Re-Evaluating a Proposed Mechanism for Ion Modulation in Kainate Receptors

Abstract

Glutamate is the major excitatory neurotransmitter in the brain, activating ionotropic receptors named for their selectivity to the agonists NMDA, AMPA, and kainate (KARs). The KAR subunit GluK2 requires external ions, in addition to glutamate, to produce a detectable current, making it unique among iGluRs. Interestingly, the non-decaying GluK2 mutant Y521C/L783C (YC/LC) functions in the absence of external ions, and has been used to suggest that ions initiate desensitization. We investigated this hypothesis through electrophysiological recordings performed on outside-out patches excised from HEK 293 cells. Unitary openings of YC/LC were extremely brief and sporadic, suggesting that desensitization is intact. Meanwhile, its agonist potency relationship was consistent with the equilibrium current of wild-type (WT) receptors, which is comprised of channels cycling between active and desensitized states. Molecular dynamics simulations of YC/LC showed that sodium quickly departs the cation binding pocket, in contrast to WT GluK2, where sodium is more stable. As a result, we decided to study GluK2 E524G, a mutant that disrupts the cation pocket. In most cases, glutamate only elicited measurable responses from E524G following application of the allosteric modulator concanavalin-A (conA), a lectin that potentiates the WT equilibrium current, and YC/LC to a lesser extent. Surface expression studies relying on TIRF microscopy and a pH-sensitive probe were used to confirm that poor functionality, not trafficking defects, account for the small responses of E254G and YC/LC. Moreover, WT GluK2 exposed to conA exhibits increased channel openings during equilibrium, suggesting both mutants principally reside in desensitized states. Our data argues that a reduction in cation binding coincides with desensitization, rather than inhibition of desensitization, supporting the perspective that external ions serve as KAR co-activators.