A Real-Time Device for Constructing Virtual Ion Channels in Living Cells

0085177 whiteintroduction。神经和肌肉细胞中的电活动是由离子通道群产生的，这些离子通道响应跨膜电压和/或关键化学物质浓度的变化而“打开”（打开或关闭）。在研究电活动背后的生物物理过程时，科学家和工程师依赖于两种基本的记录配置。在第一种记录配置中，通常被称为电流箝位，研究人员控制净跨膜电流（即通过细胞膜的电流）的量，并测量跨膜电压。在第二个记录配置中，称为电压箝位，研究人员使用电反馈电路来控制跨膜电压并测量跨膜电流。电流夹紧对于描述给定神经或肌肉细胞产生的电活动模式是有用的；电压箝位对于研究特定电活动模式下的生物物理机制是有用的。最近，第三种非常有用的记录配置已经开发出来，其中跨膜电流和跨膜电压都不是受控变量。相反，研究人员使用一种复杂的记录系统来实时模拟与给定数量的虚拟离子通道相关的电导率。这种记录模式，称为动态箝位，允许研究人员阻止原生离子通道，并用虚拟模拟物取代它们，其特性可以精确控制。动态夹紧和其他基于实时计算的协议实现了全新的实验类别，例如，应用的刺激可以模拟系统中（被阻塞的）动态组件的行为，从而用于明确确定被模拟组件对整体行为的影响。动态夹紧和其他实时实验技术作为重要的研究工具显示出巨大的前景。例如，动态夹紧可以用于研究计算机设计的药物制剂的电效应，甚至在药物在实验室开发之前。然而，到目前为止，这项技术的影响受到三个相互关联的困难的影响。首先，其设计的技术复杂性超出了大多数终端用户的技能，而且还没有人提供一个灵活、强大的交钥匙系统。其次，现有的动态箝位系统没有考虑到电压门控离子通道看似随机（即概率）的性质。增加这种能力将使研究人员能够研究一系列全新的令人兴奋的问题，这些问题目前还无法解决。第三，现有的动态箝位系统无法解释测量或假设的离子通道空间分布。具体的目标。本提案的具体目标是：(1)完成随机动态钳（SDC）系统的构建，该系统可用于研究活细胞中噪声虚拟电压和配体门控离子通道的作用；(2)创建基于web的支持系统，帮助最终用户采用和使用SDC系统进行动态夹紧和其他实时实验应用；(3)开发表示远离细胞体记录位点的虚拟离子通道的方法；(4)使用新的SDC系统来测试关于突触源噪声和电压门控离子通道噪声在限制神经元可靠性方面的相对重要性的特定假设。在课堂教学和研究项目的背景下，该项目将对研究生和本科生的教育产生影响。具体目标1和3的创新将扩展动态夹紧方法，以考虑随机和空间分布的通道。研究人员的支持（目标2）将是开发交钥匙系统的关键一步。该系统将很容易适应实时计算技术在许多生物医学工程中的应用。该装置的现场测试（目标4）将推动神经生物学领域向更定量、更面向信息的方向发展。特别是，他们将推进神经元可靠性的生物物理基础研究，这在理解神经系统使用的编码策略中起着重要作用。如果没有实时计算技术，这些进步实际上是无法实现的。

0085177WhiteIntroduction. Electrical activity in nerve and muscle cells is generated by populations of ion channels that "gate" (open or close) in response to changes in transmembrane voltage and/or the concentrations of crucial chemicals. In studies of the biophysical processes underlying electrical activity, scientists and engineers rely upon two basic recording configurations. In the first recording configuration, commonly referred to as current clamping, the researcher controls the amount of net transmembrane current (i.e., current across the cell membrane) and measures transmembrane voltage. In the second recording configuration, called voltage clamping, the researcher uses an electrical feedback circuit to control transmembrane voltage and measures transmembrane current. Current clamping is useful for characterizing the patterns of electrical activity generated by a given nerve or muscle cell; voltage clamping is useful for studying the biophysical mechanisms underlying a particular pattern of electrical activity.More recently, a third very useful recording configuration has been developed, in which neither transmembrane current nor transmembrane voltage is the controlled variable. Instead, the researcher uses a sophisticated recording system to mimic in real time the electrical conductance associated with a given population of virtual ion channels. This recording mode, called dynamic clamping, allows the researcher to block native ion channels, and replace them with virtual analogs, the properties of which can be controlled precisely. Dynamic clamping and other real-time-computing-based protocols enable entirely new classes of experiments, in which (for example) the applied stimulus can mimic the behavior of a (blocked) dynamic component of the system, and thus be used to determine unequivocally the effects of the mimicked component of overall behavior.Dynamic clamping and other real-time experimental techniques show enormous promise as important research tools. Dynamic clamping could be used, for example, to study the electrical effects of computer-designed pharmaceutical agents even before the agents are developed in the laboratory. So far, however, the impact of this technique has been educed by three interrelated difficulties. First, the technical complexities of its design are beyond the skills of most end-users, and no one has yet provided a flexible, powerful, turn-key system. Second, existing dynamic clamp systems do not account for the seemingly stochastic (i.e., probabilistic) nature of voltage-gated ion channels. Adding this capability would allow researchers to attack entirely new sets of exciting problems that are as yet unapproachable. Third, existing dynamic clamp systems cannot account for measured or assumed spatial distributions of ion channels.Specific Aims. The specific aims of this proposal are (1) to complete construction of a stochastic dynamic clamp (SDC) system that can be used to study the actions of noisy virtual voltage- and ligand-gated ion channels in living cells; (2) to create a web-based system of support to help end-users adopt and use the SDC system for dynamic clamping and other real-time experimental applications; (3) to develop methods for representing virtual ion channels that are remote from the recording site in the cell body, and (4) to use the new SDC system to test specific hypotheses regarding the relative importance of noise from synaptic sources and noise from voltage-gated ion channels in limiting neuronal reliability.The project will have educational impact at both the graduate and undergraduate levels, in the context of the classroom and research projects. Innovations from Specific Aims 1 and 3 will extend the dynamic clamp method to account for stochastic and spatially distributed channels. The investigators' support (Aim 2) will be a crucial step in developing a turn-key system. The system will be easily adaptable to apply techniques of real-time computation in many biomedical engineering applications. Field tests of the device (aim 4) will push the field of neurobiology in a more quantitative, information-oriented direction. In particular, they will advance the study of the biophysical underpinnings of neuronal reliability, which play a fundamental role in the understanding of coding strategies used by the nervous system. Such advances are not practically achievable without real-time computing technology.