Model Synthetic Channel Assemblies

描述（由申请人提供）：本项目的目标是设计、开发和实施离子选择性合成通道形成肽的使用。这种技术的主要应用将是治疗功能丧失的离子通道疾病，如囊性纤维化。上皮细胞单层作为小溶质分子（包括无机离子和药物）在身体隔室之间移动的屏障。离子通过紧密调节的离子特异性转运蛋白和通道的组合穿过上皮顶膜和基底外侧膜;可以发生穿过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，从而导致发病和死亡。多年来，这个实验室一直在开发合成肽，这些肽形成具有不同程度阴离子传导和选择性的孔。这些合成肽序列与脊髓甘氨酸受体（GlyR）α 1-亚基的成孔跨膜区段（M2）远缘相关。我们假设理想的通道形成序列应该：1）作为单体具有高水溶性; 2）没有可检测的抗原性;3）在低溶液浓度下结合并然后快速分配到生物膜中; 4）在膜中经历超分子组装以形成具有高离子通量的孔;以及5）显示生理学相关的阴离子选择性。除了最终目标阴离子选择性之外，所有这些特性都已经实现。我们现在正在探索不同的方法来提高Cl-（PCl）相对于Na+或K+的选择透过性。用我们的de novopore处理的细胞能够很好地耐受它们，净阴离子通量由反离子转运的自然调节控制。在目的中，我们提出调节孔暴露和肽-肽界面残基，其决定孔环境、通道几何形状和通道尺寸，以产生一系列孔，其限定Cl-相对于单价阳离子的渗透选择性范围，保留高阴离子渗透速率。将引入特定的氨基酸替换以调节孔衬里残基的氢键结合能力或静电性质。其他替代品将改变孔的长度和刚度。这些修改的效果将通过电生理学和结构（CD & NMR）研究结合计算机建模进行监测。

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.