Ca²⁺/CaM/CaMK? Modulates Na_V1.1

Objective:Voltage-gated sodium channels?VGSCs?are basic signaling molecules in excitable cells and are molecular targets for local anesthetic agents and anti-epileptic agents.Na _V1.1 is widely expressed in cell bodies and axon initial segments of neurons in the brains.A sequence within C-terminal of Na _V1.1 contains a classical calmodulin?CaM?-binding IQ?isoleucine and glutamine?domain,which has been involved inCa²⁺signal transduction and alter the activity in response to changes in free Ca²⁺concentration([Ca²⁺]).It has been demonstrated that Ca²⁺plays a crucial role in the physiology of mammalians and is involved in the regulation of many intracellular processes ranging from gene transcription to neurotransmitter release.CaM is an ubiquitous regulatory protein in mammals.The CaM molecule is composed of two homologous lobes?N-and C-lobe?,in which each lobe contains two Ca²⁺-binding EF-hands.The N-and C-lobe,interconnected by a-helix linker,are quite similar in structure,but the Ca²⁺-affinity of C-lobe is 3-10 times higher than that of N-lobe.It has been found that Ca²⁺/CaM modulates VGSCs activity,but the molecular mechanism how Ca²⁺/CaM binds to Na _V1.1 is unclear.CaM also acts as an activator of Ca²⁺/CaM-dependent kinase ??CaMK??.CaMK? is a multifunctional serine and threonine protein kinase activated by elevated intracellular Ca²⁺.Activated CaMK? and the subsequent maintenance of constitutive activity through autophosphorylation at threonine residue 286?Thr286?are thought to play a major role in synaptic plasticity.Phosphorylation of VGSCs by CaMK? dynamically regulates the expression,function and localization of these ion channels.A total of 70Ser/Thr phosphorylation sites in Na _V1.2 and 28 Ser phosphorylation sites in Na _V1.1have been identified in the previous studies,mostly on C-terminal of?subunit.However,the effect of CaMK? on the CaM binding to Na _V1.1 IQ domain has not been elucidated yet.Na _V1.1,revealing the regulatory mechanisms of Ca²⁺/CaM/CaMK? axis on VGSCs.Our study revealed a possible pathogenesis for ion channel disease,such as epilepsy,pain,provided the theoretical and experimental basis for potential cure target.Methods:1.cDNA construction and site-directed mutagenesisThe cDNA corresponding to the IQ domain of Na _V1.1?IQ,amino acids 1909-1936?was generated by PCR using the cDNA of human Na _V1.1 as the template.The human CaM and its truncated proteins,N-lobe?a.a.2–80?and C-lobe?a.a.76–148?were subcloned from HEK293 cells.The CaMK? mutant T286D?CaMK?T286D?was created with rat CaMK??cDNA as a template.All the mutants were constructed by site-directed mutagenesis using a Quickchange?kit?QIAGEN?.The above DNAs were individually ligated into pGEX-6P-1 H320 expression vectors.2.Expression and purification of recombinant GST fusion peptidesThe vectors were transformed into Escherichia coli BL21?DE3?to express the target peptides as glutathione-S-transferase?GST?fusion proteins.The bacteria were cultured in LB liquid medium at 37? overnight.Then the bacteria were induced with 1 mM isopropyl-1-thio-?-D-galactopyranoside?IPTG?and continued incubating for 4 hours at 37? before harvesting.The ultrasonic technique was used to harvest GST-fusion peptides.Then the fusion peptides were purified by using Glutathione Sepharose 4B beads?GS-4B,GE Healthcare?.The GST regions of CaM and its mutants were cleaved with PreScission Protease?GE Healthcare?.GST-IQ,CaM and its mutants were quantified by Enhanced BCA Protein Assay Kit.The proteins were preserved at-20?.3.GST pull-down assayGST-fusion IQ or its mutant?2–4?g?was immobilized on GS-4B and incubated in 300?L of Tris buffer with CaM or its mutants for 4 h at 4? under agitation in the presence of different Ca²⁺concentrations.Bound CaM?or its mutant?and IQ?or EQ?were resuspended in 5×SDS-PAGE loading buffer and resolved in 15%SDS–PAGE gels.Proteins were stained by Coomassie brilliant blue R?CBB?.Photoshop software and Image J were used to digitize protein bands.The optical density values were converted to protein contents using respective correction factors.The GST-fusion IQ?for control?immobilized to GS-4B beads?40?L?was phosphorylated in an assay reaction?0.4?M CaMK?T286D?in Tris buffer containing1 mM MgCl₂,and 1 mM Na₂ATP for 30 min at 30?.The dephosphorylation was achieved by adding 5 unit/ml calf intestinal alkaline phosphatase?CIP;New England Biolabs,Ipswich,MA,USA?into the reacting mixtures incubating at 37? for 30 min.4.Computational dockingA homology model of Na _V1.1 IQ was constructed based upon the solved crystal structure of IQ motif of Na _V1.2 using the Create Homology Model tool in Discovery Studio 2017?Biovia?.The N-and C-lobe of CaM were derived from the crystal structure of Ca²⁺/CaM-Ca _V1.2 IQ domain complex.Docking studies between Na _V1.1IQ and CaM N-or C-lobe were performed in Discovery Studio using the Dock Proteins protocols.The ZDOCK protocol was used for docking the IQ motif of Na _V1.1to N-or/and C-lobe of CaM,and subsequently,the RDOCK protocol was applied for the further refinement of 10 best docked poses.5.Statistical analysisCurve-fitting of the total bound ligand?CaM and its mutants?was performed with the software SigmaPlot 12.0.Bound ligand?Y?was fitted with the following Hill's equation,for the one-site fitting model Y=B_max·X/?K_d+X?,for the two-sites model,a sum of two Hill's equations was integrated to assume independent binding,Y=B_max1·X/(K_d1+X)+B_max2·X/(K_d2+X),where B_max1,B_max2,K_d1,and K_d2 represents total B_max and K_d,respectively and X is the concentration of free ligand,and K_d is the apparent dissociation constant,B_max is the maximum binding for each binding site.The data are presented as mean±S.E?n=4?.One-way ANOVA was applied.The SPSS 22.0software was used to evaluate the statistical significance.P<0.05 by One-way ANOVA test was considered statistically significant,P<0.01 by One-way ANOVA test was considered statistically greater significant.Results:1.In this study,CaM and Na _V1.1 IQ plasmids were successfully constructed and thebinding of CaM and Na _V1.1 IQ was detected.2.CaM and Na _V1.1 IQ mutants and CaM truncated proteins were successfullysynthesized and purified in this experiment.3.In the presence of Ca²⁺,the binding of IQ to Ca²⁺/CaM increased with the increasingconcentration of Ca²⁺.4.In the absence of Ca²⁺,the binding of IQ to apoCaM was much higher than that ofCa²⁺/CaM.5.The binding of CaM and Na _V1.1 IQ mutant,IE or EQ,diminished.6.As for the binding of CaM mutant and Na _V1.1 IQ,in the absence of Ca²⁺,B_max ofCaM₁₂ binding to Na _V1.1 IQ is greater than that in the presence of Ca²⁺.B_max ofCaM₃₄ binding to Na _V1.1 IQ reached maximum amount under 500nM Ca²⁺.In themeanwhile,the binding of CaM₁₂₃₄ to Na _V1.1 IQ are similar under different Ca⁽2+concentrations.7.The parameters of C-lobe binding to Na _V1.1 IQ were higher than that of N-lobe inthe absence of Ca²⁺,whereas the parameters of N-lobe binding to Na _V1.1 IQ werehigher than that of C-lobe in the presence of Ca²⁺.8.The ZDock score and E_RDock for the optimal N-lobe orientation docking intoNa _V1.1 IQ domain are 11.28 and-23.3091kcal/mol,respectively,whereas theseparameters for the best interaction of C-lobe with IQ domain are 10.66 and-26.2142kcal/mol,separately.When the N-and C-lobe were together docked into the Na _V1.1IQ domain,the best ZDock score and lowest E_RDock are 12.78 and-30.7219kcal/mol,respectively.9.CaMK? phosphorylation increased the binding of CaM to Na _V1.1 IQ.10.The binding of Ca²⁺/CaM to the Na _V1.1 IQ domain remains the same amount aftertreatment with CaMK? due to the mutation of potential phosphorylation sites.Conclusion:1.The binding of Ca²⁺/CaM to IQ was in a concentration-and Ca²⁺-dependent manner in the presence of Ca²⁺.2.ApoCaM has the highest affinity with IQ domain and Na _V1.1 IQ binds preferably to ApoCaM.3.C-lobe is the predominant domain in apoCaM interacting with Na _V1.1 IQ domain and N-lobe is the predominant domain in Ca²⁺/CaM interacting with Na _V1.1 IQ domain.4.The wild-type CaM?wt CaM?is the most stable condition binding to Na _V1.1 IQ compared to that of C-lobe and N-lobe.5.CaMK? facilitating the binding of Ca²⁺/CaM to Na _V1.1 IQ domain may due to one or several phosphorylation sites in T1909,S1918 and T1934 of Na _V1.1 IQ domain.This study explored the modulation effect of CaMK?/CaM on Na _V1.1,revealing the regulatory mechanisms of Ca²⁺/CaM/CaMK? axis on VGSCs.Our study elucidated a possible pathogenesis for ion channel disease,such as epilepsy,pain,provided the theoretical and experimental basis for potential cure target.