GABA-B Receptor Modulation of Calcium Currents

描述(由申请人提供)：该实验室的长期目标是加强我们对神经递质如何调节钙通道活动以及这种调节的功能后果的理解。哺乳动物中枢神经系统中的主要抑制性神经递质--氨基丁酸(GABA)可通过激活GABAB受体来减弱N型钙电流。我们实验室的一个独特发现是，在新生大鼠海马神经元中，GAAB受体的激活促进了L型电流的发生。了解这种促进作用的机制和功能意义将对药物成瘾、癫痫、失眠和焦虑等疾病的治疗产生重大影响，这些疾病通常使用与GABA能系统相互作用的化合物进行治疗。需要验证的中心假设是GABAB受体通过直接蛋白激酶C(PKC)介导的Cav 1.2或1.3的磷酸化来促进钙电流。具体目标1是通过确定GAAB受体的激活是否通过特定的蛋白激酶C亚型促进L型电流来继续我们对信号转导机制的研究。这将通过使用全细胞膜片钳记录和钙成像来测试信号通路的各种抑制剂来实现。PKC异构体的激活将通过观察从胞浆到膜的转位和通过Western印迹分析异构体的磷酸化来测试。具体目的#2是确定L钙电流的增强是否是通过磷酸化事件激活沉默的L钙通道。这些实验将利用单通道记录以及蛋白质印迹分析来观察L类钙通道的磷酸化。这些实验将提供关于L电流易化的信号转导机制的知识，并将提供关于全细胞电流易化发生的具体机制的信息。更好地了解GABA-B受体的功能可能会允许对GABA能系统进行更微妙的操作，从而以更少的副作用更好地设计药物。 公共卫生相关性：本提案中概述的实验将增加我们对抑制性神经递质GABA如何通过调节电压依赖性钙通道影响神经元功能的理解，特别是在新生儿早期。与GABA受体相互作用的药物化合物通常用于治疗焦虑、癫痫、失眠等，但这些化合物也会导致嗜睡、智力减慢和复视等副作用。通过增加我们对GABA具体作用的了解，将有可能设计出更好的药物来治疗这些类型的疾病。

DESCRIPTION (provided by applicant): The long range goal of the laboratory is to enhance our understanding of how neurotransmitters modulate calcium channel activity and the functional consequences of this modulation. The main inhibitory neurotransmitter in the mammalian central nervous system, ?-aminobutyric acid GABA) is known to attenuate N-type calcium currents by activation of GABAB receptors. A unique finding in our laboratory is that L-type current is facilitated in response to GABAB receptor activation in the neonatal neurons of the rat hippocampus. Understanding the mechanisms and functional significance of this facilitation will have significant consequences for the treatment of diseases such as drug addiction, epilepsy, insomnia and anxiety that are routinely treated with compounds interacting with the GABAergic system. The central hypothesis to be tested that GABAB receptors facilitate calcium current by a direct protein kinase C (PKC) mediated phosphorylation of Cav 1.2 or 1.3. Specific aim #1 is to continue our studies on the signal transduction mechanism by determining if GABAB receptor activation facilitates L-type current through a specific PKC isoform. This will be accomplished by testing various inhibitors of the signaling pathway using whole cell patch clamp recording and calcium imaging. The activation of PKC isoforms will be tested by observing translocation from the cytosol to the membrane and phosphorylation of the isoforms using Western blot analysis. Specific aim #2 is to determine whether the enhancement of L-type calcium current is through activation of silent L-type calcium channels via a phosphorylation event. These experiments will utilize single channel recording as well as Western blot analysis to observe phosphorylation of the L-type calcium channels. The experiments outlined in this proposal will provide knowledge about the signal transduction mechanism underlying the facilitation of L-type current and will provide information on the specific mechanism by which the facilitation of whole cell current occurs. A better understanding of the GABA-B receptor function may allow more subtle manipulation of the GABAergic system allowing for better design of pharmaceuticals with fewer side effects. PUBLIC HEALTH RELEVANCE: The experiments outlined in this proposal will increase our understanding of how the inhibitory neurotransmitter GABA affects neuronal function by modulation of voltage dependent calcium channels, particularly in the early neonatal period. Pharmaceutical compounds that interact with GABA receptors are routinely prescribed for anxiety, epilepsy, insomnia, etc. but these compounds also cause side effects such as drowsiness, mental slowing, and double vision. By increasing our understanding of the specific effects of GABA, it will be possible to design better drugs for these types of disorders.