FUNCTIONAL ANALYSIS OF CFTR

本研究的目的是了解结构/功能关系 在CFTR中，其缺陷导致囊性纤维化（CF）的蛋白质。 做 这个我们就研究这个复杂分子的疏水区， 因为CFTR的这一部分与Cl最密切相关， CF通道功能缺失。 将提出两个广泛的问题。 一 一组实验将确定对Cl通道重要的元素 活性通过植入半胱氨酸残基作为受体， 半胱氨酸特异性试剂。 这些变体的Cl通道功能 将在非洲爪蟾卵母细胞中进行评估。 在保留了 功能，我们将更仔细地研究那些渠道活动 被亲水性半胱氨酸修饰剂不可逆地阻断。 对这些候选突变体库的分析应确定 CFTR C1通道周围的跨膜片段和 特定的残留物排列在通道孔中。 为了补充这些研究，平行实验将使用诱变 以鉴定CFTR中可能作为作为盐桥相互作用的残基。 的 这种工作的起源与关于作用的相互矛盾的报告有关， 第一个跨膜片段中的两个带电残基（E92和K95） 的CFTR。 我们相信，如果这两个相互作用， 彼此以一种直接的方式。 为了验证离子 对在这个和其他领域的功能相关，我们将增加 用阳离子残基取代阴离子配偶体的电荷密度 (and反之亦然），并检查CFTR变体的表型。 卵母细胞 为了确定最初的残基是否与一个 另一个，然后我们将取代它们，一次一个，用中性氨基 acids. 在后面的这些试验中，盐桥的直接证据将 如果只有在带相反电荷或中性时才发现功能， 残留物存在。 总之，这些实验应该大大增加我们对 膜结构域的CFTR，从而更好地掌握如何CFTR Cl通道的功能和调节。

The goal of this study is to understand structure/function relationships in CFTR, the protein whose defect causes cystic fibrosis (CF). To do this, we will study the hydrophobic sector of this complex molecule, because this sector of CFTR is most closely associated with the Cl channel function absent in CF. Two broad questions will be asked. One set of experiments will identify elements important to Cl channel activity by implanting cysteine residues to serve as receptors for cysteine-specific reagents. The Cl channel function of such variants will be evaluated in Xenopus oocytes. Among variants that retain function, we will examine more closely those in which channel activity is irreversibly blocked by hydrophilic cysteine-modifying agents. Analysis of a pool of such candidate mutants should identify the subset of transmembrane segments surrounding the CFTR Cl channel and the specific residues lining the channel pore. To complement these studies, parallel experiments will use mutagenesis to identify residues that may interact as salt bridges in CFTR. The genesis of such work relates to conflicting reports regarding the role of two charged residues (E92 and K95) in the first transmembrane segment of CFTR. We believe the present data are resolved if these two interact with one another in a direct way. To test the hypothesis that an ion pair is functionally relevant in this and other areas, we will increase charge density by replacing the anionic partner with a cationic residue (and vice versa) and examine the phenotype of the variant CFTRs in oocytes. To determine whether the original residues interact with one another, we will then replace them, one at a time, with neutral amino acids. In these latter trials, direct evidence for a salt bridge will arise if function is found only when oppositely charged or neutral residues are present. Together, such experiments should add significantly to our understanding of the membrane domain of CFTR, leading to a better grasp of how the CFTR Cl channel functions and is regulated.