Effects Of Junctophilin2 On SK2 Channel Function In HEK293 Cell

1 Background and objectivesSmall-conductance Ca²⁺-activated K⁺channels?SK,KCa₂?almost existsin every excitable cells,and can be activated by intracellular Ca²⁺.According to the different genes encoding,SK channel is divided into three subtypes,SK1,SK2 and SK3.All three subtypes are voltage non-dependent and can be specifically blocked by apamin.The expression of SK1 and SK2 of cardiomyocytes is mainly in the atrial myocytes,and SK2 is the most sensitive to apamin.Studies showed that the atrial myocytes'repolarization can be prolonged in the mice knocked out SK2 channels,and the susceptibility to the rapid arrhythmia of atrial fibrillation and atrial fibrillation also increased.So,the research of the regulatory mechanism of the SK channels may provide some treatment basis for supraventricular tachyarrhythmia.The junctophilin?JPH,JP?family is the major structural protein involved in formation of junctional membrane complexes?JMCs?.It may keep a fixed distance between the cell membrane and sarcoplasmic reticulum,and provide a bridge for the signal transduction in excitable cells between plasma membrane ion channels and intracellular calcium release channels to regulate Ca²⁺homeostasis,which aids to excitation-contraction coupling in skeletal muscle and cardiac muscle cells.The JPH includes an N-terminal region,an alpha helix-like region,and a C-terminal region.The N-terminal region contains an MORN domain of 8 repeats.It may be divided into 2 regions:MORN1?MORN motifs I-VI?and MORNII?MORN motifs VII to VIII?are predicted to be domains coupled to ion channels on the surface of cell membranes.JPHs have four subtypes,JPH1,JPH2,JPH3 and JPH4,among which JPH1 exists in skeletal muscle,and JPH2 mainly exists in myocardium,and JPH3 and JPH4 are mainly distributed in the brain.Studies have shown that the knockout of JPH3 and JPH4 genes can decouple the SK2 channel and RyR2 in the cerebellum pukken,and RyR2 no longer has a regulatory effect on SK2 channels.The previous research work of our lab has demonstrated that there was an interaction of JPH2 with the SK channels in cardiomyocytes and transfected HEK293 cells by Co-ip and GST pull-down.They were also detected in the T tube membrane of the atrial and ventricular myocytes,and on transfected HEK293 cell membranes.These results suggest that there is interaction between SK2 and JPH2 in vitro and in vivo.And when we knocked down the JPH2 gene in mature mouse myocardial cells,the expression of SK2 did not reduce in cardiomyocyte,but the patch clamp experiments indicated that knockdown of JPH2 caused a significant decrease in the density of I_K,Ca,Ca of SK2 channels and reduced the amplitude of the Ca²⁺transient.Therefore,it is possible that JPH2 regulates the I_K,Ca in cardiomyocytes by affecting intracellular calcium homeostasis.In order to further verify the effect of JPH2 on the function of SK2 channels,Liposome was used to transiently transfect SK2 plasmid,the SK2+JPH2 co-expression plasmid and the SK2+JPH2-mutant?S101R and Y141H in MORN domain?co-expression plasmid into HEK293 cells.Western blot was used to detect the expression of SK2 channels and JPH2,and Whole-cell patch clamp method was used to detect the effect of JPH2 on I_K,Ca of SK2 channels in vitro.2 Materials and Methods1)Amplification and extraction of recombinant plasmidThe SK2 full-length plasmids?p SK2-IRES-EGFP?,SK2+JPH2 co-expression plasmids?pSK2-IRES-EGFP+JPH2?,the SK2+JPH2-mutant co-expression plasmid(pSK2-IRES-EGFP+JPH2^S101R+Y141H)had been constructed successfully and used in our laboratory.2)Transfection of HEK293cellsThree groups of plasmids were transferred into cultured HEK293 by liposome transient transfection.The cells were divided into the following three groups according to experimental requirements:Group A:HEK293 cells transfected with SK2 plasmid aloneGroup B:HEK293 cells transfected with SK2 and JPH2 co-expression plasmid?pSK2-IRES-EGFP+JPH2?GroupC:HEK293 cells transfected with SK2 and JPH2-mutated co-expressionplasmid(pSK2-IRES-EGFP+JPH2^S101R+Y141H)3)Western blot analysisThe total proteins of the three groups of transfected cells were extracted after48h,and the expression of SK2 and JPH2 were detected with specific antibodies.4)SK2 channel current(I_k,Ca)recordingThe whole cell patch clamp was used to record the apamin sensitive I_k,Ca for the three groups of transfected HEK293 cells after 48h.5)Statistical analysisDate are presented as mean±S.M.E.Channel Currents of Recording and Analyzing with OriginPro 8.0 Software.Statistical software SPSS21.0 was used to analyze the experimental data,and make the voltage current curve.The significant differences among the groups were compared by one-way ANOVA.A p value<0.05was considered to be statistically significant.3 Results1)HEK293 cells transfected with SK2 plasmid,SK2+JPH2 co-expression plasmid and SK2+JPH2-mutanted co-expression plasmid all expressed green fluorescence via the fluorescence microscopy.2)The JPH2 and SK2 proteins in each group of HEK293 cells were detected by Western blotting.There is no significant difference in the SK2 protein level among three transfected HEK293 cells.No JPH2 expression was detected in HEK293 cells transfected with pSK2-IRES-EGFP,but no significant difference of JPH2 expression in HEK293 cells transfected with pSK2-IRES-EGFP+JPH2 and pSK2-IRES-EGFP+JPH2S101R+Y141H.3)In the HEK293 cells,I_k,Ca current recorded by whole-cell patch clamp technique.There is no statistically significant difference of the apamin-sensitive Ik,Ca current between pSK2-IRES-EGFP group and pSK2-IRES-EGFP+JPH2group.Compared to pSK2-IRES-EGFP and pSK2-IRES-EGFP+JPH2 ^S101R+Y141H groups,the apamin-sensitive Ik,Ca current was significantly increased in the HEK293 cells transfected with pSK2-IRES-EGFP+JPH2 plasmids.4 ConclusionThe expressions of SK2 and JPH2 are detected HEK293 cells,JPH2 can increase the apamin-sensitive Ik,Ca current in vitro.