Potassium Chloride Cotransport Regulation in Red Cells

镰刀状红细胞(SS RBC)进入循环后会迅速脱水。这会恶化流变行为，并促进Hb S聚合，这高度依赖于Hb S的浓度。一种有希望的治疗方法是通过增加红细胞水合和减少镰状细胞的方式来影响膜离子转运体。SS红细胞中KCl共转运蛋白(KCC)的高表达和KCC对细胞体积的异常反应都可能是SS RBC脱水的原因之一。本课题主要研究SS RBC中细胞体积及其异常对KCC活性的翻译后调控。新的 这些技术将KCC激活后的密度变化与网织红细胞的流式细胞仪检测相结合，以评估网织红细胞的流量和最终达到的血红蛋白浓度，这反映了KCC的体积设定点(VSP)。具体目的1比较SS和AA中的KCC，研究VSP与KCC的生理刺激--细胞体积、pH、细胞镁和尿素--的关系，以检验SS患者的VSP相对于AA患者是异常的假设，以及这些异常是由SS RBC中已知的氧化损伤引起的。SC和CC代表也将被审查。特定目的2重点研究在红系细胞中发现的几种KCC1剪接异构体的功能特性，验证这些异构体对体积刺激的反应不同的假设。KCC1的cdna构建将是暂时的 利用CMV启动子驱动的pcDNA3载体在培养的人胚胎肾(HEK)细胞中表达。KCC活性将被测量为依赖氯的、对欧巴因和布美他尼不敏感的(86)Rb摄取，并评估对体积刺激(低张肿胀和N-乙基马来酰亚胺)的反应。具体目标3将确定hKCC1中的音量响应元件。一系列N端和C端胞质结构域逐渐截断的hKCC1蛋白将在HEK细胞中表达，并分析基础和体积刺激的KCC活性。定点突变将针对选定的丝氨酸/苏氨酸 在hKCC1的细胞质区域中的残基，以检验这些残基中的一个(或多个)被磷酸化以保持基础(未被刺激)的KCC活性，并被去磷酸化以通过体积刺激影响KCC的激活。这些研究将阐明KCC容量调节的分子基础及其在SS RBC中的异常，并导致改善治疗以减轻镰状细胞疾病的细胞脱水。

Sickle red blood cells (SS RBC) dehydrate quickly upon entering the circulation. This worsens rheological behavior and facilitates Hb S polymerization, which is highly dependent on Hb S concentration. A promising therapeutic approach is to influence membrane ion transporters in ways that increase RBC hydration and decrease sickling. High levels of expression of KCl cotransport (KCC) in SS RBC compared to AA RBC and abnormal response of KCC to cell volume may both contribute to SS RBC dehydration. This project focuses on the post-translational control of KCC activity by cell volume and its abnormalities in SS RBC. New techniques combine density shifts after KCC activation with flow cytometric detection of reticulocytes to assess the KCC flux in reties and the final hemoglobin concentration achieved, which reflects the volume set point (VSP) of KCC. Specific Aim 1 compares KCC in SS and AA reties, examining the relationship between VSP and physiological stimuli of KCC---cell volume, pH, cell Mg, and urea--to test hypotheses that the VSP in SS retics is abnormal relative to AA retics and that these abnormalities result from the oxidative damage known to occur in SS RBC. SC and CC retics will also be examined. Specific Aim 2 focuses on the functional properties of several splicing isoforms of KCCl identified in erythroid cells, testing the hypothesis that these isotypes differ in their response to volume stimuli. KCCl cDNA constructs will be transiently expressed in cultured human embryonic kidney (HEK) ceils using a pcDNA3 vector driven by the CMV promoter. KCC activity will be measured as Cl-dependent, oubain-and-bumetanide-insensitive (86)Rb uptake, and the response to volume stimuli (hypotonic swelling and N-ethylmaleimide) assessed. Specific Aim 3 will determine the volume responsive elements in hKCCl. A series of hKCCl proteins with progressive truncations of the N- and C-terminal cytoplasmic domains will be expressed in HEK cells and analyzed for basal and volume-stimulated KCC activity. Site-directed mutagenesis will target selected serine/threonine residues in the cytoplasmic domains of hKCCl to test the hypothesis that one (or more) of these residues is phosphorylated to maintain basal (unstimulated) KCC activity and dephosphorylated to effect KCC activation by volume stimuli. These studies will elucidate the molecular basis of KCC volume regulation and its abnormalities in SS RBC, and lead to improved therapies to mitigate cellular dehydration in sickle cell disease.