BIOPHYSICAL AND MOLECULAR MECHANISM OF POTASSIUM CHANNEL

钾通道的生物物理和分子机制

基本信息

批准号：
2184315
负责人：
LUO LU
金额：
$ 10.35万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-01 至 1998-07-31
项目状态：
已结题

项目摘要

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.

在除兴奋性组织以外的组织中也发现了电压门控 K+ 通道细胞，但它们在非兴奋细胞中的功能，包括造血细胞对细胞知之甚少。 P.I.发现了一个类似摇床的通道在人成髓细胞（ML-1 细胞）中。使用全细胞贴片的初步数据钳位技术证明该通道是电压门控的并且可以由 12-O-十四烷酰佛波醇-13-乙酸酯 (TPA) 激活，已知诱导该细胞系的分化。经过治疗后 TPA的分化诱导剂量，电流的动力学失活在 6 小时内发生根本改变。这个频道改变发生在分化表型出现之前。基于根据这些初步研究，P.I.具有异源 K+ 通道向非洲爪蟾卵母细胞注射来自 ML-1 细胞的 mRNA。 P.I.现在建议进一步表征该 K+ 通道并探讨其在细胞增殖和分化中的作用。这将是通过以下具体目标来实现： (i) 进一步了解渠道特点和监管使用单通道膜片钳在不同配置下获得； (二) 卵母细胞中表达的 K+ 通道的进一步表征将是使用两微电极电压钳和膜片钳获得技术； (iii) 编码初级核苷酸的全长 cDNA 将获得通道基因的序列。这将实现使用噬菌斑杂交与来自 ML-1 细胞的 cDNA 文库和非洲爪蟾卵母细胞中的表达。聚合酶链反应（PCR）也将用于引发和扩增杂交实验的探针。 K+通道的阳性克隆将被测序并表达于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 细胞中 K+ 通道的功能，以便更好地理解它在细胞增殖和分化中的作用。这可能有意义，不仅对于理解正常细胞的分化，也为了了解如何诱导这一过程来抑制恶性白血病细胞（例如 ML-1）中的生长。