Assaying the energetics of NMDA receptor pore opening

The NMDA receptor converts transient glutamate release into an electrical and biochemical signal that integrates neuronal activity and results in changes in synaptic connectivity and strength. A variety of approaches are used to address the linkage between neurotransmitter binding and ion channel opening, a key feature common to all ligand-gated ion channels. φ-value or REFER analysis, an approach derived from chemical transition state theory, can characterize the energetics and dynamics of ion channel activation. Here, we describe an approach to apply φ-value analysis to the unique features of NMDA receptor activity. The analysis is based on high-resolution single-channel recordings of single-channel patches and kinetic analysis. We further detail analytical approaches to performing burst analysis that is essential to accurately outline the multicomponent activation pathway leading to pore opening. Overall, this approach allows to quantitatively chart the allosteric cascade, at the level of single amino acids, as it propagates from the neurotransmitter-binding domain to the ion channel in NMDA receptors.