Pharmacological Characterization of Chimeric NMDA Receptors in Xenopus

NMDA receptors (NMDARs) play a critical role in synaptic plasticity and cognitive function by mediating both ionotropic and non-ionotropic signaling. During postnatal development, NMDA receptors undergo a subunit shift from GluN2B to GluN2A, a transition associated with synaptic maturation and the emergence of mature cognitive function. While previous research has explored the physiological and behavioral consequences of this transition, the specific mechanisms driving these changes remain unclear. In particular, the relative contributions of ionotropic and non-ionotropic signaling have not been fully defined. Ionotropic signaling involves calcium influx through the receptor channel upon ligand binding, while non-ionotropic signaling refers to intracellular signaling cascades initiated by conformational changes in the receptor that occur independently of ion flow. This study aims to dissect these signaling pathways by utilizing chimeric GluN2 subunits engineered to separate ionotropic and non-ionotropic functions. Chimeric constructs are currently being subcloned into Xenopus laevis–compatible vectors for functional expression in oocytes. This work has involved preparation of the pGEMHE-membrane-EGFP backbone through bacterial culture and plasmid isolation via alkaline lysis mini prep, followed by gel electrophoresis and spectrophotometric analysis to assess plasmid integrity and purity. Restriction digests were performed to linearize the plasmid and confirm backbone identity. In parallel, GluN2A, GluN2B, and chimeric constructs have been cultured from glycerol stocks and are currently undergoing PCR amplification with construct-specific primers. Following amplification, the inserts will be digested, purified, and ligated into the vector. Preliminary results confirm successful plasmid preparation, and insert amplification and ligation are ongoing. Once subcloning is complete, the constructs will be injected into Xenopus laevis oocytes, followed by two-electrode voltage clamp (TEVC) recordings to measure receptor responses to varying concentrations of glutamate and glycine, as well as pharmacological modulators of NMDA receptors. Statistical analyses using two-way ANOVA will compare current amplitudes across receptor type (GluN2A, GluN2B, ABc, BAc) and treatment condition (agonist/modulator concentration), with the goal of identifying distinct electrophysiological profiles associated with each subunit composition. This study will characterize how differences in GluN2 subunit composition and intracellular domain identity affect NMDA receptor-mediated signaling in an isolated system.

Hello all. I am Diborah Gutema and this is my video presentation for my project, Pharmacological Characterization of Chimeric NMDA Receptors in Xenopus laevis Oocytes.

NMDA receptors are ion channels located on neurons that allow calcium ions to enter the cell when activated by the neurotransmitter glutamate. This calcium signaling, known as ionotropic signaling, is critical for synaptic plasticity, learning, and memory. NMDA receptors can also engage in non-ionotropic signaling, where conformational changes in the receptor trigger internal signaling pathways without ion movement. Each receptor is composed of two GluN1 subunits and two GluN2 subunits. A developmental shift occurs where GluN2B subunits are gradually replaced by GluN2A, a transition essential for synapse maturation.
Understanding how these subunits contribute to ion flow and conformational signaling is the focus of our project.

To investigate how different regions of NMDA receptor subunits contribute to signaling, we are working with chimeric GluN2 constructs developed by Dr. Dumas’s lab. These chimeras are engineered by swapping specific intracellular domains between the GluN2A and GluN2B subunits. In doing so, we can separate the functional contributions of individual regions, such as the intracellular tail, to ion flow and to non-ionotropic signaling. By studying receptors with these controlled domain swaps, we aim to determine which portions of the subunit structure are responsible for differences in calcium permeability, activation properties, and downstream signaling. This semester, we focused on preparing the DNA constructs necessary for expressing these receptors in Xenopus laevis oocytes for future functional testing.

The overall goal of this project is to express wild-type and chimeric NMDA receptors in Xenopus laevis oocytes and compare their ionotropic signaling properties using two-electrode voltage clamp recordings. By analyzing how domain swaps between GluN2A and GluN2B affect receptor function, we aim to better understand the molecular basis of NMDA receptor signaling. This semester, we focused on preparing high-quality plasmid DNA, optimizing restriction digests, and initiating PCR amplification of the GluN2 receptor inserts to prepare for future subcloning and expression studies.

First, upon receiving the plasmid pGEMHE-membrane-mEGFP, we transferred a sample from the backstab into a 3 mL bacterial culture, which was incubated overnight at 37 degrees Celsius for 16 to 24 hours. The plasmid includes a Xenopus laevis promoter sequence, which enables later expression in oocytes. Following incubation, we isolated and purified the plasmid DNA from the bacterial culture using a alkaline lysis mini prep protocol. To ensure the integrity and purity of the plasmid, we assessed DNA quality using agarose gel electrophoresis to check for intact plasmid structure and spectrophotometry to measure the 260/280 absorbance ratio.

Next, we performed restriction digests to prepare the plasmid for future subcloning. We used the enzyme NheI to linearize the plasmid and carried out diagnostic digests to prepare for the later excision of the GFP segment originally present in the vector.

In parallel, we grew bacterial cultures containing the DNA for GluN2A, GluN2B, ABc, and BAc constructs. Using these templates, we initiated PCR amplification with construct-specific primers to selectively amplify the inserts. PCR amplification is currently ongoing. Once complete, we will purify the amplified products and verify insert size by gel electrophoresis before moving on to the next phase of subcloning.

After the inserts are fully amplified and purified, we will digest them with restriction enzymes to create compatible ends with the plasmid vector. We will then use a DNA ligase enzyme to join the inserts and vector together, creating new plasmids that carry either the wild-type or chimeric NMDA receptor sequences. Some ligation reactions will be performed in-house, while others may be sent for commercial cloning depending on efficiency. Sequence verification will follow to confirm successful ligation.

Following sequence confirmation, we will synthesize capped RNA transcripts from the recombinant plasmids using in vitro transcription. These RNA molecules will then be injected into individual Xenopus laevis oocytes, allowing the cells to produce functional NMDA receptors for electrophysiological testing.

Two to three days after RNA injection, we will perform two-electrode voltage clamp recordings, a technique that holds the membrane potential constant while measuring ionic currents. By applying glutamate and glycine, we will evaluate receptor function based on current amplitudes, activation and deactivation kinetics, and dose-response characteristics. Comparing wild-type and chimeric receptors will help us determine how specific subunit regions influence NMDA receptor ionotropic signaling.

This semester, we focused on growing bacterial cultures, isolating and purifying plasmid DNA, troubleshooting purification and digestion protocols, and beginning PCR amplification of the NMDA receptor inserts. These steps are critical for setting up RNA synthesis, oocyte injection, and functional testing. Moving forward, we aim to complete subcloning, synthesize RNA, and characterize receptor function using TEVC recordings.

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!