The Effect And Mechanism Of CaM In Regulating Voltage-gated Sodium Channel Na_v1.2 Mutation R1902C In Autism

Objective:Voltage-gated sodium（Na _V）channels underlie the rapid upstroke of action potentials in excitable cells as well as are critical determinants of modulating intracellular ion concentrations and influencing signaling pathways.As yet,nine mammalian Na _V isoforms have been recognized to form the VGSCs superfamily.Among them,Na _V1.2 is vital for propagation of action potentials along unmyelinated axons discovered mainly in nerve cells and muscle.Calmodulin（CaM）is a fundamental protein for many eukaryotic signal transduction pathways as one of the Ca²⁺-binding proteins.CaM identifies two intracellular sites in the majority of VGSCsα-subunits in a Ca²⁺-dependent manner.CaM molecule contains two lobes,each of which（N-or C-lobe）includes two Ca²⁺-binding sites—EF-hands.CaM binds to many ion channels including VGSCs and voltage-gated calcium channels（VGCCs）through IQ（isoleucine and glutamine）domains.All nine VGSC isoforms contain the classical CaM-bound IQ domains.The sequence at the C-terminus of Na _V1.2 contains IQ domains,which is associated with Ca²⁺signal transduction and modifies the activity in reaction to adjustments in the intracellular free Ca²⁺concentration([Ca²⁺]).Autism is a psychological condition with approximated heritability of 90%and one-third of people with autism experience seizures.Autism susceptibility loci have been located near a group of VGSC gene clusters.Mutations in SCN2A lead to generalized epilepsy with febrile seizures plus（GEFS⁺）.Additionally,mutations in SCN2A are essential hazard elements for autism spectrum disorder and infantile seizures.It has been shown that compared with the wild-type Na _V1.2 IQ domain,the autism-related mutation in the Na _V1.2 IQ_R1902C motif affects the binding between CaM and Na _V1.2 IQ_R1902Cmotif.Previous studies have shown that the Na _V1.2 mutant channel（R1902C）shows a continuous increase in Na⁺current.However,the detailed mechanism of the binding of Ca²⁺/CaM to the Na _V1.2 IQ_R1902C motif under various Ca²⁺concentrations is still unclear.In addition,the CaM’s constituent proteins（N-and C-lobe）that linked to each other by anα-helix linker are structurally very comparable.However,the binding ability between C-lobe and Ca²⁺is 10-25 times more than that of the N-lobe.We previously discovered that the CaM’s constituent proteins（N-and C-lobe）have lobe specificity when regulating Na _V1.1 channel.Nevertheless,lobe specificity modulation of CaM on the Na _V1.2 IQ motif has not been well demonstrated.In our study,we checked the binding between CaM and Na _V1.2 IQ_R1902Cmotif at various Ca²⁺concentrations by molecular biology methods,demonstrating different binding characteristic between CaM and Na _V1.2 IQ_{（wt and R1902C）}motif.We also found lobe-specific interactions between CaM and Na _V1.2 IQ_R1902C motif under different Ca²⁺conditions.Our study offers a new mechanism for the modulation of Na _V1.2 IQ motif including R1902C,an autism-associated mutation by Ca²⁺/CaM.Materials and Methods1.Pull-down assayThe corresponding c DNA of wild type IQ motif of human Na _V1.2（1901Lys-1927Lys）was generated by PCR.Subcloning of human CaM as well as truncated protein N-lobe and C-lobe from HEK293 cells.Construction of Na _V1.2 IQ_R1902C mutant was performed using the Quickchange TM kit（QIAGEN）for site-directed mutagenesis.The vectors were amplified in Escherichia coli BL21（DE3）expressing a fusion peptide to a target protein was glutathione-S-transferase（GST）.The GST region of fused CaM was removed by cleavage using Pre Scission Protease（GE Healthcare）.GST-IQ,CaM and lobes of CaM were quantified by Bradford method using bovine serum albumin（BSA）as the standard and with correction factors:0.83（GST-IQ）,1.22（CaM）,2.74（N-lobe of CaM）,and 0.74（C-lobe of CaM）.For the CaM binding assays,the immobilized GST-IQ or its mutants（2–4μg）were incubated with various[CaM]（0.07,0.21,0.7,1.4,2.1μM）for 4 h at 4℃in 300μL of Tris buffer（p H=8.0）in the existence of various Ca²⁺concentrations([Ca²⁺]≈free:Tris buffer,5m M EGTA,and p H=8.0;100n M:Tris buffer,5m M EGTA,4.7883m M Ca Cl₂ and p H=8.0;500n M:Tris buffer,5m M EGTA,5.096m M Ca Cl₂ and p H=8.0;2 m M:Tris buffer,0.1M Ca Cl₂ and p H=8.0).Afterwards,the reaction mixtures were gently washed twice by using the same wash buffer as above.Bound CaM was eluted with 5×SDS-PAGE loading buffer and resolved in 15%SDS-PAGE gels.2.Computer molecular dockingIn Discovery Studio 2017,we used the Create Homology Model tool to derive the crystal structure of the IQ motif of Na _V1.2 from Na _V1.2 IQ-CaM complex（PDB:2KXW）.In Discovery Studio（DS）,we used the ZDOCK and RDOCK protocols to perform docking studies.In our case,the higher the ZDOCK score is,the better interaction the docking pose should represent.Thereafter,10 topmost scoring poses from ZDOCK were selected for energy minimization in the RDOCK protocol using the CHARMM force field.Finally,the top poses with the lowest RDOCK energies（E＿RDOCK）were advanced for our binding interface analysis shown in the molecular modeling figures.The combination of ZDOCK and RDOCK procedures facilitates a better definition of a superior interaction of Na _V1.2IQ motif with CaM’constituent proteins（truncated N lobe and C lobe）.For individual interactions shown in the figures,docking results are demonstrated as solid ribbons of the best poses of N-/C lobe-IQ complex with the lowest RDOCK interactions energies.3.Statistical analysisThe gel image obtained in the pull-down assay of protein was converted into a molar amount based on the molecular mass by Image J.Data was fitted to theoretical curves using the software Sigma Plot 12.0（version 12,Sigma-Aldrich,Beijing,China）.For the binding between C lobe and Na _V1.2 IQ_wt motif,we chose the one-site fit model,and the Hill equation:y=Bmax·x/（Kd+x）;for combinations other than the binding between C lobe and Na _V1.2 IQ_wtmotif,we chose the two-sites model,and the Hill equation:y=Bmax1·x/（Kd1+x）+Bmax2·x/（Kd2+x）（Kd:apparent dissociation constant）.Statistical significance of differences was evaluated using assessed by ANOVA followed by Tukey test（Graph Pad Software,San Diego,CA）,and P<0.05 was considered statistically significant.Results1.We verified that the Na _V1.2 IQ_R1902C motif can bind to CaM.The MOE data are as follows:the ZDOCK score and E＿RDOCK of the optimal orientation of the N-lobe and Na _V1.2 IQ_R1902C motif are 11.78 and-48.67 k J/mol,respectively,while the interaction of the C-lobe and the Na _V1.2 IQ_R1902C motif is 11.72 and-33.77 k J/mol respectively.In addition,the best ZDOCK score and E＿RDOCK of CaM and Na _V1.2 IQ_R1902C are 13.86 and-68.36 k J/mol.2.We further detected the binding properties of CaM and Na _V1.2 IQ_R1902C motif. Interestingly,the Na _V1.2 IQ_R1902C motif and CaM have the largest binding at 500 n M [Ca²⁺].However,the combination between the CaM and IQ_wt motif is U-shaped,the highest when[Ca²⁺]≈free,and the lowest when 100 n M[Ca²⁺].Therefore,compared with IQ_wt motif,the Ca²⁺dependence of the binding between CaM and IQ_R1902C motif almost disappeared,and the binding to IQ_R1902C increased at 100 and 500 n M[Ca²⁺].3.Through the pull-down assay,we further detected the binding between the N lobe and C lobe of the CaM constituent protein and the Na _V1.2 IQ_R1902Cmotif.The R1902C mutant weakened its ability to bind to the N lobe at specific[Ca²⁺]concentrations (including≈free and 2 m M[Ca²⁺]).Compared with the Na _V1.2 IQ_wt motif,the mutation of R1902C reduced the ability to bind to C lobe.4.After we mutated R1902 in the Na _V1.2 IQ_wt motif to cysteine C,it is easy to form disulfide bonds between adjacent amino acids,so we added dithiothreitol（DTT）as a reducing agent to repeat experiment.The result is that whether or not to add DTT does not affect the binding between CaM and Na _V1.2 IQ_R1902C motif.Conclusion1.Compared with IQ_wt,the binding between CaM and IQ_R1902C increased at 100 and 500n M[Ca²⁺].2.In the IQ_R1902C mutant,the Ca²⁺dependence of CaM domain binding almost disappeared.3.Both of the two constituent proteins of the CaM domain（truncated N lobe and C lobe）can bind to the IQ_R1902C mutant and mainly interact with the C lobe.4.Compared with the Na _V1.2 IQ_wt motif,the mutation of R1902C reduced the binding ability to the C lobe of CaM.5.After adding the reducing agent DTT,it will not affect the binding between CaM and the Na _V1.2 IQ_R1902C motif.In summary,our research reveals a new mechanism for the regulation of the CaM domain of the IQ_R1902C motif（autism-related mutation）,which provides a new direction for the generation and development mechanism of autism and the search for the target of combined antiepileptic and autistic drugs.