| Voltage-gated calcium channels(VGCC)are large,transmenbrane multiprotein complexes that are located in brain,heart,smooth muscle and endocrine cell.These channels are central to cardiac action potential propagation,neurotransmitter and hormone release,muscle contraction and calcium-dependent gene transcription.The voltage-sensitive pores of all VGCC types are formed by so-called al subunits.Ten?1 subunits isoforms encoded by separated by separate genes.According to their different characteristics of electrical physiology and pharmacology and molecular structure are divided into three families,namely Cavl,Cav2 and Cav3.Voltage-gated L-type calcium channels(LTCCs)form the Cavl channel family,which comprises the isoforms Cav1.1,Cav1.2,Cav1.3 and Cav1.4.Although expression of Cav1.1 and Cav1.4 is mainly restricted to skeletal muscle and the retina,Cav1.2 is widely expessed in many tissues,including neurons and other electrically excitable tissues(heart,smooth muscle),sensory(inner ear,retina)and endocrine(pancreatic beta,adrenal chromaffin)cells.People initially think that Cavl.2(a 1C)L-type Ca2+channel represents the main Ca2+channel isoform in the heart while Cavl.3(?1D)L-type Ca2+ channel is known to be expressed mainly in neurons and neuroendocrine cells.But later the scientists have reported that Cav1.3 is also expressed in atrial myocytes.Moreover,Cav1.3 is preferentially expressed in atrium,sino-atrial node and the atrioventricular node compared to ventricular myocytes.Indeed,the importance of Cavl.3 isoform is underscored by the findings that Cavl.3 null mutant mice(Cavl.3-/-)show significant alteration in atrial excitability and atrial fibrillation,as well as sinoatrial and atrioventricular nodes dysfunction.L type calcium channels(LTCCs)are composed by ?1,?,?2,? subunits.Because pharmacology and gating properties of the channel are largely determined by alpha 1 subunit,there are a lot of researches focused on ?1 subunit.In recent years,the scientists are particularly pay attention to the long carboxyl terminus of al subunit.more and more experiments reported that the C terminus of Cav1.1 and Cav1.2 is proteolytically cleaved.In skeletal muscle cells,the distal C terminus of Cav1.1 is noncovalently associated with the truncated channel.In neurons and cardiac myocytes,the C terminus of Cavl.2 which is predicted to be a 75 kDa protein can translocate into nucleus and function as a transcription factor.Due to the isoform of Cavl.2 andCav1.3 channels share approximately 75%sequence homology.It hints whether Cav1.3 C-terminus is also truncated from the full length protein.Recently professor Lu reported for the first time that the C terminus of Cav1.3 can translocate into the nucleus and act as a transcriptional regulator for MLC2 expression.MLC2 protein physically interacts with SK2 channels and facilitates the membrane localization of SK2 channels.However the function effect and pathophysiological significance of C-terminal nuclear transport remain to be further research.So our lab developed an antibody to an unique epitome(2025-2140Aa)in the C-terminus(C-term Ab)of Cav1.3 to explore the function effect and pathophysiological significance of C-terminal nuclear transport.Western blotting of HEK293 cells expressing Cav1.3 lysates using the C-term Ab showed a 250KDa band(full length Cav1.3)and a 75KDa band(C-terminal fragment,endogenous Cav1.3 C-terminus is predicted to be a 75 kDa protein).In contrast,an antibody recognizing an epitope(841-910Aa)in the II-III cytoplasmic loop of Cavl.3(N-term Ab)detects a 250kDa band(full-length Cavl.3)and a 175kDa band(C-terminally truncated Cav1.3).Western bloting of total,cytoplasmic and nuclear homogenates from mice atrial using the C-term Ab showed a 75kDa band in all three components,whereas using the N-term Ab showed a 250kDa band in the total and cytoplasmic homogenates.Immunofluorescence of HEK293 cells expressing Cav1.3,HL-1 cells and atrial myocytes using the C-term Ab also showed the nuclear staining.By exogenous transfection experiments further confirmed that 1901-2141 amino acids of Cavl.3 channel was necessary for its nuclear localization.At the same time,we found the nuclear translocation of C-terminus of Cavl.3 would inhibit the transcription and expression of Cavl.3 Ca2+ channel.The data from immunofluorescence,RT-PCR and western blot showed the nuclear localization of the C terminus of Cav1.3 was regulated by chalges in[Ca2+]i.Decreasing the free extracellular Ca2+ did not affect the nuclear localization of Cav1.3-ct-eGFP-C1.conversely,increase the activity of Ca2+ channels which can enhance intracellular Ca2+ resulted in a significant decrease in the nuclear fluorescence.In the same way,the nuclear localization of the C terminus was age-dependent.Across the life span the nuclear localization of the C terminus of Cav1.3 increased steadily and significantly with age.TAC surgery and ISO-induced fresh isolated neonatal rat atrial myocytes were used to explore pathophysiological significance of C-terminal nuclear transport.Under transverse aortic constriction(TAC)induced-cardiac hypertrophic condition,the expression of Cav1.3 C-terminus was significantly increased compared with the sham group.While at the cellular level,neonatal atrial myocytes which were stimulated by ISO showed the same results.Taken together,our data demonstrated that the C-terminus of Cav1.3 L-type Ca2+channel can be truncated and translocate into the nucleus and the nuclear localization of the C terminus was age-dependent and was regulated by[Ca2+]i and myocardial hypertrophy.At the same time,the nuclear translocation of C-terminus of Cavl.3 had auto-inhibitory function.Future studies will evaluate C-terminus of Cav1.3 contributions to cellular hypertrophy and the regulation of C-terminus of Cav1.3 nuclear-cytosol shuttling. |
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