The Mechanisms That Snapin Regulates The Nuclear Trafficking Of Cav1.3-CT

Calcium signal plays an important role in cells,including short-term effectssuch as excitation-contraction coupling,secretion function,and long-term effectssuch as gene transcription,cell proliferation and cell death.Ca2+ influx via L-typecalcium channels(LTCCs)is critical for the conversion of electrical signals intointracellular biochemical events.LTCCs are multi-subunit protein complexescomposed of pore-forming ?1 subunit and several auxiliary subunits including ?,?2?and ?.Most of the pharmacological and gating properties are determined by ?1 subunit which incorporates the conduction pore,the voltage sensors and gating apparatus,and most of the known sites of channel regulation by second messengers and drugs.Four genes encoding ?1 subunits have been identified,including Cav1.1,Cav 1.2,Cav 1.3 and Cav1.4.There are two types of LTCCs in the heart,Cav 1.2 andCav1.3,which play a critical role in shaping action potential of myocardium,generating spontaneous electrical activity of sinoatrial node and the excitation-contranction coupling in the heart.Thus,LTCCs abnormality in either structure or function may lead to heart diseases,such as cardiac arrhythmia,myocardial hypertrophy and heart failure.Recently,it has been found that the C-terminus of LTCCs can function as a transcription factor to regulate the expression of itself and other genes.For example,in skeletal muscle,the C-terminus of Cav1.1 channel(?1S)can interact with AKAP15 and PKA to regulate its biological function.The C-terminus of Cav1.2 transports into the nucleus,which regulates its activity of the promoter regions to inhibit protein expression of full-length Cav1.2 channel,resulting in reduction of the calcium current density(ICa,L).In addition,the C-terminus of Cav 1.3 regulates the expression of myosin light chain(MLC2)to promote the localization of type 2 small conductance potassium channel(SK2)on the plasma membrane.However,the mechanisms that the long C-terminus of LTCC translocates into nucleus remain elusive.Interestingly,we have indentified a novel Cav1.3-binding protein,Snapin,which is a soluble N-ethylmalemide-sensitive fusion attachment protein receport(SNARE)associated protein.More importantly,Snapin can translocate into nucleus in the HL-1 cells.Recently,a study demonstrated that SNAREs regulated nuclear fusion in yeast.Based on those evidence mentioned above,therefore,we asked the questions:does Snapin regulate the nuclear import of Cav1.3 C-termius(Cav1.3-CT)?If so,what are the mechanisms underlying?To answer these questions,in present study,we performed experiments in HEK293 cells and HL-1 cells using cell and molecular biological technologies.The main results are as follows:1.The dominant fragments of nulcear import were identified.A variety of Cav1.3-CT with different lengths like(C421,C381,C286,C95,C50,C45)respectively were combined with pEGFP-C1 through molecular cloning,and those recombinant plasmids were transfected into HEK293 cells to perform immuneflorescence experiment.It was found that C421,C381,C95 and C50 were detected with stronger fluorescence signals in the nucleus,so were C286 and C45 in the cytoplasm,which demonstrated that 1830-1880aa was critical segment for the nulcear transport of Cav1.3-CT.2.To detect the subcellular contribution of Snapin,in present study,immunofluorescence and Western Blot were performed.As a result,Snapin was detected in both of nucleus and cytoplasm in mouse atrial myocytes and HL-1 cells.3.The interaction between Cavl.3-CT and Snapin was confirmed.Co-imunoprecipitation was performed in the lyses obtained from adult mouse atrial myocytes or HEK293 cells,proving that Snapin interacted with Cav1.3-CT.Interestingly,Snapin and Cav1.3-CT were both translocated in the nuclus,which presented that Snapin was likely involved in the nulcear import of Cav1.3-CT.4.Snapin was involved in regulating the Cav1.3-CT nuclear transportation.It:was found that Snapin had an influence on the whole-length protein expression of Cav1.3 channel.Overexpression of Snapin didn't lead to remarkable change of protein expression levels,while they were largely detected in the nucleus.The Cav 1.3-CT decreased a lot in the nucleus following the specifically Snapin siRNAinterference.Therefore,it was certain that Snapin regulated the Cav1.3-CT nuclear transportation.5.The mediation of Snapin in the Cav1.3-CT nuclear import is independent of functional sites.Co-IP was peformed in the overexpression of Snapin and Cav1.3-CTwith different lengths like C95,C50 and C45.The results showed that Snapin combined with three truncations,which were confirmed by yeast two hybrid(Y2H)assay.Hence,Cav1.3-CT nuclear import was not regulated by interacting with Snapin specifically.6.The mediation of Snapin in the Cav1.3-CT nuclear transportation is dependent on its phosphorylation by PKA.We constructed the Snapin mutantsincluding S50A,S50D(phosphorylation site by PKA),T117D and T117A(phosphorylation site by LRRK2)and these mutants were transfected into HEK293 cells to perform immuneflorescence experiment.These findings proved that S50Dimproved the nuclear import of Cav1.3-CT,while the others had no effect.It was suggested that Snapin regulated the Cav1.3-CT nuclear transport via PKA-regulatedphosphorylation.It has been reported that ISO can increase the activity of cAMP-PKA via stimulating ? adrenaline.In this case,we found that the Cav1.3-CT expression in nuclear increased by one fold after ISO treatment in HL-1 cells.The results confirmed that PKA-regulated Snapin phosphorylation impoved the Cav1.3-CTnuclear transport.Taken together,this study concluded that 1830-1880 amino acids were the key fragments for Cav1.3-CT nuclear transport and PKA-regulated Snapin phosphorylation played an essential role in nuclear transport of Cav1.3-CT.