Sickle Red Cell K+ Transporter Genetics in S. cerevisiae

描述（由申请人提供）： 镰状细胞病患者致密红细胞的脱水状态 加速脱氧诱导的HbS聚合， 这些致密细胞的优先镰状化。KCC K-Cl活性 协同转运蛋白和IK 1 K（ca）通道几乎介导了所有这些过程 脱水这些K外排途径的药理学阻断抑制了 镰状细胞病小鼠模型的体外细胞脱水，以及 患者，可能带来临床效益。了解离子的机制 K-Cl协同转运蛋白的转运和K离子通过IK 1 K（ca）的渗透 通道是开发安全的药理学阻断剂的关键， 效力和特异性。结构-功能关系研究，使用 KCC K-Cl协同转运蛋白和 K（ca）IK1。虽然这些研究成果丰硕，但进展相对缓慢。 在该R21申请中提出的实验将利用S. 酿酒酵母缺乏高亲和力K离子吸收，以执行 K-Cl协同转运蛋白的广泛、无偏倚的结构-功能研究， IK 1钾通道。KCC 1的甄选系统已经过验证。一 IK 1的筛选系统正在开发中。拟议的实验将 加速产生和选择表现出功能丧失的突变体， 功能获得表型。此外，该系统应允许开发 的高通量筛选抑制剂钾离子吸收这些途径。 这些目标将通过追求以下具体目标来实现：1） 验证、表征和标准化trk 1 delta/trk 2 delta的生长挽救 S.在非允许生长条件下通过表达cDNA 编码KCC K-Cl共转运蛋白。2)定义KCC 1的结构区域 对离子转运、阳离子和阴离子选择性、抑制剂 敏感性，最近发现的显性阴性表型，和急性 调控这将通过饱和诱变确定的 KCC K-Cl共转运蛋白的亚区，并筛选突变体的能力， 以拯救trk 1delta/trk 2delta S的生长。酿酒的。选择突变体将是 在非洲爪蟾卵母细胞和哺乳动物细胞中进一步研究。3)（暂定目标）： 时间允许的话，改进、验证和标准化 trk1delta/trk2delta S.酿酒酵母通过在非允许条件下表达 编码哺乳动物IK 1 K（ca）通道的cDNA。

DESCRIPTION (provided by applicant): The dehydrated state of the densest erythrocytes in sickle cell disease accelerates deoxygenation-induced polymerization of HbS and consequent, preferential sickling of these dense cells. Activity of the KCC K-Cl cotransporters and the IK1 K(ca) channel mediates nearly all of this dehydration. Pharmacological blockade of these K efflux pathways inhibits cellular dehydration in vitro in mouse models of sickle cell disease, and in patients, may bring clinical benefit. Understanding the mechanisms of ion transport by K-Cl cotransporters and of K ion permeation through the IK1 K(ca) channel is critical to development of safe pharmacological blockers of higher potency and specificity. Structure-function relationship studies using site-directed mutagenesis are underway for KCC K-Cl cotransporters and for the K(ca) IK1. Though productive, these studies are relatively slow. Experiments proposed in this R21 application will utilize strains of S. cerevisiae deficient in high-affinity K ion uptake in order to perform extensive, unbiased structure-function studies of K-Cl cotransporters and the IK1 K(ca) channel. A selection system has already been validated for KCC1. A selection system for IK1 is under development. The proposed experiments will accelerate production and selection of mutants exhibiting loss-of-function and gain-of-function phenotypes. In addition, the system should permit development of a high throughput screen for inhibitors of K ion uptake by these pathways. These goals will be achieved through pursuit of the following Specific Aims: 1) To validate, characterize, and standardize growth rescue of trk1delta/trk2delta S. cerevisiae in nonpermissive growth conditions by expression of cDNAs encoding KCC K-Cl cotransporters. 2) To define structural regions of KCC1 important to ion transport, to cation and anion selectivity, to inhibitor sensitivity, to a recently discovered dominant negative phenotype, and to acute regulation. This will be achieved by saturation mutagenesis of defined subregions of KCC K-Cl cotransporters, and screening mutants for their ability to rescue growth of trk1delta/trk2delta S. cerevisiae. Select mutants will be further studied in Xenopus oocytes and mammalian cells. 3) (Provisional Aim): Time permitting, to improve, validate, and standardize growth rescue of trk1delta/trk2delta S. cerevisiae by expression in nonpermissive conditions of cDNA encoding the mammalian IK1 K(ca) channel.