K-Cl COTRANSPORTERS IN RAT NERVOUS SYSTEM

描述(由申请人提供)：长期目标是了解神经元C-稳态的细胞和分子基础。神经元的细胞内[CI-]([Cl-]i)是决定跨质膜的Cl-电化学梯度的关键因素，因此决定了配体门控阴离子通道的反应(即去极化与超极化)，如GABA-A受体(GABAARs)。尽管由GABA受体介导的快速GABA能传递在成人中主要是超极化和抑制，但它不是静态的，在生理和病理生理上都经历了显著的极性变化。我们推测，GABAAR电压响应中的这种极性变化是主要神经元“氯泵”，即Na-K-CI共转运体(NKCC)和K-CI共转运体(KCC)转运能力变化的直接结果。我们已经鉴定并鉴定了一种神经元特异性的K-CI共转运蛋白(KCC2)亚型，它主要作为一种氯离子挤出机制发挥作用。这项建议有四个具体目标：1)阐明KCC2的运作。我们将测试KCC2作为神经元[CI-]和外部[K+]的“动态缓冲器”的功能模型，确定[Cl-]i的变化如何调节KCC2的活性，并测试KCC2的动力学模型。2)研究KCC2对土壤中铵离子的迁移。初步研究表明，KCC2对NH4+有迁移作用。我们将使用pH敏感的荧光染料，通过NH4+转运来表征单个培养神经元中KCC2的活性。3)阐明KCC2急性调节的分子机制。我们假设KCC2的急性调节涉及其磷酸化状态的变化。我们将使用KCC2蛋白在自然和异源表达系统中解决这一假设。4)确定膜转运在调节KCC2转运能力中的作用。使用自然和异源表达系统，我们将把表面KCC2密度的变化与转运蛋白活性联系起来。我们的结果将为理解神经元的氯稳态以及在生理学和病理生理学中观察到的快速GABA能传递中极性转移的细胞和分子机制提供合理的基础。

DESCRIPTION (provided by applicant): The long term objective is to understand the cellular and molecular basis for neuronal C - homeostasis. Intracellular [CI-] ([Cl-]i) of neurons is important because it is a key factor in determining the Cl-electrochemical gradient across the plasma membrane and therefore dictates the response (i.e., depolarizing vs. hyperpolarizing) of ligand-gated anion channels, like GABA-A receptors (GABAARs). While fast GABAergic transmission mediated by GABAARs is predominantly hyperpolarizing and inhibitory in the adult, it is not static and undergoes remarkable shifts in polarity in both physiology and pathophysiology. We hypothesize that such polarity shifts in the voltage response of the GABAAR are the direct result of changes in the transport capacities of the major neuronal "Cl- pumps", i.e., the Na-K-CI cotransporter (NKCC) and K-CI cotransporter (KCC). We have identified and characterized a neuron-specific isoform of the K-CI cotransporter (KCC2) that functions primarily as a Cl- extrusion mechanism. This proposal has four specific aims; 1) To elucidate the operation of KCC2. We will test a functional model of KCC2 as a "dynamic buffer" of neuronal [CI-] and external [K+], determine how changes in [Cl-]i modulate KCC2 activity, and test a kinetic model for KCC2. 2) To characterize ammonium transport by KCC2. Preliminary studies show that NH4+ is translocated by KCC2. We will use NH4+ translocation to characterize KCC2 activity in individual cultured neurons using pH-sensitive fluorescent dyes. 3) To elucidate the molecular mechanism of acute regulation of KCC2. We hypothesize that acute regulation of KCC2 involves changes in its phosphorylation state. We will address this hypothesis using KCC2 protein in both native and heterologous expression systems. 4) To determine the role of membrane trafficking in regulating KCC2 transport capacity. Using both native and heterologous expression systems, we will correlate changes in surface KCC2 density with transporter activity. Our results will provide a rational basis for understanding neuronal Cl- homeostasis as well as the cellular and molecular mechanisms responsible for polarity shifts in fast GABAergic transmission observed in physiology and pathophysiology.