Developing a reconstructing system that compensates for a missing link in the regulation of L-type Ca channels in cardiac myocytes

通道活性通过cAMP依赖性激酶的磷酸化而增强：磷酸化的最大激活可以使Ca^2+电流的峰值幅度增加到对照电流的5-6倍。由于在外源表达系统中转导L型钙通道基因不能通过cAMP依赖性磷酸化重建L型钙通道的调控，阻碍了阐明L型钙通道调控的分子机制的发展。我们假设，异源表达系统缺乏一个调节因子，在控制活动的L-型钙通道从心脏起源，应该存在于天然心脏细胞。因此，我们的策略是将L型钙通道基因导入心肌细胞本身，以完全重建L型钙通道的调节。为了实现这些目的，我们有两个问题需要克服，1）区分人工引入的Ca通道的功能与最初表达的Ca通道的功能， 关于我们 心肌细胞; 2）获得含L型钙通道基因的功能性病毒载体。对于第一种，我们测试了突变的L型Ca通道的离子选择性，例如EEKA或DEKA型，其离子选择性过滤器的部分从野生型的EEEE改变。结果表明，这两个突变体对Na^+和Cs^+的渗透性都提高了10倍。DEKA型的Ba^2+渗透性消失，而EEKA型的Ba^2+对Cs^+的渗透性高56倍。DEKA型获得性抗La^<3+>阻滞。利用这种Na^+选择性钙通道突变体，我们将能够在河豚毒素存在的情况下选择性地记录心肌细胞中转导的L型钙通道的电流。第二种方法是将CaV1.2基因插入缺失E1/E3区的腺病毒载体中。不幸的是，由于插入片段的大小（8 kb），我们无法在HEK 293细胞中扩增病毒颗粒。Ganeasn AN等的最近报道指出，他们通过构建除E1/E3区外还缺乏纤维基因的腺病毒载体，可以成功地在心肌细胞中转染Ca_V1.2基因（BBRC 749- 754，2005）。我们希望引进他们的技术，以进一步发展我们的钙通道研究。少

The channel activity is augmented by phsophorylation through cAMP-dependent kinase : maximum activation of phosphorylation can increase peak amplitude of Ca^<2+> currents up to 5-6 folds of the control current. The fact that inability to reconstruct the regulation of L-type Ca channels through cAMP-dependent phosphorylation by transducing L-type Ca channel genes in heterologous expression system hampers the development of elucidating molecular mechanisms in the regulation of L-type Ca channels. We assume that heterologous expression systems lack a regulatory factor in controlling the activity of L-type Ca channels from cardiac origin which should exist in native cardiac cells. Thus, our strategy to fully reconstruct the regulation of L-type Ca channel is transducing L-type Ca channel genes into cardiac myocyte itself. In order to achieve these objects, we have two problems to overcome, 1) differentiating function of artificially introduced Ca channels from those originally expressed in … More cardiac myocytes, 2) obtaining functional virus vectors containing L-type Ca channel gene. For the first one, we tested the ion selectivity of mutated L-type Ca channels, such as EEKA or DEKA type of which the portion of the ion selective filter was changed from EEEE of wild type. The results indicated that both mutants have 10 times higher permeability of Na^+ against Cs^+. Ba^<2+> permeability was abolished in DEKA type, while EEKA type has 56 times higher permeability of Ba^<2+> against Cs^+. DEKA type acquired resistance to La^<3+> block. With using this Na^+-selective Ca channel mutants, we would be able to selectively record currents from transduced L-type Ca channels in cardiac myocytes in the presence of tetrodotoxin. For the second one, we could insert Ca_V 1.2 gene in the adenovirus vector that lacks E1/E3 region. Unfortunately, we were unable to amplify virus particles in HEK293 cells due to the size of insert (8kb). A recent report of Ganeasn AN et al.indicated that they could successfully transduce Ca_V1.2 gene in cardiac myocytes by constructing adenovirus vector that lacks fiber gene in addition to E1/E3 region (BBRC 749-754,2005). We are hoping to introduce their technology for further development of our Ca channel study. Less