Abstract
Calcium-calmodulin-dependent protein kinase (CaMKII) is a molecule involved in several cell processes including plasticity related to learning and memory. Activation of NMDA-type glutamate receptors results in translocation of CaMKII to synapses. However, there are at least two distinct mechanisms by which glutamate-dependent CaMKII translocation occurs: one well-studied process resulting from whole-cell glutamate stimulation and one resulting from brief, local glutamate application. Unlike the relatively fast CaMKII translocation seen following whole-cell glutamate delivery (seconds), local application results in CaMKII translocation that occurs gradually within 6–10 min. This locally-induced translocation of CaMKII requires L-type Ca2+ channel co-activation but does not rely on GluN2B receptor subunit expression, unlike translocation following whole-cell application of glutamate. The current study examined if nucleotide binding is necessary for locally-induced CaMKII translocation, similar to CaMKII translocation resulting from whole-cell glutamate application. Three different mechanisms of inhibition were employed: staurosporine (ATP inhibitor), CaMKII(281–302) peptide inhibitor and expression of the K42M mutation. Locally-induced CaMKII translocation was moderately suppressed in the presence of either the broad-spectrum kinase inhibitor staurosporine (100 nm) or the CaMKII(281–302) peptide inhibitor. However, expression of the catalytically dead K42M mutation that prevents ATP-binding to CaMKII, significantly inhibited locally-induced translocation. Thus, CaMKII translocation following brief, local glutamate application requires nucleotide binding, providing support for future research into the molecular mechanisms of this distinct form of CaMKII translocation.
Highlights
Calcium-calmodulin-dependent protein kinase (CaMKII) is involved in several cellular processes and is abundantly expressed in neurons (Erondu and Kennedy, 1985)
In dendritic regions where pre-stimulation GFP-CaMKII expression was diffuse, local glutamate plus glycine stimulation resulted in the gradual appearance of GFP-CaMKII expressing puncta while previously bright puncta frequently showed an increase in fluorescence intensity, occasional decreases in fluorescence at these early-observable puncta were seen (Figure 1C)
These results demonstrate that both CaMKII activation and nucleotidebinding are necessary for locally-induced CaMKII translocation
Summary
Calcium-calmodulin-dependent protein kinase (CaMKII) is involved in several cellular processes and is abundantly expressed in neurons (Erondu and Kennedy, 1985) It is well-established that whole-neuron activation with glutamate or NMDA results in rapid translocation of CaMKII to synapses (within seconds) resulting from calcium influx through activated NMDA receptor channels (Shen and Meyer, 1999; Shen et al, 2000; Thalhammer et al, 2006). Similar to whole-cell activation protocols, CaMKII translocation resulting from a single local puff of glutamate requires Ca2+/CaM activation and calcium influx via NMDA receptor channels (Rose et al, 2009). Unlike CaMKII translocation following whole-cell stimulation, locallyinduced CaMKII translocation requires L-type calcium channel activation but not CaMKII-GluN2B binding (Rose et al, 2009; She et al, 2012) indicating that the molecular mechanisms of locally-induced synaptic CaMKII translocation differ somewhat from those involved in CaMKII translocation resulting from whole-cell activation
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