Interaction Of Junctophilin 2 With A Small Conductance Ca²⁺-activated K⁺ Channel Subtype 2 ?SK2? And Ryanodinereceptors

Small conductance Ca²⁺-activated K⁺channels or SK channels,are almost widely expressed on the serolemma in all excitable cell,and are activated by intracellular Ca²⁺.The SK channels consist of four subtypes,SK1,SK2,SK3,encoded by the genes KCNN1-KCNN3 and an intermediate-conductance Ca²⁺-activated K⁺channels,is called SK4.SK1?SK2 and SK3 all are expressed in cardiac myocytes,including mouse,rat and humans,their fundamental roles contribute to the repolarization of the cardiac action potential?AP?.Some studies show SK1 and SK2 are mainly expressed in the atrium,and the mouse of SK2 knockout displayed AP prolongation and atrial fibrillation?AF?.Hence,the understanding of the SK2 channels in the regulation of the cardiac excitability has increasingly become the focus of new therapeutic strategies for the treatment of cardiac cardiac arrhythmia.Ca²⁺from two pathways in the cell can activate SK2 channels,and SK2 channels are opened.The voltage-gated Ca²⁺channels?VGCCs?on the plasma membrane is one,the other is ryanodine receptors,a Ca²⁺release channel,locates in the endo/sarcoplasmic reticulum?ER/SR?.Some studies have reported that Cav1.3 L-type VGCCs of myocardial membrane is coupled with SK2 channels through?-actinin,and regulate SK2 channels.So,these studies suggest that SK2 channels may not be directly interacted with Ca²⁺channels.The experimental evidence in our lab has shown that SK2 channels on plasma membrane have functional coupling with SR RyR2 receptor channels in cardiomyocytes.But the middle molecules that SK2 and RyR2 are coupling are not clear.Recent studies show Junctophilin?JPH,JP?has a functional coupling with the SK channels and RyRs receptors in neuron.JP is a protein family of the junctional membrane complex?JMC?which provides a bridge for the signal transduction in excitable cells between plasma membrane ion channels and intracellular calcium release channels by anchoring the endo/sarcoplasmic reticulum?ER/SR?to the plasma membrane.There are four kinds in mammals,including JP1^JP4.Among JPs,JP1 is predominantly expressed in skeletal muscle,JP2 is expressed in cardiac,skeletal and smooth muscles,and JP3 and JP4 are mainly present in nervous system.The knockout of JP3 and JP4 can decouple the SK2 channel and RyR2 in the cerebellum pukken,and RyR2 no longer has a regulatory effect on SK2 channels.The knockout of JP1 and JP2can cause impaired communication between the L-type VGCCs and SR calcium release channels in skeletal muscle cells,and result in excitation-contraction coupling disorder and losing its function.That is to say,JP2 dysfunction has been linked to defects in integral ion channel expression,abnormal intracellular Ca²⁺handing,and heart disease.Other people's results in our laboratory also showed that the use of RNA interference techniques to reduce the expression of JP2 in cultured neonatal rat cardiomyocytes could lead to decrease in the density of I_K,Ca.So,whether there is an interaction of JP2 with the SK channels and RyR2 receptors,and whether JP2 gene might modulate the SK channels function in cardiac myocytes,now there is no reported.In this study,at first,we used mass spectrometry and bioinformatics to screen proteins that interact with JP2 in myocardium.Secondly,we used the adult mouse heart or HEK293 cells to perform reciprocal Co-Immunoprecipitation?CO-IP?and GST pull-down experiments to verify the interaction between JP2 and SK2 channels,JP2and RyR2,and three constructs contained JP2 cDNA fragments(JP2_1-253,amino acid residues 1-253;JP2_216-350,residues 216-350;JP2_340-696,residues 340-696)were prepared to verify the specific residues within JP2 for binding with the cardiac SK2channel and RyR2.Finally,after siRNA knockdown of JP2 gene was performed,the intracellular Ca²⁺transient was measured using an IonOptix photometry system,and the expressions of calcium-related proteins in myocardium were detected by Western blot.Methods1.The healthy adult female or male C57BL/6 mice,weight 20-25g,were used in this study.2.Cardiac SR vesicle isolation and detectionThe cardiac SR vesicle isolation was performed in adult C57BL/6 mice with a minor modification,and detected the expression of SK2,JP2,and RyR2 proteins.The myocardial tissue protein was used as a positive control.3.Mass spectrometryThe cardiac SR vesicles lysates were incubated with anti-JP2 antibody or IgG antibodies of mouse sources overnight at 4°C,followed by an additional incubation with Protein A/G PLUS Agarose to prepare the immune complexes.After the immune complexes were performed electrophoresis and stained by coomassie bright blue,we chosed the different target protein strips as samples to carry out mass spectrometry analysis in Peking University medical department.4.Bioinformatics analysisAccording to the literature,we screened the proteins,categorized them withthe Geneontology website,and found the interaction between them using the String website.5.The prepared immune co-precipitation complex was further tested with specific SK2 and RyR2 antibodies by Western blot.6.The construction of the prokaryotic expression vectorFull length mouse JP2 cDNA?accession number,NM₀01205076.1?was inserted at the BamHI/XhoI sites of the pGEX-4T-1 vector to generate pGEX-4T-1-JP2.Three constructs contained cDNA fragments(JP2_1-253,amino acid residues 1-253;JP2_216-350,residues 216-350;JP2_340-696,residues 340-696)were subcloned into the glutathione S-transferase?GST?fusion vector pGEX-4T-1 with the BamHI/XhoI sites to generate pGEX-4T-1-GST-JP2-N1,pGEX-4T-1-GST-JP2-N2 and pGEX-4T-1-GST-JP2-C.And two enzyme cutting and sequencing were used to identify the recombinant plasmid.7.The expression and purification of the GST fusion proteinsGST,GST-JP2 fusion protein or GST constructs of GST-JP2 were transformed into Escherichia coli BL21 cells.The bacteria expression was induced with 0.2 mM isopropyl-1-thio-?-D-galactopyranoside?IPTG?at 26°C?40 rpm?overnight.And the fusion protein was purified by GST.BindTM resin8.The construction and transfection of pSK2-IRES-EGFP and pJP2-pCDNA-EGFP plasmidsFull length mouse SK2 cDNA?accession number AY258141?was subcloned into pIRES-EGFP to obtain pSK2-IRES-EGFP.Full-length cDNA of JP2 was subcloned into pcDNA3.1 vector to obtain pCDNA3.1-JP2.Two enzyme cutting and sequencing were used to identify the recombinant plasmid.Lipofectamine 2000 reagent?Invitrogen?was used to transfect HEK293 cells with pSK2-IRES-EGFP,pJP2-pCDNA-EGFP plasmids according to the manufacturer's instructions.9.GST pull-down analysisGST,GST-JP2 or GST-JP2 constructs premixed with glutathione-sepharose 4B were incubated with the mouse heart lysates or with the HEK 293 cell lysates overnight at 4°C.The pull-downed complexes were eluted from the glutathione-agarose by10mM reduced glutathione in TBS?pH 8.0?.The bound proteins were tested with specific SK2 and RyR2 antibodies by Western blot.10.CO-IPThe solubilized proteins isolated from HEK293 cells transfected with pSK2-IRES-EGFP and pJP2-pCDNA-EGFP were incubated with anti-JP2,or anti-SK2 antibodies overnight at 4°C,followed by an additional incubation with protein G Sepharose.Immune co-precipitation complex was tested with specific JP2and SK2 antibodies by Western blot.11.Venous injection of adenovirusA total dose of 1×10⁹ PFU adenovirous with control siRNA?Ad-NC?and JP2siRNA?Ad-siJP2?were delivered into mice by tail vein injection respectively,and single cardiac myocyte isolation were carried out 7 days after injection12.After siRNA knockdown of JP2 gene in adult mouse was performed,the expressions of JP2 and SK2 in myocardium were detected by Western blot.13.Ca²⁺transient measurementsThe isolated infected adult cardiac myocytes of Ad-NC and Ad-siJP2 performed with gradient calcium,and loaded with 2?M fura 2-AM in the dark at room temperature for 30 min.To evoke[Ca²⁺]i transients,the cells were stimulated at 1Hz by field stimulation applied by two parallel platinum electrodes.The intracellular Ca²⁺transient was measured using an IonOptix fluorescence photomultiplier system.The data was analyzed using IonOptix's IonWizard 6.6 software.14.After siRNA knockdown of JP2 gene in adult mouse was performed,the expressions of calcium-related proteins in myocardium were detected by Western blot.Results1.The results of Western blot showed cardiac SR vesicles lysates can be detected the expressions of SK2,JP2 and RyR2.2.The cardiac SR vesicles lysates were incubated with anti-JP2 antibody or IgG antibodies of mouse sources to prepare samples for mass spectrometry.After the immune complexes were performed electrophoresis and stained by Coomassie bright blue,we choose 13 different target bands by observing the proteins belts of control group and experiment group,and found 35013 proteins interacted with JP2 using mass spectrometry analysis.But 90 proteins were selected,including SK2 and RyR2 protein.By information analysis,the protein was further classified,and the interaction between them was determined.3.The prediction results in String site showed that the direct homologous proteins of JP2 and RyR2 can be expressed in other species,JP2 might have interaction with RyR2.At present,the area of mice SK2 CaM?sequence for 655-785?could only be built homologous model.So,we could not predict whether there was interaction between SK2 and JP2.Western blot results showed that SK2 and RyR2 selectively bound to endogenous JP2 in cardiac SR vesicles lysates.4.GST pull-down analysis with an antibody against SK2 revealed that GST-JP2fusion proteins were able to pull down the endogenous SK2,and GST-JP2-N1?aa1-253?,but not GST-JP2-C?340-696?and GST alone,was able to pull down the endogenous SK2 protein.GST-JP2-C fusion proteins were able to pull down the endogenous RyR2.A similar pulldown assays were performed on the HEK293 cells transfected with the SK2 channel.When three GST fused constructs were immobilized on glutathione column and then incubated with solubilized proteins prepared from the cell lysates,GST-JP2-N1?aa 1-253?selectively bound to SK2.5.We performed a similar set of CO-IP experiments in HEK293 cells transfected with plasmids containing SK2 and JP2.The results showed JP2 was immunoprecipitated with an antibody against SK2.In the same way,SK2 was immunoprecipitated with an antibody against JP2.This result indicates an interaction between JP2 and SK2 in vitro.6.The adenovirous with control siRNA and JP2 siRNA were delivered into mice by tail veins injection respectively.Western blotting analysis revealed that the level of JP2 expression in the adult mouse cardiac tissure transduced with Ad-mediated si RNA specifically targeted against JP2 for 7 days was reduced to 47%and 48%,respectively,of the levels observed in the noninfected adult cardiac tissure?Ctrl-NT?and infected scramble siRNA?Ad-NC?cardiac tissure.But no significant change in expression levels of the SK2 channel was examined in the adult cardiac tissure infected with Ad-siJP2 compared with the controls.7.The Ca²⁺transient in the single adult mouse cardiac myocytes infected with Ad-siJP2 for 7days through the caudal vein injection was measured using an IonOptix fluorescence photomultiplier system.siRNA knockdown of JP2 significantly decreased the amplitude of the[Ca²⁺]i transient,and increased its time to 50%peak compared with Ad-NC.Whereas the resting calcium levels and the time for Ca²⁺transient to decay was unchanged in Ad-siJP2 cardiac myocytes compared with the controls.We measured RyR2-dependent SR store Ca²⁺content by caffeine.The adult cardiac myocytes infected with Ad-shJP2 showed a significantly lower caffeine-evoked Ca²⁺release compared to control cells.So,This result indicated that knockdown of JP2 significantly decreased caffeine-mediated SR Ca²⁺store levels and this could account for reduced the[Ca²⁺]i transients amplitude.8.We next explore whether knockdown of JP2 affects the expression of calcium-handling proteins.There was no significant alteration in the expression of RyR2,SERCA2,Cav1.2 LTCC and NCX1 in Ad-siJP2 cardiac cells compared to Ad-NC.ConclusionThe present data provide evidence that the interaction between JP2 and SK2channels,JP2 and RyR2 is present in the native mouse heart tissue.JP2 interacts with SK2 via its MORN motif at the N-terminus,and interact with RyR2 via its C-terminal.JP2 and SK2 were immunoprecipitated with solubilized proteins from HEK293 cells expressing heterologous proteins,indicating that the MORN motifs within the N-terminus of JP2 and SK2 interact directly.Functional experiment reveals that interfering RNA silencing of endogenous JP2 caused a significantly reduced the amplitude of the Ca²⁺transient in the mice cardiaomyocytes.Knockdown of JP2effects on the[Ca²⁺]i transient and its dynamics may predominantly reflect a slowed down of RyR2 release rate.While the expressions of RyR2,SERCA2,LTCC and NCX1 did not change significantly.As a result,JP2 is the key coupling molecule of RyR2 and SK2 channel in the cardiomyocytes,JP2 may control the function of SK2channels by regulating intracellular Ca²⁺.