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。上皮单分子层作为小溶质分子（包括无机离子和药物）在体隔室之间运动的屏障。离子通过严格调控的离子特异性转运体和通道穿过上皮的顶端和基底膜；在紧密连接处可以发生有限的扩散。任何通道的功能丧失都会导致电解质和体液失衡，从而导致发病率和死亡率。多年来，本实验室一直致力于开发合成多肽，使其形成具有不同程度阴离子传导和选择性的孔。这些合成的肽序列与脊髓甘氨酸受体（GlyR） a1亚基的成孔跨膜段（M2）有远亲关系。我们假设理想的通道形成序列应该：1)作为单体具有较高的水溶性；2)无可检测到的抗原性；3)在低溶液浓度下与生物膜结合并迅速分解成生物膜；4)在膜上进行超分子组装，形成具有高离子通量的孔；5)表现出生理上相关的阴离子选择性。除了最终目标阴离子选择性外，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Cl- (PCl)相对于Na+或K+的电选择性。用我们的新孔处理过的细胞对它们的耐受性很好，其净阴离子通量由反离子运输的自然调节控制。在Aims中，我们建议调节孔暴露和肽肽界面残基，这些残基决定了孔环境，通道几何形状和通道大小，以产生一系列孔，这些孔定义了相对于单价价的Cl-的热选择性范围，并保留了高阴离子渗透率。将引入特定的氨基酸替代物来调节孔隙衬里残基的氢键能力或静电性能。其他替代品将改变长度和刚性。这些修饰的效果将通过电生理和结构（CD和NMR）研究结合计算机建模来监测。

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. 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.