Exploration of KCC2: Role in Schizophrenia

描述(由申请人提供)：这是针对NIMH RFA#PA-00-073的一份试探性/开发性拨款(R21)提案。 K-CI联合转运蛋白(KCC2)是一种神经元特异性转运蛋白，广泛分布于中枢神经系统。KCC2在神经元中具有双重功能：维持低的细胞内氯([CI]I)和缓冲大脑中的外部钾([K]O)浓度。在功能上，KCC2与GABAA受体偶联，GABAA受体的激活允许阴离子(主要是CI-)流入神经元，产生GABA的主要突触功能抑制。GABAA受体的激活是否导致抑制或兴奋取决于其逆转电位值(EGABAA)。反转电位值主要由跨膜的CI的电化学梯度决定；KCC2在调节神经元[Cl]i稳态方面起着关键作用。因此，KCC2调节GABAA受体的生理功能，即正常成年CNS神经元的突触抑制，同时也调节正常发育或新生神经元的突触兴奋，并可能以一种不适应的方式调节受疾病影响的神经元。GABA能系统是大脑中的主要抑制系统，据报道在精神分裂症(SCZ)中有故障。SCZ是一种复杂的精神障碍，其特征是认知功能和行为的衰弱障碍，可能是包括GABA能系统在内的多个中枢神经传递系统功能障碍所致。然而，KCC2转运蛋白的表达和功能尚未在精神分裂症患者的大脑或精神分裂症的动物模型中进行研究。我们建议探索KCC2在慢性苯环利定(c-PCP)大鼠精神分裂症模型中的作用。这些实验将检验一种假设，即在长期服用PCP的大鼠中，KCC2功能下调和/或其在内侧前额叶皮质(MPFC)神经元中的表达发生变化。长期以来，mPFC一直与精神分裂症的发病机制有关。具体目的有三：1)比较正常和c-PCP大鼠前额叶皮质GABAA受体的突触生理学；2)测定正常和c-PCP大鼠突触和非突触部位GABAA受体逆转电位(EGABAA)的值；3)测定KCC2的蛋白表达水平，同时研究其拮抗剂速尿对其的药理作用。这项拟议的研究结果可能会为考虑精神分裂症的新发病机制提供动力，同时也为更有效的药物治疗提供基础。

DESCRIPTION (provided by applicant): This is an exploratory/development grant (R21) proposal in response to NIMH RFA#PA-00-073. The K-CI Co-Transporter (KCC2) is neuronal specific and widely distributed in the central nervous system. KCC2 serves a dual function in neurons: the maintenance of low intracellular chloride ([CI]i) and the buffering of external potassium ([K]o) concentrations in the brain. Functionally, KCC2 is coupled with GABAA receptors whose activation allows anions (predominantly CI-) to flow into neurons, producing GABA's principal synaptic function - inhibition. Whether GABAA receptor activation leads to inhibition or excitation depends upon the value of its reversal potential (EGABAA). The reversal potential value is primarily determined by an electrochemical gradient of the CI across the membrane; KCC2 plays a pivotal role in regulating neuronal [Cl]i homeostasis. Thus, KCC2 regulates the physiological functions of GABAA receptors, namely, synaptic inhibition in normal adult CNS neurons, while also synaptic excitation in normal developing or neonatal neurons, and possibly - in a maladaptive manner - in disease affected neurons. The GABAergic system, the main inhibitory system in the brain, is reported to malfunction in schizophrenia (SCZ). SCZ is a complex mental disorder characterized by a debilitating disturbance of cognitive function and behavior that may result from a dysfunction of multiple CNS neurotransmission systems including the GABAergic system. However, the expression and function of the KCC2 transporter have not been studied in schizophrenic brains or in animal models of schizophrenia. We propose to explore the role of KCC2 in a chronic phencyclidine (c-PCP) rat model of schizophrenia. The experiments will test the hypothesis that in rats treated chronically with PCP, KCC2 is functionally down-regulated and/or its expression is altered in neurons of the medial prefrontal cortex (mPFC). The mPFC has long been implicated in the pathogenesis of schizophrenia. The specific aims are threefold: namely, 1) compare the synaptic physiology of GABAA receptors in the prefrontal cortex of normal and c-PCP rats; 2) determine the value of GABAA receptor reversal potentials (EGABAA), for GABAA receptors at synaptic and non-synaptic sites in normal and c-PCP rats, respectively; and, 3) determine the levels of protein expression of KCC2 while investigating its pharmacological manipulation with an antagonist, furosemide, in normal and c-PCP rats. Results from the proposed study may provide an impetus to consider a novel pathogenesis for schizophrenia, while at the same time providing a basis for a more effective pharmacotherapy.