BIOPHYSICAL AND MOLECULAR MECHANISM OF POTASSIUM CHANNEL

钾通道的生物物理和分子机制

• 批准号: 2184314
• 负责人: LUO LU
• 金额: $ 9.6万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184314
• 关键词: Xenopus; Xenopus oocyte; alternatives to animals in research; cell differentiation; cell growth regulation; clone cells; complementary DNA; gene expression; molecular biology; molecular cloning; nucleic acid hybridization; phosphorylation; polymerase chain reaction; potassium channel; protein kinase; protein structure function; site directed mutagenesis; transfection; voltage /patch clamp; voltage gated channel

Voltage-gated K+ channels have been found in tissues other than excitable cells, but their functions in non-excitable cells, including hematopoietic cells is poorly understood. The P.I. has discovered a shaker-like channel in human myeloblastic (ML-1 cells. Preliminary data using whole-cell patch clamp technique demonstrate that this channel is voltage-gated and can be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) which is known to induce differentiation in this cell line. After treatment with a differentiation-inducing dose of TPA, the kinetics of the current inactivation become radically altered within 6 hours. This channel alteration is prior to the appearance of differentiating phenotype. Based on these preliminary studies, the P.I. has heterologously the K+ channel by injection of Xenopus oocytes with mRNA from ML-1 cells. The P.I. now proposes to further characterize this K+ channel and to explore its role in cell proliferation and differentiation. This will be accomplished through the following specific aims: (i) A further understanding of the channel characteristics and regulation will be obtained using single channel patch clamp at different configurations; (ii) A further characterization of the expressed K+ channel in oocytes will be obtained by using the two-microelectrode voltage clamp and patch clamp techniques; and (iii) The full length cDNA encoding the primary nucleotide sequence of the channel gene will be obtained. This will be accomplished using both plaque hybridization to a cDNA library from ML-1 cells and expression in Xenopus oocytes. Polymerase chain reaction (PCR) will also be used to prime and to amplify probes for the hybridization experiments. Positive clones of the K+ channel will be sequenced and expressed in oocytes and mammalian cells. The long-term goal of the project is to study, at the molecular level, functioning of the K+ channel in ML-1 cells in order to better understand its role in cell proliferation and differentiation. This may have significance, not only for understanding differentiation in normal cells, but also for understanding how to induce this process to suppress malignant growth in leukemic cells, such as ML-1.

电压门控的K+通道已在可激发的组织中发现 细胞，但它们的功能在不可消除的细胞中，包括造血细胞 细胞知之甚少。 P.I.已经发现了一个像摇床一样的频道 在人粒细胞（ML-1细胞。使用全细胞斑块的初步数据 夹具技术表明该通道是电压门控的，可以是 被12-O-四烷酰基-13-乙酸盐（TPA）激活，已知 诱导该细胞系中的分化。 用 TPA的分化诱导剂量，电流的动力学 失活在6小时内发生了根本改变。 这个频道 改变是在出现区分表型之前的。 基于 关于这些初步研究，P.I。通过 用ML-1细胞注入爪蟾卵母细胞。 P.I.现在建议进一步表征此K+通道和 探索其在细胞增殖和分化中的作用。 这将是 通过以下特定目标完成：（i）进一步 了解渠道特征和调节将是 在不同的配置下使用单通道贴片夹获得； （ii） 卵母细胞中表达的K+通道的进一步表征将是 通过使用两微电极电压夹和斑块夹获得 技术； （iii）编码主要核苷酸的全长cDNA 将获得通道基因的序列。 这将完成 使用ML-1细胞和 爪蟾卵母细胞的表达。 聚合酶链反应（PCR）也将 用于启动和扩增杂交实验的探针。 K+通道的正克隆将被测序并在 卵母细胞和哺乳动物细胞。 该项目的长期目标是在分子水平上研究 ML-1单元中K+通道的功能以更好地理解 它在细胞增殖和分化中的作用。 这可能有 意义，不仅用于理解正常细胞的分化，还 而且还为了了解如何诱导这一过程抑制恶性肿瘤 白血病细胞（例如ML-1）的生长。