Distinct subunits in heteromeric kainate receptors mediate ionotropic and metabotropic function at hippocampal mossy fiber synapses

Heteromeric kainate receptors (KARs) containing both glutamate receptor 6 (GluR6) and KA2 subunits are involved in KAR-mediated EPSCs at mossy fiber synapses in CA3 pyramidal cells. We report that endogenous glutamate, by activating KARs, reversibly inhibits the slow Ca2+-activated K+ current I(sAHP) and increases neuronal excitability through a G-protein-coupled mechanism. Using KAR knockout mice, we show that KA2 is essential for the inhibition of I(sAHP) in CA3 pyramidal cells by low nanomolar concentrations of kainate, in addition to GluR6. In GluR6(-/-) mice, both ionotropic synaptic transmission and inhibition of I(sAHP) by endogenous glutamate released from mossy fibers was lost. In contrast, inhibition of I(sAHP) was absent in KA2(-/-) mice despite the preservation of KAR-mediated EPSCs. These data indicate that the metabotropic action of KARs did not rely on the activation of a KAR-mediated inward current. Biochemical analysis of knock-out mice revealed that KA2 was required for the interaction of KARs with Galpha(q/11)-proteins known to be involved in I(sAHP) modulation. Finally, the ionotropic and metabotropic actions of KARs at mossy fiber synapses were differentially sensitive to the competitive glutamate receptor ligands kainate (5 nM) and kynurenate (1 mM). We propose a model in which KARs could operate in two modes at mossy fiber synapses: through a direct ionotropic action of GluR6, and through an indirect G-protein-coupled mechanism requiring the binding of glutamate to KA2

Co-assembly of two GluR6 kainate receptor splice variants within a functional protein complex

Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function