The Critical Role Of CaM Acetylation In Ca²⁺ Signaling And Synaptic Plasticity

Synaptic plasticity is crucial for several brain functions such as perception,learning and execution.Most if not all forms of synaptic plasticity are initiated by an increase of intracellular Ca²⁺,which triggers downstream signaling pathways including gene transcription,local protein synthesis,as well as acute machineries such as receptor trafficking and protein phosphorylation.Protein phosphorylation is the most studied PTM and occupies acentral role in regulating signal transduction pathways,metabolism and other cellular processes.By contrast,acetylation was relatively ignored for the 30years following its discovery.A remarkable series of congruent observations in the past20 years has brought protein acetylation and the enzymes that control it back to the forefront of cellular regulatory mechanisms.Lysine acetylation of proteins is catalysed by lysine acetyltransferases(KATs),which transfer the acetyl group of acetyl-Co A to the?-amino group of an internal lysine residue.A lot of studies have shown that KATs acetylates multiple lysines of histones,neutralizing the positive charge of histone lysine.Thus,histone acetylation results in chromatin decondensation,thereby allowing access to transcription factors and other transcription co?activators.However,the acetylation of synaptic proteins in LTP is underestimated.We investigate the acetylation and acetylation transferase of synaptic proteins in synaptic plasticity and learning by using mass spectrometry,biochemical experiments,electrophysiological methods,behavioral tests,etc.Results are as follows:First,In analyzing the acetylome of mouse forebrain,we unexpectedly found acetylated synaptic proteins.In particular,Calmodulin(CaM),a calcium sensor critical to synaptic plasticity,is acetylated on three lysine residues that are conserved across species.By using site-specific antibodies for each of the three acetylated lysines,we demonstrate that CaM acetylation is increased within minutes of LTP induction in a manner dependent on NMDA receptor activation.Second,to test the role of acetylated CaM in synaptic plasticity,we breed 3KR-Cam1 kocnk in mice.The field potential recording of the 3KR-Cam1 mice showed the decrease of LTP.And,intracellular injection of 3KQ-CaM(mimic CaM)in WT mice has the same result.These results show that acetylation of CAM is important in LTP.Thrid,GST-pull down and in vitro acetylation experiments confirmed that SRC3interacts with and acetylates CaM in a calcium-dependent manner.Next,SRC3mutation and knockdown reduced LTP and impaired contextual fear conditioning.By using a specific inhibitor of SRC3,we showed that SRC3 is necessary for LTP induction.SRC3 KAT domain can resuce the decrease of Src3^+/-mouse LTP which confirmed that the importance of SRC3 KAT activity in LTP.Intracellular injection of 3KQ-CaM(mimic CaM)can resuce impaired LTP by SI-2.This illustrates the important role of the acetylation of CAM by SRC3 in synaptic plasticity.Last,we investigate the performance of their learning tasks in order to detect whether the Src3^+/-and 3KR-Cam1/+mice are impaired.In contextal fear learning tasks,both Src3^+/-and 3KR-Cam1/+are impaired.These illustrate the important role of the acetylation of CaM and SRC3 in learning.Lateral ventricle injected with the SRC3inhibitor SI-2 impairs fear learning of WT mice.However,LV-3KQ-Cam can resuce the impaired fear learning by SI2.These results demonstrate the important role of the acetylation of CAM by SRC3 in fear learning.Acetylated CaM promotes the CaM-CaMKII?interaction and is more potent to activate CaMKII?,a protein kinase critical for synaptic plasticity than wild type CaM.Mutation of the acetylated lysine residues in CaM impairs CaMKII?activation and synaptic plasticity.Our study reveals that the acetylation of CaM by SRC3 play an important role in synaptic plasticity and learning in first time.Our result provides new insight for the regulation of synaptic plasticity and learning by acetylation of proteins and extranuclear KAT.