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。神经元的胞内[CI-] （[Cl-]i）很重要，因为它是决定跨质膜的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（如GABA-A受体（GABAARs））的反应（即去极化与超极化）。虽然GABAARs介导的gabaergy快速传递在成人中主要是超极化和抑制性的，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。我们假设GABAAR电压响应中的这种极性变化是主要神经元“Cl-泵”，即Na-K-CI共转运体（NKCC）和K-CI共转运体（KCC）运输能力变化的直接结果。我们已经确定并表征了K-CI共转运蛋白（KCC2）的神经元特异性异构体，其主要功能是Cl-挤压机制。这项建议有四个具体目标；1)阐明KCC2的操作。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)表征KCC2对铵的输运。初步研究表明，NH4+通过KCC2转运。我们将使用ph敏感的荧光染料，利用NH4+易位来表征单个培养神经元中的KCC2活性。3)阐明KCC2急性调控的分子机制。我们假设KCC2的急性调控涉及其磷酸化状态的变化。我们将使用KCC2蛋白在原生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。使用原生和异源表达系统，我们将把表面KCC2密度的变化与转运蛋白活性联系起来。我们的研究结果将为理解神经元Cl-稳态以及在生理和病理生理中观察到的gaba能快速传递中极性变化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.