Mapping the multi-domain allosteric network for CKAMP44 modulation of AMPA receptors

Most fast excitatory neurotransmission is mediated by AMPA-type ionotropic glutamate receptors, whose gating properties are fine-tuned by a wide range of auxiliary subunits. While bulky, multi-pass auxiliary subunits such as transmembrane AMPAR regulatory proteins (TARPs) and cornichons (CNIHs) dominate current structural models of receptor regulation by forming a stabilizing girdle around the ligand-binding and transmembrane domains, the modulatory mechanisms of simpler, single-pass transmembrane proteins like cysteine-knot AMPA receptor-modulating protein 44 (CKAMP44), also known as Shisa9, remain a critical missing link in understanding allosteric control. To identify the molecular drivers of CKAMP44 regulation, we utilized our previously characterized library of reciprocal chimeric receptors containing swapped domains between the CKAMP44-sensitive AMPAR subunit GluA2 and the CKAMP44-insensitive kainate receptor GluK2. Using whole-cell patch-clamp electrophysiology, we demonstrate that CKAMP44 modulation is not localized to a single interface but relies on a distributed allosteric network. The findings indicate a novel and essential role for the receptor's amino-terminal domain (ATD) in facilitating the CKAMP44-mediated enhancement of desensitization entry, thereby pinpointing a new site for gating regulation. Furthermore, we find that CKAMP44 functionally rescues previously non-conductive transmembrane-carboxy-terminal chimeras, highlighting a critical role in stabilizing compromised receptor assemblies. Conversely, the modulation of receptor's recovery from desensitization appears mechanistically distinct, governed primarily by the stability of the ligand-binding domain and transmembrane (TMD) interface. We propose a hierarchical dual-action model of allosteric regulation wherein the canonical gating machinery of the ligand-binding and transmembrane domains cassette is distinctly fine-tuned by distal interactions at the ATD, establishing a newly identified regulatory layer for auxiliary subunit engagement.