Ca²⁺/CaM Modulates Na_V1.2 IQ

Introduction:Epilepsy?EP?is a transient brain disorder caused by abnormal discharge of neurons in the brain.There are many causes of epilepsy.Multiple studies have proven that mutations in voltage-gated sodium channel are the most important cause of idiopathic epilepsy and refractory.Autism spectrum disorders?ASD?,is a widespread neurodevelopmental disorder.Genetics generally believes that autism is caused by chromosomal abnormalities and gene mutations.Epilepsy autism spectrum disorder is a common neurological disease,and often has a family inheritance.Expression genes SCN1A and SCN2A of epilepsy autism disease are located on autosome 2q24. SCN2A-encoded Na_V1.2 is one of the voltage-gated sodium channel?VGSC?subtypes.Na_V1.2 exists in the central nervous system in large numbers,including axonal unmyelinated bundles of mature neurons,axonal initiation segments,and Lang's nodules,which play an important role in evoking action potentials and transmitting action potentials to excite neurons.Calmodulin?CaM?is an important calcium ion binding protein in the body,regulates the intracellular calcium ion concentration,and participates in a variety of biological regulatory pathways.In terms of structure,it is composed of two highly homogeneous spherical regions,namely C-lobe and N-lobe,and an?-spiral linker connecting the two lobes.The spatial structure is dumbbell-shaped.Each lobe contains two Ca²⁺binding sites?also known as EF-hand?,and each EF-hand structure can bind one Ca²⁺,but the Ca²⁺binding capacity of C-lobe and N-lobe are different.Previous studies have shown that the affinity of C-lobe and Ca²⁺is about 5-10 times higher than that of N-lobe.The C-terminal of some voltage-dependent calcium channel subtypes and voltage-gated sodium channel subtypes have a classical sequence that can specifically bind to CaM.The isoleucine-glutamic acid sequence is named IQ?Isoleucine-Glutamine?motif.Existing studies have proven that CaM can bind to NaV1.1 IQ and regulate the VGSCs ion activity,but so far,the mechanism of CaM-Na_V1.2 interaction and binding and the structure-specific binding mode are not clear.This experiment studies the interaction between CaM and the functional specific amino acid sequence-IQ motif on Na _V1.2,and uses it to clarify the specific binding mechanism of CaM and Na_V1.2,resulting in the occurrence of ion channel diseases such as epilepsy and autism.Mechanism clarification lays a certain theoretical and practical foundation.Methods:1.cDNA constructionUsing the human cDNA sequence of Na _V1.2 as a template,the corresponding Na_V1.2 IQ motif?IQ?was prepared by PCR technology.The primers were designed by VectorNTI software.Human CaM and its truncated proteins N-lobe?a.a.2-80?and C-lobe?a.a.76-148?were cloned from cDNA of HEK293T cells.The three kinds of DNA were inserted into pGEX-6P-1 H320 vector for expression and purification.2.Primary hippocampal neuron cultureThe newborn Wistar rat pups were taken out within three days of birth,and their heads were anesthetized,and the brain was immediately taken out and soaked in Hank's solution at 4°C.The hippocampus was dissected out under an ice bath under a microscope,and it was washed with refrigerated cell culture solution?DME/F-12?.At37°C,trypsin digests the hippocampal tissue for 10 minutes.After digestion,it is uniformly mixed with the self-made hippocampal neuron culture solution and inoculated on a petri dish.After 24 hours,every 24 hours,half the volume of the neuron culture medium was changed.Observe the state of neuron growth closely within a week.3.Protein expression and purificationThe constructed three vectors were transformed into E.coli BL21?DE3?for targeted expression of proteins.Take 300?L each of the bacterial strain and ampicillin?AMP?and add them to 400 mL of 1×LB broth.Shake at 37°C in a water bath at 125 r/min overnight.The next day,the UV spectrophotometer measured an OD600 between 0.8 and1.0,and 1 mM isopropyl-1-thio-?-D-galactopyranoside?IPTG?and 50 mg/mL AMP.After incubating for four hours,divide the bacterial solution into several 50mL centrifuge tubes.Centrifuge and discard the supernatant.Resuspend the bacterial cell pellet with pre-chilled PBS buffer.In an ice bath,lyse the bacteria by sonication to get the IQ.After the bacterial solution of CaM and its truncated protein was sonicated,it was purified by incubation with glutathione-S-transferase?GST?fusion protein and glutathione agarose4B globin?GS-4B?.PreScission Protease was used to digest the GST region to obtain purified CaM or its truncated protein.The protein concentration of the purified CaM or its truncated protein was calculated by a concentration-density calibration curve using a BSA protein quantification kit,and the resulting proteins were stored at-80°C.4.Molecular docking technologyThe 3D configuration of Na_V1.2 IQ and CaM and its truncated proteins?N-lobe,C-lobe?were derived by using the homology model building tool of Discovery Studio2017 software.We used the Dock Proteins protocol's ZDOCK protocol and RDOCK protocol simulation refinement in Discovery Studio software to obtain the best connection method with the lowest energy.The solid band in each molecular docking result represents the molecular structure.5.Double-labeled immunofluorescencePrimary neuron slides were fixed,Triton X-100 was perforated and closed with 5%BSA blocking solution.Incubate with a 30-fold diluted rabbit anti-Na_V1.2 and a 100-fold diluted mouse anti-CaM primary antibody in a wet box at 4°C overnight.After washing with PBS,the cells were incubated with 100-fold diluted FITC fluorescent secondary antibodies?anti-rabbit IgG?and TRITC-labeled secondary antibodies?mouse IgG secondary antibodies 200-fold diluted?at room temperature for 1 h.Glycerol coverslips,and the co-localization expression of the two proteins was photographed under a fluorescence microscope.The negative control was the same as above without the addition of primary antibody.6.Interactions detected by GST Pull-Down technologyGST fusion IQ was fixed on GS-4B and incubated with CaM in[Ca²⁺]Tris buffer.After four hours,it was washed twice with Tris-Buffer solution corresponding to the calcium ion concentration,and the incubation was terminated.After incubation,GST-IQ and CaM protein complexes were added with SDS loading buffer and denatured by heating.After centrifugation,the supernatant was added to a 15%polyacrylamide gel for electrophoretic separation.After electrophoresis,Coomassie Brilliant Blue R?CBB?was used for staining.After decolorization,protein bands were digitized by using Photoshop software,and then the gray value was quantified by ImageJ software.After correction,it was converted into protein molarity for statistical analysis.7.Western BlotAfter incubation,15?L of SDS loading buffer was added to the complex of GST-IQ and CaM protein,and the mixture was denatured by heating.After centrifugation,the supernatant was separated for electrophoresis.After the electrophoresis completed,a transfer sandwich was made,protected from light and wet,and blocked for 2 hours.The primary antibody was 800-fold diluted mouse-derived anti-CaM antibody at 4°C overnight,washed three times for ten minutes with TBST,and incubated with HRP goat anti-rabbit IgG secondary antibody 1:5000),TBST was washed three times for 10 min.ECL luminescent liquid is mixed 1:1 to emit light.Results:1.This experiment successfully constructed CaM and its truncated proteins?N-lobe,C-lobe?and Na_V1.2 IQ plasmid,and successfully separated and purified them into CaM protein,N-lobe protein,C-lobe protein and Na_V1.2 IQ protein.2.This experiment successfully detected the co-localized expression of Na V1.2 and CaM in hippocampal neurons.3.The molecular docking results parameters were as follows:?1?CaM and Na_V1.2binding parameters:ZDock14.62,E?RDock-57.18 kJ/mol?2?N-lobe and Na_V1.2binding parameters:ZDock13.02,E?RDock-47.16 kJ/mol?3?Binding parameters of C-lobe and Na_V1.2:ZDock12.44,E?RDock-49.17 kJ/mol4.The binding amount of Ca²⁺/CaM and Na _V1.2 IQ increased with the increase of calcium ion concentration and CaM concentration.5.The binding amount of apo CaM(Ca²⁺free form calmodulin)to Na_V1.2 IQ was significantly higher than that of Ca²⁺/CaM and Na_V1.2 IQ.6.The binding amount of CaM's C-lobe and Na _V1.2 IQ under[Ca²⁺]?free condition was significantly higher than that of other Na_V1.2 IQ under the concentration of calcium ion,and the binding amount when 100 nM[Ca²⁺]was Lowest;the binding amount of CaM's N-lobe and Na_V1.2 IQ under the condition of 500 nM[Ca²⁺]was significantly higher than that of Na V1.2 IQ under the concentration of calcium ion,and the binding amount of 2mM[Ca²⁺]was lowest.At four calcium ion concentrations([Ca²⁺]?free,100 nM,500nM,and 2 mM),the Bmax of C-lobe and Na_V1.2 IQ of CaM were greater than the Bmax of N-lobe and Na_V1.2 IQ.Conclusion:1.CaM can bind to Na _V1.2 IQ.2.The binding free energy of CaM and Na_V1.2 IQ is the smallest,the score is the highest,and it is the most stable binding form.3.The combination of Ca²⁺/CaM and Na_V1.2 IQ has CaM concentration dependence and calcium ion concentration dependence.4.Apo CaM(Ca²⁺free form calmodulin)has a higher affinity for the channel than Ca²⁺/CaM for the channel,which is better than Ca²⁺/CaM combined with Na_V1.2 IQ.5.The affinity of C-lobe to the channel is higher than the affinity of N-lobe to the channel,and it is the main functional area for CaM to bind to the channel.This study reveals the mechanism of action between calcium ions,CaM,and Na V1.2,clarifies the main structural regions of CaM regulating Na_V1.2 channels,and provides research directions for plasma channel diseases such as autism spectrum disorder disorders with epilepsy.It would provide a theoretical basis for the study of drug therapeutic targets for the interaction between calmodulin and VGSCs.It also would provide a theoretical basis for in-depth study of the mechanism of disease caused by ion channel gene mutations.