Identification Of Interaction Between SK2 Channel And Junctophilin2 By Yeast Two-hybrid System

Backgrounds and ObjectivesThe small conductance Ca²⁺-activated K⁺channel is the only non-voltage-dependent K⁺channel that is activated by intracellular Ca²⁺,and is present in almost all excitable cells.According to SK channel sensitivity to apamin?bee venom peptides?it can be divided into four different subtypes,including SK1,SK2,SK3 and SK4.Some studies have shown that SK2 channels can express in myocardial cells,and it is an important ion channel involved in the end of the repolarization of action potentials which is a sensitive period in the occurrence of clinical arrhythmia.The abnormal function of the SK2 channel may be one of the mechanisms that induce arrhythmia such as atrial fibrillation and atrial flutter,leading to changes in cardiac function.Therefore,it is of great significance to study the pathological mechanism of arrhythmia such as SK2 channel.The functional SK2 channels are composed of 4 alpha subunits.Each subunit is divided into the transmembrane region?S1-S6?,a pore formation region,cytoplasmic C-terminus and N-terminus,and the C-terminus contains the binding area of Calmodulin?CaM?.Ion channels are complexes composed of multiple subunits,each containing pore forming units and ancillary subunits which regulates the function of the channel by interacting with a number of supporting proteins and regulatory proteins.To investigate the molecular regulation mechanism of SK2 channels,we observed the interaction and co-localization of SK2 and Junctophilin2?JP2?in both myocardium and transfected HEK293 cells.Moreover,JP2 did not affect the expression of SK2 protein in HEK293 cells,but increased expression on the cell membrane.The knockdown of JP2 significantly reduced the density of I_K,Ca of SK2channels in mature mouse myocardial cells.In recent years,studies have found that Junctophilin?JP,JPH?is an important protein molecule involved in the formation of junctional membrane complex?JMC?.Its main function is to maintain a fixed distance between the cell membrane and the sarcoplasmic reticulum,and to maintain intracellular Ca²⁺homeostasis,it may provide a structural basis for signal transduction between the cell membrane and the sarcoplasmic reticulum.JPs have four subtypes,JP1,JP2,JP3 and JP4,among which JP1 and JP2 mainly exists in skeletal muscle and myocardium.The structure of JP 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.The latter is divided into 2 regions:MORN1?MORN motifs I-VI?and MORN2?MORN motifs VII to VIII?are predicted to be domains coupled to ion channels on the surface of cell membranes.The studies of our laboratory in vitro and in vivo have suggested that there is an interaction between JP2 and SK2.However,very little is known about whether JP can interact with SK in the membrane through the N-terminal MORN domain,and about which region of SK can interact with JP?In this experiment,SK2 and JP2 were divided into three fragments according to the structure.The yeast two-hybrid bait expression plasmids?pGBKT7-SK2-X?were constructed,including pGBKT7-SK2-N?aa 1-145,containing N-terminus?,pGBKT7-SK2-M?aa 141-390,contains transmembrane segments?,pGBKT7-SK2-C?aa 380-580,containing C-terminus?;and the prey expression plasmids?pGADT7-JP2-Y?were constructed,such as pGADT7-JP2-N?aa 1-253,including MORN1 region?,pGADT7-JP2-M?aa 216-350including MORN2 region?and pGADT7-JP2-C?aa 340-696?.These recombined plasmids were identified by using PCR,double enzyme digestion and gene sequencing.Afterwards,the yeast two-hybrid technique was used to verify the region of interaction between SK2 and JP2 in vitro.This provides a theoretical basis for understanding the molecular regulation mechanism of SK2 channels.Materials and Methods1.Construction of Yeast two-hybrid bait expression plasmids and prey expression plasmidsAccording to the complete gene sequences and structural features of SK2 and JP2,SK2 and JP2 were divided into three fragments.The pGADT7and pGBKT7vector plasmids were double digested with restriction enzymes Ndel and BamHI to construct the recombinant,including pGBKT7-SK2-N,pGBKT7-SK2-M,pGBKT7-SK2-C,pGADT7-JP2-N,pGADT7-JP2-M,pGADT7-JP2-C,and the tag protein HA is attached at C-terminus.These recombined plasmids were identified by using PCR,double enzyme digestion and gene sequencing.2.Preparation of Yeast compatible cell AH109 by Lithium Acetate MethodThe monoclonal AH109 was picked from YPDA solid medium and inoculated into YPDA liquid medium.The competent yeast strain AH109 was resuspended and pelleted by centrifugation.3.Toxicity detection of recombinant yeast expression vectorRecombinant bait expression vector and empty vector pGBKT7,recombinant prey expression vector and empty vector pGADT7 were transformed into competent yeast AH109 cells by Lithium acetate-mediated method.We detected whether all recombinant expression vectors were toxic to host bacteria AH109 in SD/-Trp and SD/-Leu auxotrophic solid medium.4.Self-activation detection of recombinant yeast expression vectorTo test the autonomous activation of recombinant bait plasmids on host reporter genes,five sets of plasmids were transformed in AH109 yeast competent cells using Lithium acetate-mediated method:pGBKT7-SK2-Y+pGADT7,pGBKT7-P53+pGADT7-T?positive control?,pGBKT7-p53+pGADT7-LaminA?negative control?.Self-activation detection of recombinant bait expression vector used SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-?-gal auxotrophic solids culture medium.The same method was used to detect the self-activation of recombinant prey expression vector.five sets of plasmids were pGBKT7+pGADT7-JP2-Y?pGBKT7-p53+pGADT7-T?positive control??pGBKT7-p53+pGADT7-LaminA?negative control?5.Expression and identification of recombinant yeast expression vectorThe recombinant bait and prey expression vectors were transformed into competent yeast AH109 cells by Lithium acetate-mediated method,and pGBKT7 and pGBKT7-53 were used as controls,respectively.After bacterial proteins are extracted,HA antibody was used to detect the expression of target gene by Western blotting.6.Identification of SK2 and JP2 interactions regionThe three recombinant plasmids of pGBKT7-SK2-X were paired with the three recombinant plasmids of pGADT7-JP2-Y,respectively,and a pair of plasmid was co-transformed into the yeast AH109.The results of double-hybridization were determined by observing the presence or absence of yeast growth and blue-white coloration in SD/-Trp-Leu-Ade-His/X-?-gal auxotrophic solid medium.Results1.The double enzyme digestion and sequencing results showed that the yeast bait recombinant plasmid pGBKT7-SK2-X and the prey recombinant plasmid pGADT7-JP2-Y were successfully constructed.2.Compared with the empty carrier pGBKT7 and pGADT7 transformation group,the yeast cell clones of bait recombinant plasmid and prey recombinant plasmid were similar in volume and number,and the monoclonal OD value of experimental groups was close to the control group after 24h.The result indicates that the recombinant bait and prey expression plasmid are not toxic to the host strain AH109.3.Both the bait plasmid and the prey plasmid transformation groups were able to grow in SD/-Trp-Leu plates,whereas only positive controls could be seen the yeast cloned and blue in SD/-Trp-Leu-Ade-His/X-?-gal solid media.None of the negative control and the recombinant plasmid bait expression vector transformed group appeared to be cloned,suggesting that the recombinant plasmid could not activate the reporter gene.4.All fragments of SK2 and JP2 could express in yeast AH109 by Western Blotting after the recombinant plasmid transfection yeast AH109.5.After pGBKT7-SK2-X and pGADT7-JP2-Y co-transformed the competent yeast AHl09,no positive blue colony growth were detected,suggesting that there is no direct interaction between the SK2 protein fragment and the JP2 protein fragment.ConclusionThe yeast bait?pGBKT7-SK2-N,pGBKT7-SK2-M and pGBKT7-SK2-C?and prey?pGADT7-JP2-N,pGADT7-JP2-M and pGADT7-JP2-C?expression vectors are successfully constructed.The recombinant plasmids are identified to be suitable for yeast two-hybrid system by toxicity and self-activation detection;all fragments of SK2 and JP2 could express in yeast AH109.But there is no direct interaction between the SK2 protein fragment and the JP2 protein fragment.