Control of Calcium Channel Expression by Cellular ras Genes

电压门控离子个体发生的机制 肌肉中的通道未知。 要确定是否表达 电压门控通道依赖于有丝分裂原的撤回和生长 阻滞，如对于肌肉特异性基因的诱导通常如此 产品，BC 3 H1肌肉细胞系被用作有利的 模型系统 分化的BC 3 H1肌细胞形成功能性钙 和钠通道，通过膜片钳技术发现， 对应于骨骼肌横小管中的那些。 Ca和 在有丝分裂原撤除后约4天首次检测到Na通道。 由于细胞ras基因已被发现在肿瘤的发生发展中起作用， 转导的生长因子信号，BC 3 H1细胞已被修改 与激活的ras表达载体连接。 用缬氨酸-12转染 c-Ha-ras载体抑制功能性钙通道的形成， 钠通道超过4周。 相反，钾离子通道 既不受有丝分裂培养基的影响，也不受转染ras的影响 基因 因此，ras基因选择性地阻断了离子的表达， 通道，其诱导取决于有丝分裂原撤回。 这些 研究将扩展到阐明控制Ca 频道号 首先，分化的BC 3 H1表达的Ca通道 将彻底表征肌肉细胞。 二、增长因素 在BC 3 H1细胞中抑制肌肉特异性基因表达的药物将 在将直接效应与次级效应分离的条件下使用 细胞增殖的影响。 第三，Ca通道表达将是 在BC 3 H1肌细胞中研究，这些细胞修饰有影响 肌生成 这些研究将集中在钙通道的调控上 细胞ras基因的表达，并将分析产生的影响 通过可逆诱导的ras表达载体，通过一系列分级的 c-Ha-ras基因密码子12处的错义突变，通过显微注射 ras蛋白，并通过阻断抗p21 ras的抗体。 这项研究代表了第一个重大努力， 阐明调节特异性表达的遗传机制， 肌肉膜中的电压依赖性离子通道。 拟议 实验应该阐明细胞Ha-ras基因在细胞凋亡中的作用。 在肌肉分化过程中控制钙通道表达。

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.