Molecular Mechanisms, Modulation, and Synaptic Organization of Kainate Receptors

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Molecular Mechanisms, Modulation, and Synaptic Organization of Kainate Receptors Kainate receptors (KARs) are members of the ionotropic glutamate receptor (iGluR) family of cation channels, which also includes AMPA and NMDA receptors. They localize to synapses where they respond to L- glutamate (L-Glu) which is the most abundant excitatory neurotransmitter in the brain. KARs serve a canonical role in synaptic depolarization, and non-canonical roles in modulating L-Glu and GABA release and synapse maturation. They are involved in pain perception, epilepsy and mood disorders which has made them important drug targets. Unfortunately, our understanding of KAR molecular mechanisms is limited without structures for the most abundant KAR complexes in the brain. My research program aims to address this gap by developing molecular mechanisms for KAR gating, modulation, and organization. KARs assemble as tetramers from a pool of five subunits (GluK1-5). After assembly and positioning at the synapse, they respond to synaptic L-Glu by opening their ion channel and, under the sustained presence of L-Glu, desensitize within milliseconds to close their channel. A goal of my lab is to understand how KAR subunits are organized in the receptor, and how the subunits coordinate together to control the ion channel. My recent cryo-electron microscopy (cryo-EM) work revealed the surprising organization, symmetries, and domain interfaces of homo-tetrameric GluK2 (Meyerson et al. 2014 Nature; Meyerson et al. 2016 Nature). However, these results are of limited physiological value because native KARs in the brain are hetero-tetramers composed of GluK2 and GluK5 subunits. The GluK2/K5 hetero-tetramer has strict assembly rules and unique kinetic properties which underpin KAR physiology, but little is known of its subunit organization, or activation and desensitization mechanisms. Our preliminary data show successful expression, purification, and initial structure determination of the GluK2/K5 heteromer to 3.7 Å resolution with cryo-EM. In research Area 1 we aim to determine how GluK2/K5 organizes and functions by using a combination of single molecule fluorescence, electrophysiology, and cryo-EM. KARs do not function in isolation. Synaptic KAR complexes incorporate transmembrane Neto2 auxiliary proteins which shapes their characteristic kinetic profile. KARs also attach to C1ql2 proteins which are required for proper receptor localization. Understanding how KARs interact with these proteins is essential to bridging the gap between molecular mechanism and synapse biology and is a major research goal. Towards this end we have developed new biochemical preparations which enable detailed mechanistic interrogation on these systems. In Area 2 we will determine how KARs interact with and are modulated by C1ql2 and Neto2. I hypothesize that our work in research Areas 1 and 2 will give important insights into KAR physiology.