MODEL SYNTHETIC CHANNEL ASSEMBLIES

描述(申请人提供)：上皮细胞作为小分子移动的障碍，包括离子和药物，在身体隔间。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，外源性提供的药物，特别是亲水性或带电化合物，不会在上皮细胞中传输到任何明显的程度。我们的实验室已经开发出两种不同类别的多肽，它们促进选择性离子转运或调节阻止药物进入的上皮屏障。这些肽序列来自脊髓甘氨酸受体(M2GlyR)的成孔M2跨膜(TM)片段。多肽插入和组装形成跨膜结构，显示出不同的、依赖于序列的生理反应。它们或者选择性地增加氯的转运，或者形成非选择性的通道，瞬时地促进较大的亲水性化合物的细胞旁移动到基侧隔室。以前的序列已被提议作为一种可能的治疗干预措施，用于治疗囊性纤维化(CF)等经络病变。后者目前正在接受调查，因为它促进了亲水性抗生素通过角膜的运输。虽然人们对这些序列在天然环境中的通道性质了解很多，但对合成肽的组装膜结构(S)了解很少。用多维溶液核磁共振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。我们建议使用化学和生物物理方法来确定在模型膜和细胞中聚集形成活性结构的跨膜片段的数量。最近的研究表明，不同的膜脂组成可以改变这些多肽的二级结构。研究将使用具有预示肽螺旋结构的组合物的模型膜来进行。我们假设不同大小的膜组件可以在不同的脂质环境中稳定下来。了解在生理条件下组装的节段数量将极大地帮助未来的建模工作。还计划进行更多的实验，以检查形成阴离子选择性通道孔的序列中第17位苏氨酸环的定位和氢键强度。在这一位置上的各种氨基酸取代将评估在选择性和高通量方面优化合成通道所需的几何结构和氢键强度。这些结果将被纳入未来的渠道设计，最终目标是确定治疗结构。

DESCRIPTION (provided by applicant): Epithelia acts as barriers to the movement of small molecules, including ions 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 2 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 secondary 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.