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

描述（由申请人提供）：通过纳米级分子器件实现光对天然电压门控钠通道（NaVs）的控制，可以证明作为光感受器退行性疾病的视觉修复治疗以及基础神经科学研究是有价值的。 金纳米颗粒（Au NPs）在收集和局部耗散（作为热）可见光能量方面的有效性已得到充分证实。 再加上 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细胞外表面具有高特异性亲和力的小蛋白质的可用性，提高了通过与蛋白质配体亲和试剂缀合而定位于NaV胞外区的Au NP可以介导选择性光热NaV活化的可能性。 拟议的探索性项目旨在测试这项技术的可行性。 目标1将为项目提供直接基础。 在这里，我们将确定游离Au NP的作用（即，未与蛋白质配体缀合）对爪蟾卵母细胞、脂质双层和大鼠视网膜的分离的单个神经节细胞中的光诱导跨膜电流的影响。 Au NP对光引发的膜电流的贡献将通过比较在接近与远离纳米颗粒的吸收峰（~530 nm）的波长处由类似强度的光刺激引发的膜电流来确定。 目标1实验还将建立该光热方法所需的脉冲刺激光的合适定时/持续时间以及Au NP的水溶性所需的实验条件。 在目标2中，我们将Au NP共价偶联至蛋白质配体Ts 1，其对NaV结构域II胞外区具有高亲和力。 我们假设Au NP与膜的紧密接近以及通过将Au NP连接到该NaV配体而提供的NaV靶将是使NaV活化所需的光水平最小化的关键。 我们将构建Au NP-Ts 1缀合物，其中Ts 1的特定氨基酸残基被修饰以作为Au NP的附着位点;我们将通过电生理记录（实验类似于目标1）和通过荧光团标记的缀合物分析细胞结合来确定缀合物的最佳结构。 该项目的成功将确立所研究的基于Au NP的缀合物作为NaV光调节剂的可行性。 具体而言，它将鼓励进一步开发这种技术作为纳米假体，以使晚期感光细胞变性患者的神经节细胞中的NAV介导的光信号传导成为可能。 领导这项研究的将是大卫R。 Pepperberg，PhD. 眼科和视觉科学，伊利诺伊大学芝加哥分校（UIC））;弗朗西斯科Bezanilla博士和Stephen B. H. 肯特博士（部门） 化学、生物化学和分子生物学，芝加哥大学）;和Karol S. Bruzik，PhD. 药物化学和生药学，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).