摘要
目的 检测由缝隙连接蛋白 (connexin ,Cx) 4 3和Cx4 5组成的多种异型缝隙连接通道 (het eromultimericgapjunctionchannels ,HGJC)和磷酸化对缝隙连接 (gapjunction ,GJ)的调变作用。方法 将转染了编码为Cx4 3或Cx4 5的DNA后的Hela细胞放置在一起共同培养组成双侧和单侧异型GJ通道。显微注射若丹明 12 3(rhodamine 12 3,Rh)检测经 2 0 0nmol/L十四 (烷 )酰佛波醇乙酸酯 (12 0 tetrade canoylphorbol 13 acetae ,TPA)处理前后 ,在紫外光显示下由Cx4 3和Cx4 5所组成的不同GJ通道对荧光染料的偶联率 (couplingratio)。结果 在不同的GJ中 ,同型GJ通道Cx4 3(homotypicCx4 3,HoCx4 3)偶联率最高。从Cx4 5侧注入荧光染料的单侧异型GJ通道 4 5 (mono heteromericCx4 5 Cx4 3/4 5 ,MH4 5 )偶联率较之从Cx4 3/4 5侧注入荧光染料的MH4 5、双侧异型GJ通道Cx4 3/4 5 (bi heteromericCx4 3/4 5 ,BH4 3/4 5 )及同型GJ通道Cx4 5 (homotypicCx4 5 ,HoCx4 5 )等的偶联率是最低的。根据HoCx4 3或HoCx4 5通道的偶联率对各型通道偶联率进行标准化处理。BH4 3/4 5和MH4 3通道的偶联率均较HoCx4 3降低。对MH4 5通道来说 ,从Cx4 3/4 5侧注射的通道偶联率大于从Cx4 5侧注射的偶联率。TPA处理后HoCx4 3的偶联率降低 。
Objective The aim of this study is to determine the docking effects of the heteromultimeric combination of Cx43 and Cx45 and the phosphorylation on the permeability properties of their channels.Methods The Hela cells were transfected DNA encoding Cx43 or Cx45 colocalized to form bi-heteromeric and mono-heteromeric channels.The dye permeability of various combinations of Cx43 and Cx45 under epifluorescent illumination before and after 200 nmol/L 12-0-tetradecanoylphorbol-13-acetae(TPA)treatment was determined by microinjection with rhodamine123.Results Homotypic Cx43(HoCx 43)channels were the best coupled in the different combination of connexins.The permeability of mono-heteromeric Cx45-Cx43/45 channels(MH45)injected from the side of Cx45 is the least compared with the channels of MH45 injected from the side of Cx43/45,bi-heteromeric Cx43/45(BH43/45) and homotypic Cx45(HoCx45).We normalized the changes in coupling ratio for heteromultimeric channels according to the change in HoCx43 or HoCx45 channels.The coupling ratios for both channels of BH43/45 and mono-heteromeric Cx43-Cx43/45(MH43) were decreased compared with HoCx43 channels.For MH45 channel,the coupling ratios of channels injected from in the side of cells expressing Cx43/Cx45 were larger than those from the side of cells expressing Cx45.After TPA treatment,the coupling ratio of HoCx43 was reduced and this reduction in coupling was accentuated for channels with bi-heteromeric or mono-heteromeric connexons.Conclusion Co-expressed Cx43 and Cx45 can form bi-heteromenic and mono-heteromeric channels,which result in low gap junctional communication and insensitivity to phosphorylation.The permeability of mono-heteromeric channels depending on the direction of dye injection might relate the anatomical basic for the reentry in arrhythmia.
出处
《中华心律失常学杂志》
2003年第4期229-233,共5页
Chinese Journal of Cardiac Arrhythmias
