Structure-function relationships of the chimeric TRPM7 channel-kinase

嵌合TRPM7通道激酶的结构-功能关系

• 批准号: 8291012
• 负责人: CARSTEN SCHMITZ
• 金额: $ 27.26万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8291012
• 关键词: 14-3-3 Proteins; Adenosine Diphosphate Ribose; Affect; Antibodies; B-Lymphocytes; Biochemistry; Biological; Biological Process; Biophysics; Biosensor; C-terminal; Cell Line; Cell Proliferation; Cell model; Cell physiology; Cell surface; Cells; Cellular Membrane; Cessation of life; Complement; Complex; Development; Elongation Factor; Ensure; Environment; Event; Family; Genetic; Genetic Models; Genomics; Goals; Health; Homeostasis; Homologous Gene; Human; Hydrolase; Intervention; Ion Channel; Ions; Light; Link; Mediating; Membrane; Metabolic; Modeling; Molds; Molecular; Molecular Biology; Molecular Target; Movement; Mutation; N-terminal; Nutrient; Nutritional; Pathway interactions; Patients; Permeability; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Positioning Attribute; Process; Proteins; Regulation; Relative (related person); Role; Serine; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Subgroup; Supplementation; TRP channel; Threonine; Translations; Work; aqueous; base; cell assembly; cell growth; cyclic-nucleotide gated ion channels; design; extracellular; immune function; insight; member; monomer; mouse model; mutant; novel; novel strategies; physical model; public health relevance; research study; response; sulfated glycoprotein 2; trafficking; voltage

DESCRIPTION (provided by applicant): The carefully orchestrated movement of ions across cellular membranes is crucial to virtually every biological process. Ion channels play thereby a central role by allowing the regulated flow of relevant ions through their aqueous pore. In the past few decades, progress made in Molecular Biology and Genomics has revealed the unsuspected variety of ion channels expressed in our membranes. The family of TRP channels with more than two dozens of members in humans that function as biosensors and signal integrators is a good illustration of this development. It includes the only known examples of ionic pores fused to enzymatic regions, so-called "chanzymes". TRPM7 and its close relative TRPM6 both harbor a kinase region at their C-terminus that is capable of phosphorylating protein substrates on Serine/Threonine residues. TRPM7 is widely distributed and has been shown to be an essential and irreplaceable molecule in several genetic models, including a B-lymphocyte cell line called DT40. TRPM7-deficiency in this context results in cell growth arrest and death, unless the extracellular media is supplemented with Mg2+, suggesting a role for TRPM7 in Mg2+- homeostasis regulation. Despite these insights into TRPM7's physiological function, the significance of its kinase region remains unclear. Understanding the role of this unique domain holds the promise to shed some light on novel mechanisms of cellular adjustments to a changing ionic environment. The main goal of this proposal is to investigate the relationship between the channel and kinase portions of TRPM7 by utilizing a combination of biochemistry, genetics and biophysics approaches, allowing for a detailed function-structure relationship study. We plan to determine how altering the ionic selectivity of TRPM7 channels through pore mutations might affect its kinase activity. Furthermore the functional importance of the covalent link between kinase and channel will be assessed by studying liberated domains and through the introduction of linker structures. Finally, we also designed experiments allowing us to define molecular determinants of channel assembly and trafficking to the cell surface. PUBLIC HEALTH RELEVANCE: Ion channels form highly sophisticated pores in biological membranes allowing for the controlled movement of ions between biologically relevant compartments. Most are at the surface of the cell and therefore easily accessible for pharmacological intervention; many are validated molecular targets. Understanding the functional versatility of ion channels is therefore key to providing us with novel potential strategies of interfering with biological processes relevant to human health. The structural uniqueness of the TRPM7 protein as an ion channel/kinase fusion appears to be a reflection of its complex involvement in regulating cellular functions in response to nutrient availability. Understanding the molecular determinants of the interplay between kinase and ion channel function will therefore provide us with novel opportunities to influence processes such as cell proliferation or immune function.

描述（由申请人提供）：精心策划的离子穿过细胞膜的运动对于几乎每个生物过程都至关重要。因此，离子通道通过允许相关离子通过其水孔进行调节流动而发挥核心作用。在过去的几十年里，分子生物学和基因组学取得的进展揭示了我们细胞膜中表达的令人意想不到的各种离子通道。 TRP 通道家族在人类中拥有超过两打成员，充当生物传感器和信号积分器，这是这一发展的一个很好的例证。它包括唯一已知的与酶区域融合的离子孔的例子，即所谓的“chanzymes”。 TRPM7 及其近亲 TRPM6 均在其 C 末端含有一个激酶区域，能够磷酸化丝氨酸/苏氨酸残基上的蛋白质底物。 TRPM7 分布广泛，在多种遗传模型中已被证明是一种重要且不可替代的分子，包括称为 DT40 的 B 淋巴细胞细胞系。在这种情况下，TRPM7 缺陷会导致细胞生长停滞和死亡，除非细胞外培养基补充 Mg2+，这表明 TRPM7 在 Mg2+- 稳态调节中发挥作用。尽管对 TRPM7 的生理功能有这些见解，但其激酶区域的重要性仍不清楚。了解这个独特结构域的作用有望揭示细胞适应不断变化的离子环境的新机制。 该提案的主要目标是通过结合生物化学、遗传学和生物物理学方法来研究 TRPM7 的通道和激酶部分之间的关​​系，从而进行详细的功能结构关系研究。我们计划确定通过孔突变改变 TRPM7 通道的离子选择性如何影响其激酶活性。此外，将通过研究释放的结构域和引入接头结构来评估激酶和通道之间共价连接的功能重要性。最后，我们还设计了实验，使我们能够定义通道组装和运输到细胞表面的分子决定因素。 公共健康相关性：离子通道在生物膜上形成高度复杂的孔，允许离子在生物相关区室之间受控移动。大多数位于细胞表面，因此很容易进行药物干预；许多是经过验证的分子靶标。因此，了解离子通道的功能多样性是为我们提供干扰与人类健康相关的生物过程的新的潜在策略的关键。 TRPM7 蛋白作为离子通道/激酶融合体的结构独特性似乎反映了其复杂地参与调节细胞功能以响应营养物质的可用性。因此，了解激酶和离子通道功能之间相互作用的分子决定因素将为我们提供影响细胞增殖或免疫功能等过程的新机会。