MODES OF SINGLE NA CHANNEL GATING DURING LATE CURRENTS

这一持续项目的长期目标是描述 骨骼肌和其他组织中钠离子通道的功能。在.期间 今后五年，我们将致力于： 1.测定Na~+猝发过程中的动力学变化速度和机理 洋流。以前的工作导致了一种假设，即一种 NA+通道有时会更改为新值，这些新值派生自 每种利率的可能性分布广泛。假说 将通过测量开闭口率的分布进行检验 DPI 201-106中的常数诱导小鼠骨骼肌爆发行为 NA+通道。进一步的观测将确定爆炸的速度 对于单个通道，动力学可以改变，这与温度有关 过程，以及变化的机制。 2.测试快速失活的Na+电流是由于同质性还是由于 多变的频道。认为快速Na+电流是由于 相同的、不变的通道将通过测量平均值进行测试 单个航道在低温条件下的行为。这个 多通道贴片中每个通道的平均电流将由下式确定 用紫外光照射前后减去电流， 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，如航道的“稳定图” 开放时间，也将被用来进一步检验这一假说。 3.测量Na+通道亚电导频率和 一辈子。通道中亚水平事件的分子起源尚不清楚。 现在有几种新技术可以定量测量 这些事件的振幅和寿命分布。实验是 建议利用这些新技术来衡量 温度、细胞内和细胞外的pH值、渗透率和斑块 对亚电导水平的表达进行了删除。将使用这些数据 以完善对这些事件机制的假设。 4.检测细胞质在Na+通道功能中的作用。Na+通道 在小鼠骨骼肌中对细胞质的变化很敏感 环境。“衰败”的速度和程度以及 灭活曲线可用于各种人工细胞质的测定 解决办法。PH、离子条件、温度、渗透压的影响 压力、氧化电位和磷酸化将被检测 在斑块切除时对通道属性改变的直接影响。 如果无效，肌肉细胞质的粗提物将被测试其 活动。进一步的分离可以鉴定出这种物质。 5.比较Na+和Ca++通道的特性。多个职能部门 在这两个结构上相同的通道之间存在同源性， 包括模式选通行为、电压灵敏度陡度、 子导电平、幅度和寿命。这些常见的功能可能 根植于渠道的共同结构特征。实验 计划比较钙离子的动力学变化程度 Bay-K诱发突发期间的通道。进一步的实验将量化 膜电位、细胞质pH和温度对细胞生长的影响 钙离子通道的亚电导水平。所有这些实验都是 被设计用来测试或推导出关于 Na+和Ca++通道功能。结果将对我们的 了解各种蜂窝环境中的通道功能，以及 为了对这些渠道的初级结构和 它们的功能。

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.