MRP4 extrudes cAMP for localized regulation of calcium channel activity

描述(申请人提供)：多药耐药蛋白4(MRP4)已被证实是一种膜转运蛋白，促进肾、前列腺、肠上皮和心肌细胞中cAMP的细胞外排。MRP4与环核苷酸和外源分子在血脑屏障的运输有关，但它在神经元活动中的作用尚不清楚。在心肌细胞中，MRP4转运体似乎抑制了2肾上腺素能受体(2AR)对cAMP水平的刺激。我们假设，MRP4在神经元中起着这样的作用，它与2AR的关联可能会增强cAMP对高选择性突触信号的时空限制。在大脑中，2AR、GS蛋白、腺苷环化酶(AC)和蛋白激酶A(PKA)与Cav1.2形成复合体，对这一L类型的通道进行局部和有效的调节。由于Cav1.2约占脑内L类通道的80%，这一钙通道是研究突触可塑性的分子途径的首要靶点。当2AR被激活时，PKA在丝氨酸1700处的磷酸化增强了L类型的Cav1.2。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正在成为这个经典的2AR纳米结构域中的另一个潜在的调节因子。这项拟议的研究将确定MRP4是否与突触后位置的2AR/Cav1.2-信号复合体偶联，从而在空间上限制cAMP从胞质cAMP池进入这些复合体。在有或没有MRP4抑制或shRNA敲除(KD)的情况下，cAMP水平将通过荧光共振能量转移来监测。将确定蛋白质相互作用位点，并将使用取代MRP4的多肽来评估MRP4与2AR和Cav1.2的关联的功能作用。MRP4对Cav1.2的调节将通过监测Cav1.2通道在有或没有MRP4抑制或KD的情况下在2AR上的磷酸化来测试。MRP4的功能将通过监测突触后部位Cav1.2介导的钙瞬变来进一步测试。研究MRP4与2AR/Cav1.2纳米结构域的组装将促进对局部cAMP空间控制的更好理解。这项拟议的研究将阐明有助于L类通道上调的信号机制，从而进一步开发治疗中枢神经系统Cav1.2相关疾病的方法，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.