Nanoparticle-based Photo-activator of Voltage-gated Sodium Channels

描述(由申请人提供)：通过纳米级分子设备实现对天然电压门控钠通道(NAV)的光控制可能被证明是一种有价值的视觉修复疗法，对于光感受器退行性疾病以及基础神经科学研究都是有价值的。金纳米颗粒(Au NPs)在收集和局部消散(作为热量)可见光能量方面的有效性已经得到了很好的证实。这一点，连同 最近的证据表明，突然施加光热能量可以促进NAV介导的动作电位的产生，以及对NAV胞外面具有高特异性亲和力的小蛋白的可获得性，这增加了通过与蛋白质配体亲和剂结合定位于NAV外膜的Au NP可以介导选择性光热NAV激活的可能性。拟开展的探索性项目旨在验证该技术的可行性。AIM 1将为该项目提供直接的基础。在这里，我们将测定游离金纳米粒(即未连接到蛋白质配体)对非洲爪哇卵母细胞、脂双层和分离的大鼠视网膜单个神经节细胞光致跨膜电流的作用。Au-NP对光激发膜电流的贡献将通过比较在接近和远离纳米颗粒吸收峰(~530 nm)的波长处由相似强度的光刺激引起的膜电流来确定。目标1的实验还将建立这种光热方法所需的脉冲刺激光的适当时间/持续时间所需的实验条件，以及金纳米粒子的水溶液增溶所需的条件。在目标2中，我们将Au NP共价偶联到与NAV结构域II胞外区具有高亲和力的蛋白质配体Ts1上。我们假设，通过将Au NP连接到NAV配体上而提供的Au NP与膜及其NAV靶标的紧密接近将是最小化NAV激活所需的光照水平的关键。我们将构建Au NP-Ts1结合物，其中Ts1的特定氨基酸残基被修饰作为Au NP的附着位置；我们将通过电生理记录(实验类似于Aim 1的实验)和通过荧光团标记的结合物与细胞结合的分析来确定结合物的最佳结构。该项目的成功将确立所研究的Au-NP基共轭化合物作为NAV光调节器的可行性。具体地说，它将鼓励这项技术作为纳米假体的进一步发展，使NAV介导的光信号能够在晚期光感受器变性患者的神经节细胞中发挥作用。领导这项研究的将是David R.Pepperberg博士(系)。大学眼科学与视觉科学系。伊利诺伊州芝加哥分校(UIC)；Francisco Bezanilla博士和Stephen B.H.Kent博士(系大学化学、生物化学和分子生物学系。和卡罗尔·S·布鲁齐克博士(系药物化学和生药学专业(UIC)。

DESCRIPTION (provided by applicant): Achieving the control, by light, of native voltage-gated sodium channels (NaVs) by a nanoscale molecular device could prove valuable as a vision repair therapy for photoreceptor degenerative diseases as well as for fundamental neuroscience research. The effectiveness of gold nanoparticles (Au NPs) in collecting and locally dissipating (as heat) the energy of visible light is well established. This, together with recent evidence that a sudden application of photothermal energy can promote NaV-mediated action potential generation, and the availability of small proteins possessing high specific affiniy for the NaV extracellular face, raise the possibility that a Au NP localized at the NaV ectomain by conjugation with the protein ligand affinity reagent can mediate selective photothermal NaV activation. The proposed exploratory project is aimed at testing the feasibility of this technolog. Aim 1 will provide the project's immediate foundation. Here we will determine the action of free Au NPs (i.e., not conjugated to protein ligand) on photo-induced transmembrane current in Xenopus oocytes, lipid bilayers, and isolated single ganglion cells of rat retina. The contributio of the Au NP to the light-elicited membrane current will be determined by comparing membrane currents elicited by photo-stimuli of similar intensity at wavelengths near to vs. distant from th nanoparticle's absorbance peak (~530 nm). The Aim 1 experiments will also establish experimental conditions needed for suitable timing/duration of the pulsed stimulating light required for this photothermal approach, and for aqueous solubilization of the Au NPs. In Aim 2 we will covalently couple the Au NP to the protein ligand Ts1, which has high affinity for the NaV domain II extracellular region. We hypothesize that close proximity of the Au NP to the membrane and its NaV target provided by attaching the Au NP to this NaV ligand will be key to minimizing the light level needed for NaV activation. We will construct Au NP-Ts1 conjugates in which a specific amino acid residue of the Ts1 is modified to serve as attachment site for the Au NP; we will determine the optimal structure of the conjugate by electrophysiological recording (experiments similar to those of Aim 1) and by analysis of cell-binding by fluorophore-tagged conjugate. Success in the project will establish feasibility of the investigated Au NP-based conjugate as a NaV photo-regulator. Specifically, it will encourage further development of this technology as a nano-prosthetic to enable NaV-mediated photo-signaling in ganglion cells of patients with advanced-stage photoreceptor degeneration. Leading the research will be David R. Pepperberg, PhD (Dept. of Ophthalmology and Visual Sciences, Univ. of Illinois at Chicago (UIC)); Francisco Bezanilla, PhD, and Stephen B. H. Kent, PhD (Depts. of Chemistry, Biochemistry and Molecular Biology, Univ. of Chicago); and Karol S. Bruzik, PhD (Dept. of Medicinal Chemistry and Pharmacognosy, UIC).