MRP4 extrudes cAMP for localized regulation of calcium channel activity

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、前列腺、肠上皮和心肌细胞中cAMP的细胞挤压。MRP4参与环核苷酸和异种分子在血脑屏障的运输，但其在神经元活动中的作用尚不清楚。在心肌细胞中，MRP4转运体似乎可以抑制cAMP水平的2肾上腺素能受体（¿2AR）刺激。我们假设MRP4在神经元中具有这样的功能，其与¿2AR的关联可能会增强cAMP对高选择性突触信号传导的时空限制。在大脑中，¿2AR， GS蛋白，腺苷酸环化酶（AC）和蛋白激酶A （PKA）复合物与Cav1.2一起定位和有效调节该l型通道。由于Cav1.2占大脑l型通道的约80%，这一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在丝氨酸1700处被PKA磷酸化而增强。虽然很明显，通过cAMP传递的肾上腺素能信号在空间上被磷酸二酯酶（PDE）所包含，但MRP4正在成为这一经典纳米结构域的另一种潜在调节剂。这项拟议的研究将确定MRP4是否在突触后位点与¿2AR/ cav1.2信号复合体偶联，从而在空间上限制cAMP从细胞质cAMP池进入这些复合体。cAMP的水平将通过荧光共振能量转移在有或没有MRP4抑制或shRNA敲低（KD）的¿2AR刺激下监测。蛋白质相互作用位点将被确定，取代MRP4的肽将被用来评估MRP4与¿2AR和Cav1.2关联的功能作用。MRP4对Cav1.2的调控将通过监测MRP4是否抑制或KD时的Cav1.2通道磷酸化来测试。MRP4功能将通过监测突触后位点cav1.2介导的Ca2+瞬态进一步测试。用¿2AR/Cav1.2纳米结构域检测MRP4组装将有助于更好地理解局部cAMP的空间控制。该研究将阐明促进l型通道上调的信号机制，从而进一步开发中枢神经系统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。

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.