MRP4 extrudes cAMP for localized regulation of calcium channel activity

MRP4 挤出 cAMP 来局部调节钙通道活性

• 批准号: 8649990
• 负责人: Olivia Ruth Asfaha
• 金额: $ 3.59万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649990
• 关键词: ABCC1 gene; ATP-Binding Cassette Transporters; Accounting; Adenosine; Adenylate Cyclase; Adrenergic Agents; Adrenergic Receptor; Affinity; Alzheimer' s Disease; Anxiety; Binding Sites; Biological Assay; Blood - brain barrier anatomy; Brain; Calcium Channel; Cardiac Myocytes; Cell membrane; Complex; Couples; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Cytoplasm; Cytosol; Dendritic Spines; Diffuse; Diffusion; Disease; Excision; Figs - dietary; Fluorescence Resonance Energy Transfer; Hippocampus (Brain); Hydrolysis; Indium; Individual; Kidney; Laboratories; Link; Mediating; Membrane; Membrane Transport Proteins; Mental Depression; Microglia; Molecular; Monitor; Mus; Neuraxis; Neurons; P-Glycoproteins; Pathway interactions; Peptides; Phosphorylation; Production; Prostate; Proteins; Receptor Signaling; Regulation; Research; Role; Rotation; Senility; Serine; Signal Transduction; Site; Stroke; Synapses; Synaptic plasticity; Testing; Up-Regulation; Xenobiotics; adrenergic; base; efflux pump; extracellular; gastrointestinal epithelium; knock-down; nervous system disorder; phosphoric diester hydrolase; postsynaptic; prevent; public health relevance; receptor; receptor coupling; sensor; small hairpin RNA; spatiotemporal; therapy development

DESCRIPTION (provided by applicant): Multidrug-Resistant Protein 4 (MRP4) has been established as a membrane transporter that facilitates cellular extrusion of cAMP in kidney, prostate, gut epithelium, and cardiac myocytes. MRP4 is implicated in the transport of cyclic nucleotides and xenobiotic molecules at the blood-brain barrier, yet its role in neuronal activity remains unknown. In cardiac myocytes, the MRP4 transporter appears to curb ¿2 adrenergic receptor (¿2AR) stimulation of cAMP levels. We hypothesize that MRP4 serves such a function in neurons, where its association with ¿2AR might enhance spatiotemporal restriction of cAMP for highly selective synaptic signaling. In the brain, ¿2AR, GS protein, adenylyl cyclase (AC), and protein kinase A (PKA) complex with Cav1.2 for localized and efficient regulation of this L-type channel. With Cav1.2 accounting for ~80% of L-type channels in brain, this Ca2+ channel is a prime target for studying molecular pathways that underlie synaptic plasticity. Upon ¿2AR activation, L-type Cav1.2 becomes potentiated by PKA phosphorylation at serine 1700. While it is clear that ¿-adrenergic signaling through cAMP is spatially contained by phosphodiesterases (PDE), MRP4 is emerging as another potential regulator within this classic ¿2AR nanodomain. The proposed research will determine whether MRP4 couples with the ¿2AR/Cav1.2-signaling complex at postsynaptic sites to spatially limit cAMP access to those complexes from the cytosolic cAMP pool. Levels of cAMP will be monitored by Fluorescence Resonance Energy Transfer upon ¿2AR stimulation with and without MRP4 inhibition or shRNA knockdown (KD). Protein interaction sites will be determined and peptides that displace MRP4 will be used to evaluate the functional role of MRP4 association with ¿2AR and Cav1.2. The regulation of Cav1.2 by MRP4 will be tested by monitoring Cav1.2 channel phosphorylation upon ¿2AR with and without MRP4 inhibition or KD. MRP4 function will be further tested by monitoring Cav1.2-mediated Ca2+ transients at postsynaptic sites. Examination of MRP4 assembly with ¿2AR/Cav1.2 nanodomains will promote a better understanding of spatial control of local cAMP. The proposed research will elucidate signaling mechanisms that contribute to L-type channel up-regulation, thus furthering development of treatments for Cav1.2-associated disorders in the central nervous system, including stroke, depression, anxiety, senility and Alzheimer's disease.

描述（由申请人提供）：多药耐药蛋白 4 (MRP4) 已被确定为一种膜转运蛋白，可促进肾脏、前列腺、肠上皮和心肌细胞中 cAMP 的细胞排出。 MRP4 参与血脑屏障处环核苷酸和外源分子的转运，但其在神经元活动中的作用仍不清楚。在心肌细胞中，MRP4 转运蛋白似乎可以抑制 ¿2 肾上腺素受体 (¿2AR) 对 cAMP 水平的刺激。我们假设 MRP4 在神经元中发挥这样的功能，其中它与 ¿2AR 的关联可能增强 cAMP 的时空限制，以实现高度选择性的突触信号传导。在大脑中，¿2AR、GS 蛋白、腺苷酸环化酶 (AC) 和蛋白激酶 A (PKA) 与 Cav1.2 复合，对 L 型通道进行局部有效调节。 Cav1.2 约占大脑 L 型通道的 80%，因此该 Ca2+ 通道是研究突触可塑性分子通路的主要目标。在 ¿2AR 激活后，L 型 Cav1.2 通过丝氨酸 1700 处的 PKA 磷酸化而得到增强。虽然很明显，通过 cAMP 的 ¡-肾上腺素能信号在空间上被磷酸二酯酶 (PDE) 所包含，但 MRP4 正在成为这个经典 ¿2AR 纳米结构域中的另一个潜在调节因子。拟议的研究将确定 MRP4 是否与突触后位点的 ¿2AR/Cav1.2 信号复合物偶联，以在空间上限制 cAMP 从胞质 cAMP 池中获取这些复合物。 cAMP 水平将通过 2AR 刺激（有或没有 MRP4 抑制或 shRNA 敲低 (KD)）时的荧光共振能量转移进行监测。将确定蛋白质相互作用位点，并使用取代 MRP4 的肽来评估 MRP4 与 ¿2AR 和 Cav1.2 关联的功能作用。 MRP4 对 Cav1.2 的调节将通过监测 ¿2AR 上的 Cav1.2 通道磷酸化（有或没有 MRP4 抑制或 KD）来测试。 MRP4 功能将通过监测突触后位点 Cav1.2 介导的 Ca2+ 瞬变来进一步测试。用 ¿2AR/Cav1.2 纳米结构域检查 MRP4 组装将促进更好地理解局部 cAMP 的空间控制。拟议的研究将阐明导致 L 型通道上调的信号机制，从而进一步开发中枢神经系统中 Cav1.2 相关疾病的治疗方法，包括中风、抑郁、焦虑、衰老和阿尔茨海默病。