INTERACTIONS BETWEEN H+.1 AND CA2+1 IN MYXICOLA AXON

MYXICOLA AXON 中 H .1 和 CA2 1 之间的相互作用

• 批准号: 3399172
• 负责人: RONALD F ABERCROMBIE
• 金额: $ 14.2万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3399172
• 关键词: acidity /alkalinity; adenosine triphosphate; axon; axoplasm; caffeine; calcium binding protein; calcium metabolism; calcium transporting ATPase; cell cycle; cell differentiation; cell membrane; hormone regulation /control mechanism; inositol; inositol phosphates; ion transport; mitochondria; muscle contraction; neurofilament; neurofilament proteins; organelles; physical chemical interaction; secretion

Long-term objectives are to gain a better understanding of the regulation of both intracellular free calcium (Ca2+) i, and free protons, (H+), and the interdependence between these two ion concentrations. These ions are now known or suspected to regulate or influence numberous cell functions, including muscle contraction, neuro-secretion, ion permeability, intercellular electrical coupling, cell division and differentiation, and metabolism. Calcuium regulatory mechanisms will be examined at four loci: (a) endoplasmic reticulum, (b) plasma membrane, (c) intracellular Ca-binding proteins, and (d) mitochondria. Calcium uptake and release from the endoplasmic reticulum of neural cytoplasm iwll be characterized using a new Ca electrode that detects free Ca in the nM concentration range. ATP-dependent Ca uptake will be studied in the presence of mitochondrial inhibitors as a function of ATP, pH, and (cA2+). The effects of caffeine and inositol phosphates on Ca release from organelles close to the plasma membrane will be compared to organelles near the center of the axon. Intracellular Ca-binding proteins will be studied with special emphasis on neurofilaments. The 150,000- 160,000 molecular-weight neurofilament proteins will be isolated and the binding of Ca to these proteins will be quantitiatively examined in solutions of different pH, ionic strength, and temperature. Using limited, specific proteolytic cleavage and autoradiographic analysis, the portions of the neurofilament molecule containg the calcium-binding site will be examined. The influence of pH on mitochondria will be studied. Using tetraphenol phosphonium (TPP+), a TPP+-microelectrode, and samples of axoplasm containing mitochondria, the effect of pH on the mitochondrial membrane potential will be examined to see if this explains a previus observation concerning pH and Ca uptake. Using pH microelectrodes and isolated samples of cytoplasm, the "effective" hydrogen ion diffusion coefficient in neural cytoplasm will be determined, and the importance of fixed and mobile pH buffers in dissipating intracellular pH gradients will be evaluated.

长期目标是更好地了解 细胞内游离钙 (Ca2+) i 和游离钙的调节 质子 (H+) 以及这两个离子之间的相互依赖性 浓度。 现在已知或怀疑这些离子 调节或影响许多细胞功能，包括肌肉 收缩、神经分泌、离子渗透性、细胞间质 电耦合、细胞分裂和分化，以及 代谢。 钙调节机制将在 四个位点：(a) 内质网，(b) 质膜，(c) 细胞内 Ca 结合蛋白，和 (d) 线粒体。 钙 神经细胞内质网的摄取和释放 细胞质将使用新的 Ca 电极进行表征 检测 nM 浓度范围内的游离 Ca。 ATP依赖性 将在线粒体存在的情况下研究 Ca 的吸收 抑制剂作为 ATP、pH 和 (cA2+) 的函数。 的影响 咖啡因和磷酸肌醇对细胞器 Ca 释放的影响 靠近质膜的将与细胞器进行比较 靠近轴突的中心。 细胞内 Ca 结合蛋白将 特别强调神经丝的研究。 150,000- 将分离出 160,000 个分子量的神经丝蛋白 Ca 与这些蛋白质的结合将被定量 在不同 pH 值、离子强度和 温度。 使用有限的、特异性的蛋白水解切割和 放射自显影分析，神经丝的部分 将检查含有钙结合位点的分子。 这 将研究 pH 对线粒体的影响。 使用 四酚鏻 (TPP+)、TPP+-微电极，以及 含有线粒体的轴浆样品，pH 对 将检查线粒体膜电位以确定是否 这解释了之前关于 pH 值和 Ca 吸收的观察结果。 使用 pH 微电极和分离的细胞质样品， 神经细胞质中“有效”氢离子扩散系数 将确定，以及固定和移动 pH 值的重要性 将评估消散细胞内 pH 梯度的缓冲液。