Junctophilin2 Modulates The Surface Expression Of SK2 Channel

Backgrounds and Objectives: Small conductance Ca2+ activated K+ channels(SK channels) are activated by intracellular Ca2+ and expressed in almost all of the excitable cells. SK2 channel is highly expressed in mammal cells.It is one type of calcium-activated K+ channel that non voltage dependent and high sensitive to the change of intracellular Ca2+ concentration. When the cytosolic Ca2+ concentration increased, the SK2 channel is activated, which is realized by Ca2+ binding to the calmodulin that induces its conformational changes, leading to K+ efflux. The SK2 channel involves in the action potential repolarization, especially during the late phrase of membrane action potential repolarization, which corresponds to the relative refractory period and supernormal phase of cardiac myocytes excitability.The clinical arrhythmia often occurres during this period as well. Consequently, the SK2 channel plays an essentional role in the duration and shape formation of the cardiac action potential. The SK2 channel abnormal expression can cause the changes of the myocardial function, and may be lead to or induce arrhythmia, such as atrial fibrillation and atrial flutter.It maybe one of the mechanisms of arrhythmia. Therefore the further research of molecular regulation of SK2 channel has very important significance. Junctional membrane complexs(JMCs) are the the structural basis for theeffective cross-talk between the plasma membrane(PM) and endoplasmic/ sarcoplasmic reticulum(ER/SR). Recent studies have revealed that junctophilins are a membrane binding protein that relates to the formation of JMC and has JMC characteristics. Junctophilins are considered to keep the JMC stability through maintain a fixed distance between the plasma membrane(PM) and endoplasmic/ sarcoplasmic reticulum(ER/SR). Junctophilin 2 is a membrane binding protein within the junctional membrane complexs in cardiac myocytes, which can interact with both the plasma membrane and endoplasmic/sarcoplasmic reticulum membrane at the same time, so that the two can reach a fairly close.Therefore it can keep intracellular calcium stability as well as it is essential for realizing calcium induced calcium release(CICR) and excitation- contraction coupling process of cardiac myocytes. Within hypertrophic cardiomyopathy, heart failure and other cardiac diseases, JPH2 protein levels can be reduced, leading to the decrease of calcium induced calcium release and the loss of excitation contraction coupling in cardiac myocytes. It has been known that the expression of Junctophilin has great significance for the coupling of ion channels on the membrane surface, which is one of the pathological mechanisms of the related diseases. Our laboratory previous studies have confirmed that adenovirus mediated si RNA targeting JPH2 gene of mouse can silence the expression of JPH2 in mouse cardiac muscle cells.Moreover, it can also inhibit the expression of SK2 channel on the cell membrane. For further research interactions between JPH2 protein and the SK2 channel, this study we establishes HEK293 cells of SK2 channel expression cell line so as to explore the molecular regulation of SK2 channel and its mechanism by interaction of JPH2 protein.Materials and Methods 1. The plasmids of p SK2-IRES-EGFP, p SK2-IRES-EGFP+JPH2 and p SK2-IRESEGFP+JPH2101+141- mutants were constructed. 2. The recombinants of p SK2-IRES-EGFP, p SK2-IRES-EGFP+JPH2 and p SK2-IRES-EGFP+JPH2101+141-mutants plamids were transfected into HEK293 cellsaccording to a lipsome infection protocol, respectively. After 48 h, the expression of green fluorescent protein EGFP was observed under the fluorescence microscopy. 3. The SK2 total and membrane proteins from the HEK293 cells transfected with p SK2-IRES-EGFP, p SK2-IRES-EGFP+JPH2, p SK2-IRES-EGFP+JPH2101+141-mutants plasmid were obtained with cell lysis and cell-surface biotinylation analysis. Then they were examined by Western blot. The results were dealt with gray level analysis via Image J. The data were processed with statistical analysis by graphpad prism 5, among. The total and membrane proteins of the SK2 were compared using a two sample mean t test, P < 0.05 for differences are significant. 4. The distribution of the SK2 channel on the HEK293 cells transfected with the vectors was determined by immunofluorescence microscopy.Results: 1. The endonuclease digestion and sequence analysis show the successful constructions of p SK2-IRES-EGFP, p SK2-IRES-EGFP+JPH2 and p SK2-IRESEGFP+JPH2101+141- mutants plasmids with eukaryotic expression vector. 2. The total and the cell surface expression of the SK2 protein from HEK293 cells cotransfected with JPH2 and SK2 plasmids significantly increased compared with the HEK293 cells transfected with the SK2 channel alone. Whereas HEK 293 cells transfected with p SK2-IRES-EGFP+JPH2101+141 plasmids exhibited diminished cells surface expression when compared with cells treated with the SK2 channel alone. 3. Microscopy images show that the distribution of the SK2 channel both on the plasma membrane and in the cytosol of the cells when HEK 293 cells were transfected with SK2 plasmid alone. A marked overlap in the distribution of SK2 channel and JPH2 on the plasma membrane when HEK 293 cells were coexpressed the plasmids containing SK2 and JPH2. In contrast, when the SK2 channel was coexpressed with JPH2 mutation in HEK293 cells, the SK2 channel displayed a low level of the immunoreaction.Conclusion: JPH2 protein aids in the surface expression and proper membrane location of SK2 channel.