Control of Calcium Channel Expression by Cellular ras Genes

肌肉中电压门控离子通道的个体发生机制尚不清楚。为了确定电压门控通道的表达是否依赖于有丝分裂原退出和生长停止，就像肌肉特异性基因产物的诱导一样，BC3H1肌肉细胞系被用作一个有利的模型系统。分化的BC3H1肌细胞形成功能性钙和钠通道，通过膜片钳技术发现这些通道与骨骼肌横小管中的通道相对应。停丝原后约4天首次检测到Ca和Na通道。由于已经发现细胞ras基因在生长因子信号的转导中起作用，因此用激活的ras表达载体修饰BC3H1细胞。用valine-12 c-Ha-ras载体转染后，可抑制功能性Ca通道和Na通道的形成4周以上。相比之下，钾通道既不受有丝分裂培养基的影响，也不受转染ras基因的影响。因此，ras基因选择性地阻断离子通道的表达，离子通道的诱导取决于有丝分裂原的退出。这些研究将扩展到阐明控制Ca通道数的机制。首先，将对分化的BC3H1肌细胞表达的Ca通道进行彻底的表征。其次，抑制BC3H1细胞中肌肉特异性基因表达的生长因子将在分离细胞增殖的直接效应和次要效应的条件下使用。第三，我们将在影响肌肉发生的基因修饰的BC3H1肌细胞中研究Ca通道的表达。这些研究将集中于细胞ras基因对Ca通道表达的控制，并将分析可逆诱导的ras表达载体、c-Ha-ras基因密码子12的一系列分级错义突变、微注射ras蛋白和阻断p21 ras抗体所产生的影响。这项研究是阐明调节肌肉膜中特定电压依赖性离子通道表达的遗传机制的第一个主要努力之一。提出的实验应阐明细胞Ha-ras基因在肌肉分化过程中钙通道表达控制中的作用。

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. To determine whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle specific gene products, the BC3H1 muscle cell line is being used as an advantageous model system. Differentiated BC3H1 myocytes form functional calcium and sodium channels which, by patch clamp techniques were found to correspond to those in transverse tubules of skeletal muscle. Ca and Na channels were first detected about 4 days after mitogen withdrawal. Since cellular ras genes have been found to have a role in the transduction of growth factor signals, BC3H1 cells have been modified with an activated ras expression vector. Transfection with a valine-12 c-Ha-ras vector suppressed the formation of functional Ca channels and Na channels for more than 4 weeks. In contrast, potassium channels were affected neither by mitogenic medium nor by the transfected ras gene. Thus, the ras gene selectively blocked the expression of ion channels whose induction was contingent upon mitogen withdrawal. These studies will be extended to elucidate the mechanisms that control Ca channel number. First, Ca channels expressed by differentiated BC3H1 muscle cells will be thoroughly characterized. Second, growth factors that inhibit muscle-specific gene expression in BC3H1 cells will be used under conditions that uncouple direct effects from secondary effects of cell proliferation. Third, Ca channel expression will be investigated in BC3H1 myocytes modified with genes that affect myogenesis. These studies will focus on the control of Ca channel expression by cellular ras genes and will analyze the effects produced by a reversibly inducible ras expression vector, by a graded series of missense mutations at codon 12 of the c-Ha-ras gene, by microinjected ras proteins, and by blocking antibodies against p21 ras. This research represents one of the first major efforts to elucidate the genetic mechanisms regulating the expression of specific voltage-dependent ion channels in muscle membranes. The proposed experiments should clarify the role of the cellular Ha-ras gene in the control of calcium channel expression during muscle differentiation.