MODES OF SINGLE NA CHANNEL GATING DURING LATE CURRENTS

后期电流期间的单 NA 通道选通模式

• 批准号: 3158249
• 负责人: JOSEPH B PATLAK
• 金额: $ 15.77万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3158249
• 关键词: Anura; acid base balance; calcium channel; cell osmotic pressure; cytoplasm; electrical potential; electrolyte balance; electrophysiology; membrane channels; membrane permeability; membrane potentials; oxidation; phosphorylation; sodium channel; striated muscles; temperature; voltage /patch clamp; voltage gated channel

The long-term objective of this continuing project is to characterize the function of the Na+ channels in skeletal muscle and other tissues. During the next five years, effort will be dedicated to: 1. Measure the rate and mechanism of kinetic change during bursting Na+ currents. Previous work has led to the hypothesis that the kinetics of a Na+ channel can occasionally change to new values, which are derived from a broadly distributed range of possibilities for each rate. The hypothesis will be tested by measuring the distribution of opening and closing rate constants in DPI 201-106 induced bursting behavior on mouse skeletal muscle Na+ channels. Further observations will determine the rate at which burst kinetics can change for a single channel, the temperature dependence of this process, and the mechanism of the change. 2. Test whether fast-inactivating Na+ currents are due to homogeneous or to changeable channels. The null hypothesis that fast Na+ currents are due to identical, unchanging channels will be tested by measuring the average behavior of individual channels under conditions of low temperature. The mean current of each channel in a multi-channel patch will be determined by subtracting currents before and after illumination with UV light, which irreversibly destroys a portion (0, 1, or 2 channels) of the total channel population. Other statistical checks, such as "stability plots" of channel open times, will also be used to try to test further this hypothesis. 3. Measure alterations in Na+ channel subconductance frequency and lifetime. The molecular origins of sublevel events in channels are unknown. Several new techniques now permit a quantitative measurement of the amplitude and lifetime distributions of these events. Experiments are proposed to utilize these new techniques to measure the effects of temperature, intracellular and extracellular pH, permeations, and patch excision on the expression of subconductance levels. The data will be used to refine hypotheses for the mechanisms of these events. 4. Examine the role of the cytoplasm in Na+ channel function. Na+ channels in mouse skeletal muscle are sensitive to alterations in their cytoplasmic environment. The rate and extent of "run-down" and the shift of the inactivation curve will be used as assays of various artificial cytoplasmic solutions. The effects of pH, ionic conditions, temperature, osmotic pressure, oxidation potential, and phosphorylation will be assayed for direct effects on the alteration of channel properties upon patch excision. If ineffective, crude extracts of muscle cytoplasm will be tested for their activity. Further isolation could identify such substances. 5. Compare the properties of Na+ and Ca++ channels. A number of functional homologies exist between these two structurally homologous channels, including mode-like gating behavior, steepness of voltage sensitivity, subconductance level amplitude and lifetime. These common functions might have roots in the shared structural features of the channels. Experiments are planned to compare the extent of kinetic changeability in the Ca++ channel during BAY-K induced bursting. Further experiments will quantitate the influence of membrane potential, cytoplasmic pH, and temperature on subconductance levels of the Ca++ channel. All of these experiments are designed to test or to derive specific, detailed hypotheses about the function of Na+ and Ca++ channels. The results will be important for our understanding of channel function in a variety of cellular environments, and for modeling the relationship between these channel's primary structure and their functions.

这一持续项目的长期目标是， Na+通道在骨骼肌和其他组织中的功能。 期间 今后五年将致力于： 1. 测定钠离子猝发过程中动力学变化的速率和机理 水流 以前的工作已经导致了这样的假设，即， Na+通道偶尔会改变为新的值，这些值来自于 每个速率的可能性的广泛分布范围。 的假设 将通过测量开盘率和收盘率的分布来检验 DPI 201-106诱导小鼠骨骼肌爆发行为的常数 Na+通道。 进一步的观察将确定爆发的速度 对于单个通道，动力学可以改变，这种动力学的温度依赖性 过程，以及变化的机制。 2. 测试快速失活Na+电流是由于同质还是 可变频道 快速Na+电流是由于 通过测量平均值来测试相同的、不变的通道 在低温条件下单个通道的行为。 的 多通道贴片中每个通道的平均电流将由下式确定： 在用UV光照射之前和之后减去电流， 不可逆地破坏总通道的一部分（0、1或2个通道 人口 其他统计检查，如通道的“稳定性图” 开放时间，也将被用来试图进一步测试这一假设。 3. 测量Na+通道亚电导频率的变化， 辈子 通道中次级事件的分子起源尚不清楚。 现在有几种新的技术可以定量测量 这些事件的振幅和寿命分布。 实验 建议利用这些新技术来衡量 温度、细胞内和细胞外pH、渗透性和斑块 切除亚电导水平的表达。 数据使用 以完善这些事件的机制的假设。 4. 检查细胞质在Na+通道功能中的作用。 Na+通道 在小鼠骨骼肌中， 环境 “衰败”的速度和程度以及 灭活曲线将用作各种人工细胞质 解决方案 pH值、离子条件、温度、渗透压 压力、氧化电位和磷酸化将被测定， 直接影响斑片切除后通道特性的改变。 如果无效，将测试肌肉细胞质的粗提物， 活动 进一步的分离可以确定这些物质。 5. 比较Na+和Ca++通道的特性。 多个功能 在这两个结构上同源的通道之间存在同源性， 包括类模式选通行为，电压灵敏度的陡度， 亚电导水平振幅和寿命。 这些常见功能可能 根源在于通道的共同结构特征。 实验 计划比较Ca++中的动力学可变性程度， 通道在BAY-K诱导的爆裂。 进一步的实验将定量 膜电位、细胞质pH值和温度对 亚电导水平的Ca++通道。 所有这些实验 旨在测试或推导出关于 Na+和Ca++通道的功能。 结果将对我们的 了解各种蜂窝环境中的信道功能，以及 为了模拟这些通道的主要结构和 它们的功能。