| Small conductance Ca2+-activated K+(KCa2,SK)channels,a subfamily of KCa channels,are widely expressed in the nervous system,and cardiovascular system.Different with big conductance Ca2+ activated K+ channels(KCa1,BK)channels,SK channels are activated solely by internal Ca2+ with higher sensitivity,submicromolar concentration.Ca2+ activates SK channel by binding to the calmodulin(Ca M),which is located at the C terminus of channel,leading to K+ efflux and consequent changes in cell membrane potential upon alteration of intracellular calcium.There are three subtypes of SK channels,SK1 to SK3,in which SK2 is predominance one in atrial myocytes and contributes to the late atrial repolarization and play a key role in physiological and pathological conditions.In a SK2 knockout mouse model,the repolarization of atrial was prolonged and the susceptibility to atrial fibrillation(AF)was increased.Some studies also pointed out that the overexpression of SK3 channel shortened the repolarization of action potential and increased the occurrence of atrial fibrillation.It is suggested that SK channel can be used as a target for the treatment of cardiovascular diseases such as atrial fibrillation.However, little is known about the molecular regulation of SK channels.Junctophilins(JPs)maintains a close relationship between endoplasmic reticulum/sarcoplasmic reticulum(ER/SR)and plasma membrane in many types of cells.In mammals,this family includes four subtypes(JP1-4).JP1 is mainly expressed in skeletal muscle,JP2 exists in skeletal muscle,cardiomyocytes and smooth muscle,and JP3 and JP4 are widely located in nervous system.Studies have shown that knockout of JP3 and JP4 make Ry R2 channel lose regulation of SK2 channel in nervous system,while knockout of JP1 and JP2 in skeletal muscle and myocardium makes L-type voltage-dependent calcium channels(LTCC)uncoupled with ryanodine receptor2(Ry R2),and skeletal muscle and myocardium contract abnormally.The close relationship between JP2 and Ry R2 was observed under high-resolution optical microscope,Ry R2 channel regulated its gating through direct interaction with JP2.Knockout JP2 significantly affected the localization and function of sodium-calcium exchanger(NCX1).The binding between JP2 and Caveolin1 proteins provides the structural and functional basis for the coupling of plasma membrane and sarcoplasmic reticulum in the calcium microdomain of vascular smooth muscle,and maintains the functional coupling between Ry R2 and BK channels.The direct interaction between JP2 and Caveolin3 can regulate the maturation and development of junction membrane complex(JMC)in neonatal mice.Therefore,JP2 can be seen that as a "bridge" protein in cardiac myocytes,the effective signal transduction between JP2 and ion channels lays the foundation for sufficient calcium-induced calcium release(CICR)and normal myocardial contraction,which has important physiological significance.The MORN(Membrane Occupation and Recognition Nexus)domain,located at the N-terminal of JP2,is a specific functional domain of JP2 coupled with the plasma membrane,which is composed of the MORNI domain and the MORNII domain.However,there are different studies on the mechanism of the interaction between MORN domain and plasma membrane,which may be accomplished by binding directly to plasma membrane phospholipid molecules or by interacting with cell membrane proteins.Our previous studies have found the interaction between JP2 protein and SK2 channel in mouse myocardium,however,it is unknown which region of JP2 interacts with SK2,and whether JP2 MORN domain participates the regulation of SK2 in cardiomyocytes.In this study,according to the literature reports and structure of proteins,different fragments of plasmids of SK2 and JP2 were prepared,and the specific regions of their binding were detected by molecular biology and cell biology methods.Furthermore,the effect of JP2 MORN domain on the surface membrane of SK2 channel and its mechanism were observed,and the effect of JP2 MORN domain on SK2 potassium current was observed by functional experiments.Part Ⅰ Identification of the interaction region between JP2 protein and SK2 channelMethods 1.Double immunostaining and co-immunoprecipitation analysis were used to detect the localization and interaction of SK2 and JP2: JP2 or SK2 antibodies or homologous antiIg G antibodies were added to adult SD rat myocardial tissue or cell lysate,and protein A/G beads were added to prepare immunoprecipitation complexes.The interaction between SK2 and JP2 proteins was analyzed by Westernblot.The distribution of SK2 and JP2 protein in the H9c2 cells was observed by immunostaining.2.The eukaryotic expression vectors of SK2 and JP2 fragments were constructed: different SK2 fragments SK2-N(1-145),SK2-M(141-390)and SK2-C(380-574)were cloned into the vector p IRES2-EGFP,and the Flag/HA tag was inserted into the C-terminal.JP2 c DNA:JP2-N1(1-253),JP2-N2(216-350)and JP2-C(340-696)of different length were cloned into p EGFP-C1 vector with His tag.3.The interaction between SK2 and JP2 fragments was detected by coimmunoprecipitation: SK2-N,SK2-M and SK2-C or JP2-N1,JP2-N2 and JP2-C were transiently transfected into HEK293 cells,and the total cell proteins of each transfection group were extracted.The binding of SK2 fragment to JP2 fragment was analyzed by using specific Flag and His antibody.4.GST pull-down analysis: GST-JP2-N1,GST-JP2-N2,GST-JP2-C and control plasmids were transformed into BL21 strain,and the fusion protein was expressed and purified by IPTG.The purified protein of each group was incubated with SK2-HA fragment protein expressed in HEK293 cells.After specific elution of glutathione,the expression of HA tag in the complex was detected by Western blot.1.SK2 and JP2 could form immunoprecipitation complexes in rat cardiomyocytes and H9c2 cells,suggesting that SK2 and JP2 might interact with each other.Immunofluorescence analysis showed that SK2 and JP2 proteins were distributed in the membrane and cytoplasm of H9c2 cells,and they were clearly co-located.2.JP2-N1,JP2-N2 and JP2-C were transiently transfected into HEK293 cells with SK2-N,SK2-M and SK2-C eukaryotic expression vectors,respectively.Coimmunoprecipitation assay was carried out with specific tag His and Flag antibody.The results showed that JP2-N1 and SK2-C terminal could interact directly.No immunopositive bands were found in the other experimental groups and control groups.3.GST-pulldown assay showed that the GST-JP2-N1 fusion protein expressed in E.coli BL21 could bind to the SK2-C terminal protein expressed in HEK293 cells.ResultsPart Ⅱ JP2 MORN domain I increase the membrane expression and localization of SK2 channelMethods 1.The eukaryotic expression vectors of SK2+JP2-N1 and SK2+JP2-N1 mut point mutations were constructed: the c DNA of SK2 and JP2-N1 was subcloned into p IRES2-EGFP to obtain the SK2+JP2-N1 plasmid.According to the point mutation technique,two mutation sites N101 R and Y141 H were constructed in SK2+JP2-N1 plasmid.2.HEK293 stable cell line screening: SK2,SK2+JP2-N1 and SK2+JP2-N1 mut plasmids were respectively transfected into HEK293 cells and screened by G418.After the positive clones appeared,the target genes were extracted by limited dilution and expanded culture,and the total cell RNA,RT-PCR was extracted.3.Biotin extracted cell membrane protein: the surface of HEK293 cells transfected with plasmid and H9c2 cells infected with adenovirus were biotinylated.The biotin-bound protein was incubated with avidin beads and the membrane protein complex was eluted.The expression of SK2 channel in each group was detected by Western blot with specific SK2 antibody.4.Construction and infection of JP2 knockdown adenoviruses: H9c2 cells inoculated in petri dishes were infected with adenoviruses carrying negative control si RNA(Ad-NC)or JP2-si RNA(Ad-si JP2).The cells infected with 48-72 hours were used in the next experiment.Westernblot was used to detect the expression of JP2 m RNA and protein in infected cells of each group,and to observe whether it affected the expression of SK2 channel.5.Co-immunoprecipitation analysis was used to detect the effect of JP2 mutation on its binding to SK2 channel.6.Electrophysiological recording: HEK293 cells transfected with SK2 and SK2+JP2-N1 were selected as the object of study,and SK2 channel currents were recorded by whole-cell patch clamp.Results 1.We establish HEK293 cell lines stably expressing SK2,SK2+JP2-N1 and SK2+JP2-N1 mut.The total protein and membrane protein of SK2 channel in each transfection group were extracted and detected by SK2 antibody by Westernblot method.The results showed that compared with SK2 group,the total protein of SK2 channel in SK2+JP2-N1 group had no significant change,but the membrane protein increased significantly.There was no significant difference in total protein and membrane protein of SK2 between SK2 group and SK2+JP2-N1 mut group.Cellular immunofluorescence assay showed that SK2 was distributed in the cytoplasm of HEK293 cells transfected with SK2 plasmid.The membrane protein distribution of SK2 channel increased significantly in HEK293 cells transfected with SK2+JP2-N1 plasmid,which suggested that JP2 MORNI domain could significantly increase the membrane expression and localization of SK2 channel.2.H9c2 cells were infected with JP2 knockdown si RNA and negative control si RNA,respectively,and the total protein and membrane protein of H9c2 cells were extracted.Westernblotting showed that compared with the control group and negative control group,the total protein of SK2 channel in JP2 si RNA group had no significant change,but the membrane protein decreased significantly.3.SK2+JP2 or SK2+JP2mut plasmids were transfected into HEK293 cells,respectively,and co-immunoprecipitation experiments with specific SK2 and JP2 antibodies showed that the mutation of JP2 MORN domain significantly reduced the binding between JP2 MORN domain and SK2 channel.4.HEK293 cells were transfected with SK2 and SK2+JP2-N1 plasmid respectively.The calcium-activated potassium currents in different HEK293 cells were recorded by whole-cell patch clamp.Compared with the SK2 group,the potassium current increased significantly in SK2+JP2-N1 group.Part Ⅲ The membrane expression regulation mechanism of SK2 channel by JP2 MORN domainMethods 1.The effect of knockdown JP2 on the expression of SK2 m RNA in H9c2 cells was detected by RT-q PCR.2.Double immunostaining was used to detect the localization of SK2 channel in early endosome and Golgi apparatus in HEK293 cells transfected with SK2+JP2-N1 mut plasmid.3.Western blot was used to detect the membrane expression of SK2 channel in HEK293 cells transfected with SK2+JP2-N1 or SK2+JP2-N1 mut plasmid treated with primaquine.4.Statistical analysis: all experimental data were represented by mean ±standard error,and the figures were created by Graph Pad Prism 5 software.t-test was used for statistical analysis.The difference was significant(P < 0.05).Results 1.The trafficking of SK2 channels in early endosome and Golgi organelles was observed in HEK293 cell lines stably expressing SK2+JP2-N1 mut plasmid.The results of cellular immunofluorescence showed that the SK2 channel was obviously colocalized with the early endosome markers Rab5 and EEA1,but weakly with the Golgi body marker GM130.2.The Western blot of HEK293 cells expressing SK2+JP2-N1 and SK2+JP2-N1 mut plasmids were treated with primaquine.The results showed that the membrane proteins of SK2 channel in both groups were significantly lower than those in DMSO group.These results suggest that JP2 MORNI domain mutations significantly affect the endocytosis recycling pathway in the retrograde pathway of SK2 channels,resulting in abnormal SK2 trafficking to the cell membrane due to the aggregation of SK2 in early endosome.Conclusion JP2 MORN domain directly regulates the function and trafficking of SK2 channels through its interaction with the C terminal of SK2. |
PDF Full Text Request