Effect Of Rab5 On Large Conductance Ca²⁺-Activated K⁺ (BK_Ca) Channels Expressed In HEK293 Cells

Objective: Large conductance calcium activated potassium(BK_Ca)channel could link excitability and intercellular calcium,which were very important regulator for blood flow,urinary and immunity response.The protein of BK_Ca channel played a crucial role in the tension regulation of vascular smooth muscle.The BK_Ca channel contained 4 a subunits and b subunits,which was the key point for the protein channel playing physiological function.If there were some abnormal expression and function of BK_Ca channels,it would induce many cardiovascular diseases,such as hypertension.After being synthesized and processed in endoplasmic reticulum and Golgi apparatus respectively,BK_Ca protein should be transported to the plasma membrane to take physiological function with complicated mechanisms.As one member of the small GTPase family,Rab expresses extensively in the intracellular membrane and subcellular compartments with the role of regulating signal transduction,vesicle transportation and ion channel's activity.According to a few previous research,there may some connection between Rab and BK_Ca channels.Therefore,we aim to investigate the effects of Rab5,one of important Rab GTPases family for regulating many steps of membrane trafficking,on BK_Ca channels expressed in HEK293 cells.Methods: According to our experience of our previous research,we used HEK293 cells as the model cells.The expressing plasmid Flag-hSlo-GFP containing BK_Ca channels a subunit?hSlo1?of human vascular smooth muscle with Flag and GFP fusion tags was transfected into HEK293 cells with different types(wide type?WT?,dominant negative?DN?or constitute active?CA?of Rab5 expressing plasmid by liposome transfection.The effect of Rab5 on BK_Ca channels were demonstrated with patch clamp technique,Western blotting and Flow cytometry.Results: The main electrophysiological properties of BK_Ca channels expressed in HEK293 cells were consistent with those obtained from the native cells.In the whole cell configuration Rab5 WT and Rab5 CA significantly increased BK_Ca macroscopic currents at different membrane voltage?V_m??P<0.01 or P<0.05?.At V_m=⁺60mV,Rab5 WT and Rab5 CA significantly increased BK_Ca current density from 49.19±3.76 pA/p F?P<0.05,n=6?and 87.16±3.79 pA/pF?P<0.05,n=6?,while Rab5 DN inhibited the BK_Ca current density from 49.19±3.76 pA/pF to 34.68±3.37 pA/pF?P<0.05,n=6?.The Western blotting results showed that Rab5 WT and Rab5 CA increase the expression of membranous BK_Ca protein in HEK293.Compared with the control group,the expression of membranous BK_Ca protein was 1.24±0.08 times in Rab5 WT group and 1.42±0.13 times in Rab5 CA group.In the meanwhile,Rab5 DN decreased the expression of membranous BK_Ca protein and the relative quantity was 0.73±0.11?P < 0.05?times than that in control group.The results of FCM showed that Rab5 WT and Rab5 CA significantly increase the ratio of cells with Flag positive(Flag⁺,indicating the cells with expression of BK_Ca channels in cell membrane)to cells with GFP positive(GFP⁺,indicating the cells with expression of BK_Ca channels),and Rab5 DN significantly decrease the ratio of Flag⁺ to GFP⁺?P<0.05,or P<0.01?.Rab5 WT and Rab5 CA accelerated the trafficking of BK_Ca channel protein from cytoplasm to cell membrane and increased the expression on cell surface.Conclusions: Rab5 significantly increased the expression BK_Ca channels on cell surface and may increase channel protein trafficking to membrane in HEK293 cells.