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: 8774113
• 负责人: Olivia Ruth Asfaha
• 金额: $ 3.68万
• 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/8774113
• 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; 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（由适用提供）：已建立多药耐药蛋白4（MRP4）作为一种膜转运蛋白，可促进肾脏，前列腺，肠上皮和心肌细胞的cAMP的细胞延伸。 MRP4在循环核动肽和血脑屏障上的异种分子的运输中隐含，但其在神经元活性中的作用仍然未知。在心肌细胞中，MRP4转运蛋白似乎遏制了2个肾上腺素受体（2AR）刺激cAMP水平。我们假设MRP4在神经元中发挥了这种功能，在神经元中，其与2AR的关联可能会增强对cAMP的时空限制，以进行高度选择性的合成信号传导。在大脑中，`2AR，GS蛋白，腺苷酸酶（AC）和蛋白激酶A（PKA）与CAV1.2的蛋白激酶A（PKA）复合物，用于对此L型通道的局部和有效调节。由于CAV1.2占大脑中L型通道的约80％，该CA2+通道是研究突触可塑性构成的分子途径的主要目标。 2AR激活后，L型CAV1.2通过丝氨酸1700处的PKA磷酸化而成为潜在的。虽然很明显，通过cAMP的 - 肾上腺素能信号在空间上由磷酸二酯酶（PDE）在空间上含有，但MRP4在这种经典的2AR nanodomain中的另一个潜在调节剂中，它却被培养为另一个潜在的调节剂。拟议的研究将确定MRP4夫妇是否在突触后地点具有2AR/CAV1.2信号复合物，以在空间上限制camp进入细胞质营地池中对这些络合物的访问。在有或没有MRP4抑制或shRNA敲低（KD）的2AR刺激后，将通过荧光共振能量转移来监测cAMP的水平。将确定蛋白质相互作用位点，并将替代MRP4的Pepperides用于评估MRP4与2AR和CAV1.2的功能作用。 MRP4对CAV1.2的调节将通过监测CAV1.2在有和没有MRP4抑制或KD的情况下监测CAV1.2 CAV1.2通道磷酸化。 MRP4功能将通过监测突触后位点的CAV1.2介导的Ca2+瞬变来进一步测试。检查使用2AR/CAV1.2纳米域的MRP4组件的检查将促进对当地营地的空间控制的更好理解。拟议的研究将阐明有助于L型通道上调的信号传导机制，从而进一步发展中枢神经系统中CAV1.2相关疾病的治疗方法，包括中风，抑郁症，焦虑症和阿尔茨海默氏病。