Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K + or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within~30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core,~100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

CaMKII Phosphorylation Regulates Synaptic Enrichment of Shank3

“…Dynamic and differential Shank3 remodeling in the PSD by neuronal activity is highly dependent on the actin cytoskeleton ( Kuriu et al, 2006 ; Tao-Cheng et al, 2016 ). Since neuronal activity-dependent translocation of CaMKII to the PSD is also well characterized ( Shen and Meyer, 1999 ; Tao-Cheng, 2020 ), Shank3 S782 may be a CaMKII regulated phosphorylation site that balances the Shank3 association with the actin signaling molecules that are regulated by Shank3 S685 phosphorylation. Additional experiments are required to understand more about the possible mechanistic link between these proteins.…”

Interactions with PDZ proteins diversify voltage‐gated calcium channel signaling

“…It warns the individual to escape the pain-inducing stimulus and protects the injured tissue site during healing. Multiple lines of evidence suggest that Ca V 3 channels are involved in pain processing (Weiss & Zamponi, 2019, 2020. They help to transmit the nociceptive signal by regulating afferent fiber excitability and controlling the Ca 2+ influx in synaptic nerve terminals in the spinal dorsal horn.…”

“…They both contain a single PDZ domain that binds to a distal C-terminal sequence in the α 1 subunit of Ca V 1.3. Shank is a scaffold protein located in the PSD pallium (100-120 nm from the postsynaptic membrane; Dosemeci, Weinberg, Reese, & Tao-Cheng, 2016;Tao-Cheng, 2020) and the overexpression of shank in neurons can induce the formation of functional dendritic spines in nonspiny cerebellar neurons (Roussignol et al, 2005). Conversely, neurons from mice lacking shank have smaller dendritic spines.…”

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

“…Immunogold labeling of endogenous proteins with specific antibodies at the EM level allows localization of these proteins in intact cells [ 3 , 4 ]. Information on the distribution and quantification of these proteins under different stimulation conditions can offer important clues to their functions [ 5 , 6 ].…”