Human NMDA receptors: Functional insights into the two isoforms of the human GluN2A subunit.

Abstract

Glutamate is a major excitatory neurotransmitter in the central nervous system and plays an essential role in cognition and memory formation. This function is enacted through binding with glutamate receptors found at glutamatergic excitatory synapses. One such receptor is the N-methyl-D-aspartate receptor (NMDAR). NMDARs form ion channels and play key roles in neuronal development, synaptic communication, and mechanisms underlying learning and memory. Their function depends on the coincidence of pre- and post-synaptic activity, which relieves the voltage-dependent Mg2+ blockade and permits Ca2+ influx. While the GluN2A subunit is well-characterised, recent evidence suggests the existence of an alternatively spliced isoform, GluN2A-Short (GluN2A-S), distinguished by a truncated C-terminal domain and a unique C-terminus. Expression of constructs containing cDNA for human GluN2A results in a mixed expression of GluN2A-Long (GluN2A-L) and GluN2A-S isoforms, limiting insight into their individual contributions to function. This thesis investigates whether the two human GluN2A isoforms can be studied independently to determine their specific functional properties within the NMDAR complex. Using site-directed mutagenesis, we disrupted the splice acceptor site responsible for generating both isoforms, enabling the selective expression of GluN2A-L. Applying this tool, we examined whether GluN2A-S confers distinct functional properties to the NMDAR under both physiological and disease-associated conditions. We studied both wild-type (WT) and mutant forms of human GluN2A-S with association with neurological disease in humans. Using electrophysiology and a bioluminescence-based Ca2+ assay, we found that WT GluN2A-S contributes enhanced voltage-dependent Mg2+ blockade compared to GluN2A-L. This finding pointing at an isoform-specific function of NMDARs containing either GluN2A-L or GluN2A-S in physiological conditions.We further assessed how GluN2A-S functions in disease-associated mutations such as the epilepsy-associated GRIN2A mutation: L812M. When investigating this gain-of-function mutation, we found that GluN2A-S mitigates the decrease in voltage-dependent Mg2+ blockade but does not alleviate the increase in glutamate potency. This suggests an isoform-specific gain-of-function effect of the L812M mutation.Furthermore, the epilepsy-associated G483R mutation exhibited an isoform-dependent effect, resulting in a loss-of-function in GluN2A-L but a gain-of-function in GluN2A-S. Specifically, while the G483R mutation caused a strong decrease in Ca2+ conductance through GluN2A-L-containing NMDARs, it led to a marked increase in conductance through GluN2A-S-containing receptors.These findings underscore the clinical relevance of each individual human GluN2A isoforms. Studying them separately may provide deeper insight into their distinct contributions to NMDAR function in both synaptic physiology and neurological disease. In the context of the epilepsy-associated mutations explored in this thesis, upregulation of the human GluN2A-S isoform and/or suppression of GluN2A-L expression could be a potential strategy for managing the dysfunction imposed by L812M. Similarly, increasing GluN2A-S expression in patients carrying the G483R mutation may help rescue the loss-of-function imposed by the mutation