KINASES IN ION COTRANSPORTER FUNCTION

描述(申请人提供)：阳离子-氯离子共转运体，如Na-K-21和K-CI共转运体，在各种细胞和组织中发挥基本作用。在神经系统中，它们调节抑制性突触传递，并参与盐分和液体在上皮细胞中的移动。Na-K-2CI共转运体NKCC1在感觉神经元(DRG，嗅觉)积聚氯离子，从而促进GABA去极化反应。辅助转运体还参与内耳液的产生和脑脊液中K+的重吸收。NKCC1基因敲除导致多种表型，包括对疼痛的敏感性增加和感音神经性耳聋。阳离子-氯离子共转运蛋白受多种刺激的调节，其中大多数(如果不是全部)都集中于转运蛋白的磷酸化/去磷酸化，但对影响它们的激酶了解不多。我们最近发现了与酵母Ste20激酶家族相关的应激蛋白，它们是阳离子-氯共转运蛋白的相互作用因子。与WNK4(一种与人类高血压相关的激酶)一起，应激蛋白激酶调节辅转运体的活动。我们还发现了一种SPAK相互作用的酪氨酸激酶，它可以负向调节NKCC1的活性。为了了解这些激酶和辅助转运蛋白之间相互作用的生理意义，我们建议：1)定义应激激酶和WNK4在调节NKCC1活性中的作用；2)定义酪氨酸激酶AATYK在调节NKCC1活性中的作用。这将通过对非洲爪哇卵母细胞中共转运体和激酶突变体的功能研究以及体外磷酸化实验来实现。我们还将讨论PP1的作用及其与SPAK结合域重叠的位点的假定结合。3)利用一个表达NKCC1、Spak、OSR1、WNK4和AATYK的细胞系，通过沉默实验来研究每个激酶在共转运蛋白调节中的特定作用。这些研究将阐明阳离子-氯共转运体功能和调控的新方面，并提供更好的理解阳离子-氯共转运体与中枢神经系统相关疾病的联系。通过细胞膜运输无机离子的机制涉及多种疾病：盐耗障碍、高血压、神经退化和大脑过度兴奋。转运蛋白不是单独发挥作用的，而是受到多种其他蛋白质的严格调控，这些蛋白质也有可能参与这些疾病。详细研究调控蛋白和转运蛋白之间的相互作用对于理解这些人类疾病至关重要。

DESCRIPTION (provided by applicant): Cation-chloride cotransporters, e.g. Na-K-21 and K-CI cotransporters, play fundamental roles in a variety of cells and tissues. In the nervous system, they modulate inhibitory synaptic transmission and participate to the movement of salt and fluid across epithelia. The Na-K-2CI cotransporter, NKCC1, accumulates Cl- in sensory neurons (DRG, olfactory), thus promoting GABA depolarizing responses. The cotransporter also participates in the production of the inner ear fluid and the reabsorption of K+ from the CSF. Knockout of NKCC1 leads to multiple phenotypes including increased sensitivity to pain and sensorineural deafness. Cation-chloride cotransporters are regulated by a variety of stimuli, most of them (if not all of them) converging to phosphorylation/dephosphorylation of the transporters, but not much is known about the kinases affecting them. We have recently identified, as interactors of cation-chloride cotransporters, stress kinases related to the yeast Ste20 kinase family. Together with WNK4, a kinase that is associated with human hypertension, the stress kinases modulate the activity of the cotransporters. We have also identified a SPAK-interacting tyrosine kinase which negatively regulates NKCC1 activity. To understand the physiological significance of the interaction between these kinases and the cotransporters, we propose to 1) Define the role of the stress kinases and WNK4 in modulating the activity of NKCC1 and 2) Define the role of the tyrosine kinase AATYK in the regulation of NKCC1 activity. This will be achieved through functional studies of cotransporter and kinase mutants in Xenopus laevis oocytes and through in vitro phosphorylation experiments. We will also address the role of PP1 and its putative binding to a site that overlaps with a SPAK binding domain. 3) Utilize a cell line which expresses NKCC1, SPAK, OSR1, WNK4, and AATYK to address through silencing experiments the specific role of each kinase in the regulation of the cotransporter. These studies will elucidate novel aspects of cation-chloride cotransporter function and regulation and provide a better understanding of cation-chloride cotransporter links to CNS-related disorders. Mechanisms that transport inorganic ions across cell membranes are involved in a variety of disorders: salt wasting disorders, hypertension, nerve degeneration and brain hyperexcitability. The transporters do not function in isolation but are tightly regulated by a variety of other proteins that also have the potential to participate in these diseases. Detailed studies of the interaction between regulatory proteins and the transporters are of critical importance for understanding these human disorders.