Impact of neuronal chloride transport on treatment of seizures.

描述（由申请人提供）：在该资助的最后一个周期期间，我们确定了内向阳离子-氯共转运的阻断改变了发育中神经元中的神经元稳态氯浓度（Cl-1），从而提高了GABA介导的突触抑制的功效和新生儿癫痫发作的控制。这一发现构成了美国和欧洲新临床试验的基础。我们还发现，阳离子-Cl转运体在静息Cl-1时处于平衡。然而，我们不知道如何将这一发现与利用Cl-敏感的荧光双波长荧光团Clomeleon的实验数据相协调，Clomeleon的实验表明每个神经元都有一个独特的Cl-1，与其相邻的神经元完全不同。我们在这里解决的问题是：阳离子-Cl转运蛋白如何在如此多的不同Cl-i下保持平衡？平衡仅取决于Cl和阳离子浓度的经典观点不能解释Cl-1的变化。神经元阳离子-氯离子转运蛋白专门将水与阳离子和Cl一起移动，因此这些转运蛋白将等渗（135 mM）阳离子-Cl溶液移入和移出神经元。因此，神经元阳离子Cl转运蛋白也运输细胞质体积，这改变了细胞质的静水压力。这预示着跨膜流体静压和渗透压梯度有助于阳离子-Cl运输的自由能，从而平衡Cl-1。例如，具有大量促炎活性蛋白质的神经元应具有比具有较少蛋白质的神经元更低的平衡Cl-1。我们的主要假设是，跨神经元膜的压力梯度有助于运输的自由能，因此运输处于平衡的Cl-1。这一假说对长时间癫痫发作具有重要意义，癫痫发作诱导神经元细胞骨架的变化，增加神经元的体积，从而降低渗透压。因此，一个相关的次要假设是，尿素诱导的渗透压变化有利于阳离子、Cl-1和水通过共转运蛋白进入神经元，使得Cl-1增加，GABA信号变得兴奋。我们将测试这些假设，通过测量神经元的体积和Cl-1在稳态和离子和渗透的挑战，癫痫发作，和特定的运输抑制剂。我们将使用遗传表达克隆龙的小鼠，急性和器官型切片制备，体外和体内多光子显微镜，pH敏感染料，电生理记录和转运蛋白磷酸化研究。阳离子-Cl运输对局部压力梯度的敏感性将允许神经元保持稳定电缆特性和连接所需的令人难以置信的精确几何形状，尽管动态亚细胞和细胞间的蛋白质活性含量波动。该假说还预测，临床上可用的利尿剂和细胞骨架变化的抑制剂也可能用于治疗发育和成熟神经系统中的癫痫持续状态。 公共卫生相关性：长时间的癫痫发作会破坏皮层神经元的细胞骨架，使神经元扩张并积累所需的盐和水来填充新的细胞内空间。盐的一种成分是氯化物，它的积累导致神经递质GABA促进而不是抑制癫痫发作。我们将研究氯化物蓄积的机制和预防氯化物蓄积的策略，以改善长期癫痫发作的治疗。

DESCRIPTION (provided by applicant): During the last cycle of this grant, we established that block of inwardly-directed cation-chloride co-transport alters the neuronal steady-state chloride concentration (Cl-i) in developing neurons, and consequently improves the efficacy of GABA-mediated synaptic inhibition and the control of seizures in the newborn. This finding forms the basis of new clinical trials in the US and Europe. We also found that cation-Cl transporters are at equilibrium at resting Cl-i. However, we don't know how to reconcile this finding with data from experiments utilizing the Cl-sensitive fluorescent dual wavelength fluorophore, Clomeleon, which demonstrate that each neuron has a unique Cl-i that is quite different from its neighbors. The question we address here is: how can cation-Cl transporters be at equilibrium at so many different Cl-i? The classic view that equilibrium depends only on Cl and cation concentrations does not explain the variance in Cl-i. Neuronal cation-chloride transporters obligately move water with cations and Cl, so that these transporters move isotonic (135 mM) cation-Cl solution into and out of neurons. Thus neuronal cation-Cl transporters also transport cytoplasmic volume, which alters the cytoplasmic hydrostatic pressure. This predicts that transmembrane hydrostatic and osmotic pressure gradients contribute to the free energy of cation-Cl transport and thus the equilibrium Cl-i. For example, a neuron with lots of osmotically active protein should have a lower equilibrium Cl-i than a neuron with less protein. Our primary hypothesis is that the pressure gradient across the neuronal membrane contributes to the free energy of transport and thus the Cl-i at which transport is at equilibrium. This hypothesis has important implications for prolonged seizures, which induce changes in the neuronal cytoskeleton that increase the volume of neurons, thereby lowering the osmotic pressure. Thus a linked secondary hypothesis is that seizure-induced changes in osmotic pressure favor movement of cations, Cl-i and water into neurons via cotransporters so that Cl-i increases and GABA signaling becomes excitatory. We will test these hypotheses by measuring neuronal volume and Cl-i at steady state and in response to ionic and osmotic challenges, seizures, and specific transport inhibitors. We will use mice that genetically express Clomeleon, acute & organotypic slice preparations, in vitro and in vivo multiphoton microscopy, pH-sensitive dyes, electrophysiological recordings, and transporter phosphorylation studies. Sensitivity of cation-Cl transport to local pressure gradients would allow neurons to maintain the incredibly precise geometries needed for stable cable properties and connectivity despite dynamic subcellular and intercellular fluctuations in osmotically active protein content. The hypotheses also predict that clinically available diuretics and inhibitors of cytoskeletal changes might also be useful in the treatment of status epilepticus in both developing and mature nervous systems. PUBLIC HEALTH RELEVANCE: Prolonged epileptic seizures damage the cytoskeleton of cortical neurons, allowing the neurons to expand and accumulate the salt and water needed to fill the new intracellular space. One component of salt is chloride, and its accumulation causes the neurotransmitter GABA to promote rather than inhibit seizures. We will investigate the mechanisms of chloride accumulation and strategies to prevent it in order to improve the treatment of prolonged seizures.