Date of Award

Spring 2026

Language

English

Embargo Period

4-28-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Program

Chemistry

First Advisor

Li Niu

Committee Members

Alexander Shekhtman, Mehmet Yigit, Jia Sheng, Haijun Chen

Keywords

Glutamate receptors, kainate receptors, kinetic mechanism of channel opening, GluK2, NETO1/2

Subject Categories

Biochemistry | Biophysics

Abstract

Ionotropic glutamate receptors (iGluRs) are found both pre- and postsynaptically in neuronal membranes, and mediate the majority of fast excitatory synaptic transmission in response to the binding of glutamate, a major excitatory neurotransmitter in the central nervous system (CNS). Ionotropic glutamate receptors are divided into three distinct subtypes: kainate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartic acid (NMDA) receptors. Auxiliary proteins specific to each receptor subtype also play a role in tuning both receptor expression and gating properties, such as neuropilin and tolloid-like (NETO) proteins that modulate kainate receptors, and transmembrane AMPA receptor regulatory proteins (TARPs) that regulate AMPA receptors. Like other iGluR subtypes, kainate receptors are tetrameric assemblies of a complex array of subunit compositions. Among the five kainate receptor subunits, i.e., GluK1-5, each of the GluK1–3 subunits can form functional, homomeric channels, whereas GluK4/5 subunits only form functional receptors with at least one of GluK1–3. GluK4/5 heteromeric channels have a higher affinity for glutamate than any of GluK1–3 homomeric channels. Neuronal kainate receptors are known to both contain high affinity kainate receptor subunits (i.e., GluK4 and GluK5) and interact with auxiliary subunits, termed as NETO1 and NETO2. Dysregulation and dysfunction of kainate receptors have been implicated in a variety of neurological diseases and disorders, such as epilepsy and neuropathic pain. Therefore, understanding how these complex receptors work is significant. My thesis work is centered on both the pore-forming subunits of kainate receptors and their auxiliary NETO proteins.

One focus of my studies is the GluK5 subunit. GluK5 is thought to be the most widely expressed kainate receptor subunit. It must coassemble with a pore-forming subunit to form a functional channel. Among kainate receptors, GluK1 and GluK2 are two key pore-forming subunits. Currently, it is not known if incorporation of GluK5 into GluK1/K5 and GluK2/K5 heteromers affects the rate of channel opening in response to glutamate binding, compared with GluK1 and GluK2 homomeric channels. The lack of knowledge is due to the fact that current kinetic techniques do not have sufficient time resolution to measure the rate of receptor channel opening, which occurs in the microsecond (µs) time domain. Using a laser-pulse photolysis technique, together with “caged glutamate”, combined with whole-cell recording, I first investigated the kinetic mechanism of channel-opening of the GluK1 and separately GluK2 homomeric channels expressed in human embryonic kidney 293 (HEK-293) cells, and then investigated GluK1/K5 and GluK2/K5 heteromeric channels separately. The laser-pulse photolysis technique provides a microsecond time resolution sufficient to measure the rate of channel opening kinetics of a kainate receptor. In particular, I can determine the channel-opening rate constant, which reflects how fast a channel opens following glutamate binding to the receptor, and the channel-closing rate constant, which reflects how fast an open channel transitions back to the closed state.  Characterization of these rate constants is critical to understand synaptic neurotransmission involving kainate receptors.  In Chapter 2, I show that GluK1 homomeric channels open and close ~2-fold faster than GluK2. The rates of channel opening for each were reduced by ~2.7-fold and ~1.8-fold, respectively, when coassembled with GluK5. In contrast, GluK5 coassembly has equalized the rates of channel closing for GluK1 and GluK2, decreasing ~1.7-fold and increasing ~1.3-fold to converge on a channel closing rate of ~600 s-1, respectively. Given that GluK5 also speeds the rate of channel desensitization for both channel types by ~2.5-fold, and confers equal EC50 values, my results show that GluK5 modulates GluK1-containing and GluK2-containing heteromers similarly, indicating that its effects may not be subunit-dependent.

Kainate receptors have two auxiliary proteins, i.e., NETO1 and NETO2. When coassembled with kainate receptors, NETO proteins are previously known to enhance the macroscopic current amplitude and affect channel properties such as channel desensitization rate. However, whether a NETO protein affects the rate of kainate receptor channel opening in response to glutamate binding is not known. In Chapter 3, I separately investigated the kinetic mechanism of channel opening of GluK2 homomeric channels coexpressed with NETO1 and separately with NETO2 in HEK-293 cells. The aim was to study how fast a NETO-containing channel opens as compared to the channel without NETO, and whether different NETO proteins affect the same kainate receptor the same way. My study shows that, as compared with GluK2 homomeric channels alone, NETO1 slows the channel-opening and channel closing rate by ~2-fold, whereas NETO2 slows these rates by ~7-fold and ~3-fold, respectively. Given that NETO2 also slows the rate of channel desensitization and reduces EC50 value more significantly than NETO1, I conclude that NETO2 seems to be the more impactful auxiliary subunit on GluK2 homomeric channels.

The GluK2/K5 receptor is the most prominently expressed native kainate receptor in the CNS and is thought to complex with either one of the kainate receptor auxiliary proteins, i.e., either NETO1 or NETO2. How these NETO proteins affect the rate of a native kainate receptor channel opening is not known either. In Chapter 4, I built up from the work in both Chapters 2 and 3 and further investigated the channel-opening mechanism of GluK2/K5 heteromeric channels coexpressed with NETO1 and separately with NETO2. I found that neither NETO1 nor NETO2 affects the rate of channel opening, as compared to GluK2/K5 alone. However, the rate of channel closing is slowed by ~1.7-fold and ~2.8-fold, respectively. I found that NETO2 slows the rate of channel desensitization and impacts the EC50 value more significantly than NETO1. Both NETOs shifted the dose-response relationship to the right, however. My results support that NETO proteins modulate kainate receptor subtypes differently, thus providing insight into their putative native function. These results suggest an “inhibitory” role for NETO when a NETO complexes with the heteromeric GluK2/K5 receptor in the CNS. My work overall suggests that kainate receptors of various combinations, including heteromeric combinations with and without auxiliary subunits, show in general different kinetic rate constants for channel-opening and channel-closing processes. These rate constants thus provide new knowledge about the potential native activities of these receptor forms.

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Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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