. Using patch-clamp electrophysiology, we mapped the functional consequences of these perturbations. Our analysis reveals that the ∼50-fold difference in recovery from desensitization between GluA2 and GluK2 is a cooperative property of a tightly coupled LBD-TMD gating cassette, with the LBD's S2 lobe as a primary determinant. We also identified strict rules of functional compatibility; pairing the GluK2 TMD with the GluA2 CTD was non-functional, demonstrating a requirement for co-evolved coupling. This systematic approach uncovered emergent phenotypes by disrupting critical inter-domain interfaces, including a completely non-desensitizing receptor, demonstrating that conserved functions can arise from non-conserved structural mechanisms.. We conclude that iGluR function is not purely modular but emerges from a hierarchy of domain interactions, providing a functional map of this allostery and key insights into iGluR subfamily identity.