KINASES IN ION COTRANSPORTER FUNCTION

描述（由申请人提供）：阳离子-氯化物共转运蛋白，例如Na-K-21和K-Cl共转运蛋白，在多种细胞和组织中发挥重要作用。在神经系统中，它们调节抑制性突触传递并参与盐和液体穿过上皮的运动。Na-K-2Cl协同转运蛋白NKCC 1在感觉神经元（DRG，嗅觉）中积累Cl-，从而促进GABA去极化反应。协同转运蛋白还参与内耳液的产生和从CSF重吸收K+。敲除NKCC 1导致多种表型，包括对疼痛和感觉神经性耳聋的敏感性增加。阳离子-氯协同转运蛋白受多种刺激物的调节，其中大多数（如果不是全部的话）会聚于转运蛋白的磷酸化/去磷酸化，但对影响它们的激酶知之甚少。我们最近已经确定，作为阳离子-氯共转运蛋白的相互作用，与酵母Ste 20激酶家族相关的应激激酶。与WNK 4（一种与人类高血压相关的激酶）一起，应激激酶调节协同转运蛋白的活性。我们还确定了一个SPAK相互作用的酪氨酸激酶，负调控NKCC 1活性。为了理解这些激酶和共转运蛋白之间相互作用的生理意义，我们建议：1）确定应激激酶和WNK 4在调节NKCC 1活性中的作用; 2）确定酪氨酸激酶AATYK在调节NKCC 1活性中的作用。这将通过对非洲爪蟾卵母细胞中协同转运蛋白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PP 1的作用及其与SPAK结合结构域重叠的位点的假定结合。3)利用表达NKCC 1、SPAK、OSR 1、WNK 4和AATYK的细胞系，通过沉默实验阐明每种激酶在协同转运蛋白调节中的特定作用。这些研究将阐明新的方面的阳离子-氯离子协同转运蛋白的功能和调节，并提供了一个更好的理解阳离子-氯离子协同转运蛋白的链接到中枢神经系统相关疾病。无机离子跨细胞膜转运的机制涉及多种疾病：盐耗障碍、高血压、神经变性和脑过度兴奋。转运蛋白不是孤立发挥作用的，而是受到各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。详细研究调节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.