Model Synthetic Channel Assemblies

描述(由申请人提供)：该项目的目标是设计、开发和实现离子选择性、合成通道形成多肽的用途。这种技术的主要应用将是治疗丧失功能的离子通道疾病，如囊性纤维化。上皮单层作为小溶质分子运动的障碍，包括无机离子和药物，在身体隔室之间。离子通过严格调控的离子特定转运体和通道的组合跨越上皮根尖和基底侧膜；可以发生跨紧密连接的有限扩散。任何通道的功能丧失都会导致电解质和液体失衡，从而导致发病率和死亡率。多年来，该实验室一直在开发形成具有不同程度阴离子传导和选择性的孔的合成肽。这些合成肽序列与脊髓甘氨酸受体(GlyR)A1亚基的成孔跨膜片段(M2)有较远的亲缘关系。我们假设理想的通道形成顺序应该是：1)作为单体具有高的水溶解性；2)没有可检测到的抗原性；3)在低溶液浓度下与生物膜结合，然后快速分配到生物膜中；4)在膜中进行超分子组装以形成具有高离子通量的孔；以及5)表现出生理上相关的阴离子选择性。除了阴离子选择性这一最终目标外，所有这些性质都已实现。我们现在正在探索不同的方法来提高氯离子(PCL)相对于Na或K的渗透选择性。用我们的新孔处理的细胞对它们有很好的耐受性，净阴离子流量由反离子运输的自然调节控制。在这一目标中，我们建议通过调节孔暴露和多肽界面残基来决定孔环境、通道几何形状和通道大小，以生成一系列孔，这些孔定义了相对于单价阳离子的氯离子选择性范围，并保持了高的阴离子渗透率。将引入特定的氨基酸取代物来调节孔衬残留物的氢键能力或静电性能。其他替代物将改变孔的长度和刚性。这些修饰的效果将通过电生理和结构(CD&核磁共振)研究与计算机建模相结合的方式进行监测。 与公共健康相关：本提案中描述的生物基多肽材料来自相同的生物来源，并在膜和活细胞中进行自我组装，形成显示独特生物特性的通道孔。生产具有高阴离子选择性的新多肽将在治疗囊性纤维化等经络疾病的经络替代疗法领域具有翻译前景。

DESCRIPTION (provided by applicant): The goal of this project is to design, develop and implement the use of ion-selective, synthetic channel-forming peptides. A primary application for such technology will be the treatment of loss-of-function ion channel diseases such as cystic fibrosis. Epithelial monolayer's act as barriers to the movement of small solute molecules, including both inorganic ions and drugs, between body compartments. Ions cross epithelial apical and basolateral membranes via combination of tightly regulated ion-specific transporters and channels; limited diffusion across tight junctions can occur. Loss-of-function of any channel leads to electrolyte and fluid imbalances that result in morbidity and mortality. For many years, this laboratory has been developing synthetic peptides that form pores with varying degrees of anion conduction and selectivity. These synthetic peptide sequences are distantly related to the pore-forming transmembrane segment (M2) of the spinal cord glycine receptor (GlyR) a1-subunit. We hypothesized that the ideal channel-forming sequence should:1) have high aqueous solubility as a monomer; 2)have no detectable antigenicity;3) bind to and then partition rapidly into biological membranes at low solution concentrations; 4) undergo supramolecular assembly in the membrane to form pores with high ion throughput; and 5) show physiologically relevant anion selectivity. All of these properties have been achieved with the exception of the final goal, anion selectivity. We are now exploring distinct approaches to raise the permselectivity for Cl- (PCl) relative to either Na+ or K+. Cells treated with our de novopores tolerate them well with the net anion flux controlled by the natural regulation of counter-ion transport. In the Aims we propose to modulate pore-exposed and peptide-peptide interfacial residues that dictate the pore environment, channel geometry, and channel size to generate a series of pores that define a range of perm selectivity's for Cl- relative to monovalentcations, with retention of high anion permeation rates. Specific amino acid replacements will be introduced to modulate pore lining residues with regard to hydrogen bonding capabilities or electrostatic properties. Other replacements will alterpore length and rigidity. The effects of these modifications will be monitored by a combination of electrophysiological and structural (CD & NMR) studies in conjunction with computer modeling. PUBLIC HEALTH RELEVANCE: The bio-based peptide materials described in this proposal are derived from the same biological source and undergo self assemble in both membranes and living cells to form channel pores that display unique biological properties. Producing new peptides with high anion selectivity would have translational promise in the area of channel- replacement therapy for channelopathies such as cystic fibrosis.