Sickle Red Cell K+ Transporter Genetics in S. cerevisiae

描述(由申请人提供)： 镰状细胞病中密度最大的红细胞的脱水状态 加速脱氧诱导的HBS和随后的聚合， 对这些致密细胞的优先镰刀。KCC K-Cl的活度 共转运体和IK1 K(Ca)通道几乎参与了所有这些过程 脱水。药物阻断这些K外流途径可抑制 镰状细胞病小鼠模型的体外细胞脱水，以及 患者，可能会给临床带来好处。了解离子的作用机制 K-Cl共转运体的转运和K离子在K_1K(Ca)中的渗透 通道是开发安全的高等药物阻滞剂的关键 效力和特异度。结构-功能关系研究使用 KCC K-Cl共转运体和KCC K-Cl共转运体的定点突变正在进行中 K(Ca)Ik1.尽管这些研究富有成效，但相对缓慢。 在本R21申请中提出的实验将利用S。 缺乏高亲和力钾离子摄取的酿酒酵母 广泛的，无偏倚的钾-氯共转运体结构-功能研究和 K1K(Ca)通道。KCC1的选拔系统已经过验证。一个 IK1的选拔系统正在开发中。拟议中的实验将 加速生产和筛选具有功能缺失和 功能增益表型。此外，该系统应允许开发 高通量筛选通过这些途径吸收K离子的抑制剂。 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trk1Delta/trk2Delta的增长恢复 酿酒酵母在非允许生长条件下的cDNA表达 编码KCC K-Cl共转运蛋白。2)确定KCC1的结构区 对离子传输、对阳离子和阴离子的选择性、对缓蚀剂的重要性 对最近发现的显性阴性表型和急性 监管。这将通过定义的饱和突变来实现 KCC K-Cl共转运蛋白亚区及其功能突变体的筛选 以挽救trk1Delta/trk2Delta酿酒酵母的生长。精选的突变体将是 在非洲爪哇卵母细胞和哺乳动物细胞中进一步研究。3)(暂定目标)： 在时间允许的情况下，改进、验证和规范增长救援 Trk1Delta/trk2Delta酿酒酵母在不允许的条件下表达 编码哺乳动物IK1 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.