Electrophysiological Investigation of Ionotropic and Nonionotropic Signaling in NMDA Receptors with Chimeric GluN2 Subunits

This project focuses on understanding the functional differences between native NMDA receptors containing GluN2A and GluN2B subunits, and chimeric receptors with swapped subunit domains. Specifically, the chimeras ABc (GluN2A backbone + GluN2B carboxyl terminus) and BAc (GluN2B backbone + GluN2A carboxyl terminus) are being studied to explore how these structural modifications influence receptor activity and signaling. It is hypothesized that the ABc chimera will exhibit faster activation and higher calcium permeability than native GluN2A, while also demonstrating slower desensitization and a more negative reversal potential compared to native GluN2B. Meanwhile, the BAc chimera is expected to display slower activation, longer-lasting currents, faster desensitization, and a less negative reversal potential than native GluN2B. Using two-electrode voltage clamping in Xenopus laevis oocytes, this project aims to determine how these chimeric receptors alter ionotropic and non-ionotropic signaling pathways, with a particular focus on their roles in synaptic plasticity. Insights gained from this study could advance our understanding of NMDA receptor dynamics and inform therapeutic strategies for neurological disorders associated with receptor dysfunction.

Hello all. I am Diborah Gutema and this is my video presentation for my project, Electrophysiological Investigation of Ionotropic and Nonionotropic Signaling in NMDA Receptors with Chimeric GluN2 Subunits.

I will be presenting my research on the functional differences between NMDA receptors containing chimeric GluN2 subunits and those with native GluN2A or GluN2B subunits. This project is part of a larger effort to understand how these subunits contribute to synaptic plasticity, memory formation, and cognitive processing in the hippocampus.

NMDA receptors, which are excitatory ionotropic receptors in the hippocampus, are central to information processing and memory formation. These receptors allow calcium ions to enter the cell upon activation, triggering signaling events that modify synaptic strength and network activity. The NMDA receptor is composed of four subunits: two GluN1 subunits and, typically, two GluN2 subunits. The GluN2 subunits can be GluN2A, GluN2B, GluN2C, or GluN2D, and the type of GluN2 subunit incorporated largely determines the receptor’s properties, including its calcium conductance and its ability to engage in non-ionotropic signaling. During development, NMDA receptors shift from containing predominantly GluN2B to those containing GluN2A, a transition that is essential for the maturation of spatial learning and memory. Understanding the functional differences between these receptor types, particularly their contributions to ionotropic and non-ionotropic signaling, is a key focus of this project. To investigate these differences, we are using transgenic mice that express chimeric GluN2 subunits, developed by Dr. Dumas’s lab, which allows us to separate these two forms of signaling.

The goal of this project is to compare the functional properties of NMDA receptors containing chimeric GluN2 subunits with those containing native GluN2A or GluN2B subunits. While the chimeric receptors express well in mammalian neurons, they do not yet express in Xenopus laevis oocytes, which are an ideal model system for electrophysiological recording. To address this, we are transferring the chimeric receptor constructs into oocyte-compatible vectors. This will allow us to express the receptors in Xenopus oocytes and assess their functional differences in isolation.

First, upon receiving the plasmid pGEMHE-membrane-EGFP, we’ll transfer a sample from the backstab into a 3 mL bacterial culture, which will be incubated overnight for 16 to 24 hours. The next step involves isolating and purifying the plasmid DNA from the bacterial culture using a mini prep protocol. To ensure the integrity and purity of the plasmid, we’ll assess its concentration using agarose gel electrophoresis and spectrophotometry, checking the 260/280 ratio to confirm DNA quality.

Once the plasmid is prepared, the next step will be linearizing it using the NaeI restriction enzyme. This will make the plasmid ready for RNA synthesis. We’ll also perform diagnostic digests to confirm the plasmid’s identity by excising GFP using restriction enzymes. After that, we’ll select appropriate restriction sites for subcloning and prepare the plasmid for ligation with the NMDA receptor inserts.

Next, we’ll grow bacterial cultures of the NMDA receptor inserts, which include GluN2A, GluN2B, and the ABc and BAc chimeras. These inserts will be PCR amplified and then digested with the same restriction enzymes used on the plasmid. Afterward, the inserts will be ligated into the pGEMHE vector, either through in-house ligation or commercial subcloning, to create the final constructs.

The purified plasmid DNA will then be used for RNA synthesis, and this RNA will be injected into Xenopus laevis oocytes for electrophysiological recording.

For the final step, we will use the two-electrode voltage clamp (TEVC) setup to record the baseline and experimental responses of the oocytes to glutamate and glycine. This will help us observe the activity of both wild-type and chimeric NMDA receptors.

In summary, this project combines molecular biology techniques with electrophysiological analysis to investigate the functional differences between chimeric and native NMDA receptors. While we’ve focused on preparing the constructs for RNA synthesis and oocyte injection, the next phase will involve detailed electrophysiological recording to analyze the ionotropic and non-ionotropic signaling properties of these receptors. The insights gained from this research could contribute to a deeper understanding of how NMDA receptors function in hippocampal circuits and their role in synaptic plasticity, learning, and memory.

I’d like to take a moment to thank those who have been instrumental in this project.
Dr. Herin who has been an invaluable mentor in electrophysiology and molecular biology.
Dr. Dumas who has provided expert guidance on receptor signaling and chimeric constructs.
Hannah Zikria-Hagemeier who was essential in training me on plasmid preparation.
Finally, I’d like to thank the rest of the PBNJ Lab for their collective support through guidance and resources, which has been key to my growth as a researcher.
Thank you all for your help and support!