Mechanisms and Implications of Nanoelectroporation in Living Cells

描述（由申请人提供）： 脉冲功率技术的最新进展最终导致能够向生物组织和细胞样本等低阻抗负载提供高压纳秒持续时间电脉冲（nsEP）的新设备的工程设计。我们发现，nsEP可以作为一种独特的工具来修改活细胞中质膜的生理学和改变细胞功能。nsEP最显著的效果是打开纳米或亚纳米直径的长寿命、电压和电流敏感、整流、离子选择性、不对称孔（“纳米孔”）。这些复杂的行为通常只适用于复杂的设备，如蛋白质离子通道，并将纳米孔与常规（较大）电孔区分开来。一旦诱导，纳米孔在开放和准开放（电沉默）状态之间振荡数分钟，然后逐渐重新密封或突然分解成更大的孔，立即失去纳米孔特异性。纳米孔似乎充分配备了传统上归因于经典离子通道的某些功能;我们假设，在生理和病理条件下可能形成纳米孔，以补充离子通道作为额外的离子转运途径。 纳米孔以前曾在合成箔和平面脂质双层中报道过，但我们的工作是第一个记录活细胞中纳米孔的形成及其特性的工作。此外，我们已经建立了抑制和促进反应的内源性离子通道后，nsEP治疗，以及细胞生理学变化，由于渗透压失衡。本研究申请旨在探索活细胞中的纳米电穿孔现象，并评估这种新技术在研究和医学中的潜在应用。拟议的研究包括四个具体目标，旨在表征和改善纳米电穿孔程序;揭示允许纳米孔执行其复杂活动的机制;并阐明nsEP对质膜屏障功能和离子运输影响的机制：具体目标1：探索纳米孔形成对电脉冲物理参数的依赖性，优化纳米电穿孔程序和纳米孔检测技术。具体目标二：分析纳米孔的结构和功能特性（孔寿命，开口直径，离子选择性，电压和电流灵敏度），并揭示这些特性的机制。具体目标3：探索纳米电穿孔对经典电压门控离子通道功能的影响，以及对神经和肌肉细胞中的兴奋和动作电位传播的影响。具体目标4：探索纳米孔对质膜透水性和细胞体积控制的潜在机制。 公共卫生相关性： 本研究将集中在纳米电穿孔的新现象，这是形成稳定的，电压和电流敏感的，纳米直径的膜孔暴露于纳秒持续时间，高压电脉冲（nsEP）的活细胞。我们将专注于物理化学和生理机制，基础和确定质膜nanoelectroporation和nsEP对内源性离子通道和水代谢的影响。预期的结果将促进新的医疗和研究应用的发展，使用nsEP对细胞功能进行故意修改，特别是在神经和肌肉组织中。

DESCRIPTION (provided by applicant): Recent advances in pulsed power technology culminated in engineering of new devices capable of delivering high-voltage, nanosecond-duration electric pulses (nsEP) to low-impedance loads such as biological tissues and cell samples. We found that nsEP can be employed as a unique tool to modify physiology of the plasma membrane in living cells and alter cell function. The most remarkable effect of nsEP was opening of long-lived, voltage- and current-sensitive, rectifying, ion-selective, asymmetrical pores of nano- or sub- nanometer diameter ("nanopores"). These complex behaviors are normally expected only from sophisticated devices like protein ion channels and distinguish nanopores from conventional (larger) electropores. Once induced, nanopores oscillated between open and quasi-open (electrically silent) states for minutes, followed by either gradual resealing or abrupt breakdown into larger pores, with immediate loss of nanopore-specific properties. Nanopores appeared adequately equipped for certain functions that are traditionally ascribed to classic ion channels; we hypothesize that nanopores may form under physiological and pathological conditions to supplement ion channels as an additional ion transport pathway. Nanopores have previously been reported in synthetic foils and planar lipid bilayers, but our work is the first one to document the formation of nanopores and their properties in living cells. Furthermore, we have established both inhibitory and facilitatory responses of endogenous ion channels after nsEP treatment, as well as cytophysiological changes due to the osmotic imbalance. This Research Application is designed to explore the phenomenon of nanoelectroporation in living cells and to evaluate potential applications of this novel technique in research and medicine. The proposed study consists of four Specific Aims intended to characterize and improve the nanoelectroporation procedure; to reveal mechanisms that allow nanopores to perform their complex activities; and to elucidate mechanisms that underlie nsEP effects on plasma membrane barrier function and ion traffic: Specific Aim 1: Explore the dependence of nanopore formation on the physical parameters of electric pulses, optimize nanoelectroporation procedures and nanopore detection techniques. Specific Aim 2: Analyze structural and functional properties of nanopores (pore lifetime, opening diameter, ion selectivity, voltage and current sensitivity) and reveal mechanisms responsible for these properties. Specific Aim 3: Explore the impact of nanoelectroporation on the function of classic voltage-gated ion channels, and on the excitation and action potential propagation in nerve and muscle cells. Specific Aim 4: Explore mechanisms underlying nanoporation effect on plasma membrane water permeability and cell volume control. PUBLIC HEALTH RELEVANCE: This study will be focused on the new phenomenon of nanoelectroporation, which is the formation of stable, voltage- and current-sensitive, nanometer-diameter membrane pores in living cells exposed to nanosecond- duration, high-voltage electric pulses (nsEP). We will focus on physico-chemical and physiological mechanisms that underlie and determine plasma membrane nanoelectroporation and nsEP effects on endogenous ion channels and water metabolism. Anticipated results will promote the development of new medical and research applications using nsEP for deliberate modification of cell functions, particularly in nerve and muscle tissues.