MODEL SYNTHETIC CHANNEL ASSEMBLIES

上皮对小分子的移动起到了障碍作用，包括细胞和药物在身体隔间的运动。 离子通过严格调控的离子特异性转运体和通道穿过上皮根尖和基底侧膜。 然而，外源供应的药物，特别是亲水性或带电化合物，不会被运送到任何 上皮下可察觉到的范围。我们的实验室已经开发出两种不同类别的多肽，它们可以促进 选择性离子转运或调节阻止药物进入的上皮屏障。推导出了多肽序列 来自脊髓甘氨酸受体(M2GlyR)的成孔M2跨膜(TM)片段。肽插入物 并组装成显示不同的、依赖于序列的生理反应的跨膜结构。 它们要么选择性地增加氯的转运，要么形成非选择性的通道，瞬时地促进 较大的亲水性化合物向基底侧隔室的细胞旁运动。以前的序列有 已被建议作为一种可能的治疗干预措施，用于治疗囊性纤维化等经络疾病。后者是 目前正在调查是否促进了亲水性抗生素通过角膜的运输。虽然很多都是 对这些序列在其本地上下文中的信道属性的了解很少关于 组装膜结构(S)的合成肽。多维溶液核磁共振已被用于 研究了多肽单体在十二烷基硫酸钠胶束中的结构，并尝试了螺旋束的模拟。我们 建议确定在模型中组装形成活性结构的跨膜片段的数量 使用化学和生物物理方法的膜和细胞。最近的研究表明，不同的 膜脂成分可以改变这些多肽的二级结构。研究将使用模型进行 具有预示肽的螺旋结构的组合物的膜。我们假设不同的大小 膜组件可以在不同的脂质环境中稳定下来。关于分段数量的知识 在生理条件下组装将极大地帮助未来的建模工作。其他实验包括 计划中将检查苏氨酸环在17位的位置和氢键强度 形成阴离子选择性通道孔的序列。在这个位置上的各种氨基酸替换将评估 在选择性和高选择性方面优化合成通道所需的几何形状和氢键强度 吞吐量。这些结果将被纳入未来的渠道设计，最终目标是定义治疗 结构。

Epithelia act as barriers to the movement of small molecules, includingions and drugs, between body compartments. Ions cross the epithelial apical and basolateral membranes via tightly regulated ion-specific transporters and channels. However, exogenously supplied drugs, particularly hydrophilic or charged compounds, are not transported to any appreciable extent across the epithelium. Our labooratory has developed two different classes of peptides that promote selective ion transport or modulate the epithelial barrier that precludes drug access. The peptide sequences are derived from the pore-forming M2 transmembrane (TM) segment of the spinal cord glycine receptor (M2GlyR). Peptides insert and assemble to form transmembrane structures that display distinct, sequence dependent, physiological responses. They either selectively increase chloride transport or form non-selective channels that transiently facilitate the paracellular movement of larger hydrophilic compounds to the basolateral compartment. The former sequences have been proposed as a possible therapeutic intervention for channelopathies such as cystic fibrosis (CF).The latter are currently under investigation for facilitated transport of hydrophilic antibiotics across the cornea. While much is understood about the channel properties of these sequences in their native context little is understood about the assembled membrane structure(s) of the synthetic peptides. Multi-dimensional solution NMR has been used to investigate the structure of peptide monomers in SDS-micelles and modeling of helical bundles has been attempted. We propose to determine the number of transmembrane segments that assemble to form the active structures in model membranes and cells using both chemical and biophysical methods. Recent studies have indicated that different membrane lipid compositions can alter the seconday structure of these peptides. Studies will be conducted using model membranes with compositions that preseve the helical structure of the peptides. We hypothesize that different sized membrane assemblies could be stabilized in different lipid environments. Knowledge of the the number of segments that assemble under physiological conditions will greatly assist future modeling efforts. Additional experiments are planned that will examine the positioning and hydrogen bonding strength of a ring of threonines at position 17 in the sequences that form anion selective channel pores. Various amino acid substitutions in this position will assess the geometry and hydrogen bonding strengths required to optimize a synthetic channel with regard to selectivity and high throughput. These results will be incorporated into future channel designs with the ultimate goal of defining therapeutic structures.