Mechanistic insights to the Ca2+-dependent inactivation of NMDA receptors by Calmodulin
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Calcium has evolved to be a ubiquitous second messenger that regulates many diverse and often opposing physiological processes in eukaryotic cells. In the central nervous system, NMDA receptors are glutamate and glycine-gated ion channels that mediate large Ca2+ influx during excitatory synaptic transmission. The Ca2+ load through NMDA receptors is responsible for both synaptic plasticity, learning and memory as well as excitotoxicity during aberrant synaptic transmission. To curb excessive Ca2+ influx and fine tune the synaptic current, NMDA receptors employ calmodulin as an endogenous sensor of intracellular Ca2+ that also directly inhibits channel function. In this dissertation, I outline my empirical results that garner new insights into the mechanism of calmodulin-dependent regulation of NMDA receptors. Combining precise whole-cell measurements of calmodulin-dependent inactivation of receptor currents with mathematical modeling of Ca2+ signaling and calmodulin/channel interactions, I found that under specific conditions, the degree of inactivation can be explained by a model where calmodulin primes resting channels for inactivation in the absence of Ca2+. Given the close spatiotemporal coupling of Ca2+ sensing by calmodulin to channel activity, I investigated whether the Ca2+ influx through individual channels was sufficient to engage calmodulin-dependent inactivation of neighboring channels. Using cellattached patch clamp, I determined whether NMDA receptors in multi-channel patches gated cooperatively in a Ca2+/calmodulin-dependent manner. I found that the negative cooperativity exhibited by channels could be augmented by scaffold protein, PSD-95. Lastly, I examined the apparent insensitivity of GluN2B-containing channels to calmodulin. I found that GluN2B channels rapidly inactivate when exposed to tonic Ca2+ dialysis into the cell. To reconcile this discrepancy with the literature, I used patch clamp fluorometry to measure the kinetic mechanism of inactivation while manipulating intracellular Ca2+ levels. I found that both GluN2A and GluN2B channels inactivate by a common mechanism by which perturbations in their activation pathways lead to increased occupancies of desensitized states. The differential subtype kinetics of the active channel explains the subunit-specific manifestation of calmodulin-dependent inactivation. Overall, these findings highlight the nuances of Ca2+ regulation of NMDA receptors and provide new insight into their function in both Ca2+-dependent physiological processes and pathologies.