Local Ca2+ Nanodomains Initiate Ca2+/Calmodulin-Dependent Inactivation of NMDA Receptors

Abstract

NMDA receptors (NRs) are glutamate- and glycine- gated non-selective excitatory channels that are expressed throughout the central nervous system. They have large (50 - 70 pS) unitary conductance and a large fraction of the current (10 - 20 %) is carried by Ca2+. Specifically the NR-mediated Ca2+ influx drives critical physiological processes including synaptic plasticity and apoptosis and also reduces NR gating, a process termed Ca2+-dependent inactivation (CDI). CDI requires Ca2+ binding to calmodulin (CaM) and is typically measured as increased macroscopic current desensitization. To investigate mechanisms underlying CDI we combined mathematical models of Ca2+ diffusion with one-channel and macroscopic current recordings from HEK293 transfected with GluN1-2a and GluN2A NR subunits. First, to investigate whether CDI is produced by local rises in intracellular Ca2+, we calibrated the extent of CDI observed extracellular Ca2+ flux through the pore under conditions of high intracellular buffering (BAPTA) to the extent of CDI observed with controlled dialysis of intracellular [Ca2+]. Using a model for intracellular Ca2+ diffusion based on Neher-Stern theory and the experimentally measured CDI produced by NR-mediated Ca2+ influx, we estimated the Ca2+-sensor responsible for CDI (i.e. CaM) resides within 5 nm distance of the Ca2+ source. This is consistent with previous biochemical evidence supporting the hypothesis that CaM preassociates with the C-terminal domain of GluN1 on the C0 cassette. In addition, single-channel current recordings combined with kinetic modeling suggest that Ca2+/CaM decreases NR activity by increasing the occupancy of desensitized states. Together, these results suggests CDI is initiated by an influx of Ca2+ which activates a local population of CaM to potentiate desensitization of NRs.