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[Cl-]（[Cl-]i）是重要的，因为它是确定跨质膜的Cl-电化学梯度的关键因素，因此决定了响应（即，去极化与超极化）的配体门控阴离子通道，如GABA-A受体（GABAAR）。虽然由GABAAR介导的快速GABA能传递在成人中主要是超极化和抑制性的，但它不是静态的，并且在生理学和病理生理学中经历了极性的显著变化。我们假设GABAAR电压响应的极性变化是主要神经元“Cl-泵”转运能力变化的直接结果，即，Na-K-Cl协同转运蛋白（NKCC）和K-Cl协同转运蛋白（KCC）。我们已经确定并表征了神经元特异性K-Cl协同转运蛋白（KCC 2）的亚型，其主要起Cl-挤出机制的作用。这项建议有4个具体目的：1）阐明KCC 2的运作。我们将测试KCC 2作为神经元[Cl-]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KCC 2活性，并测试KCC 2的动力学模型。2)通过KCC 2表征铵转运。初步研究表明，NH 4+被KCC 2转运。我们将使用NH 4+易位来表征KCC 2活性在单个培养的神经元中使用pH敏感的荧光染料。3)阐明KCC 2急性调节的分子机制。我们假设KCC 2的急性调节涉及其磷酸化状态的变化。我们将在天然和异源表达系统中使用KCC 2蛋白来解决这一假设。4)确定膜运输在调节KCC 2转运能力中的作用。使用天然和异源表达系统，我们将表面KCC 2密度的变化与转运蛋白活性相关。我们的研究结果将提供一个合理的基础，了解神经元的Cl-稳态以及细胞和分子机制，负责在生理学和病理生理学中观察到的快速GABA能传递的极性转变。

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.