MRP4 extrudes cAMP for localized regulation of calcium channel activity

描述（由申请方提供）：已确定多药耐药蛋白4（MRP 4）是一种膜转运蛋白，可促进肾脏、前列腺、肠上皮和心肌细胞中cAMP的细胞外排。MRP 4与环核苷酸和外源性分子在血脑屏障的转运有关，但其在神经元活动中的作用仍不清楚。在心肌细胞中，MRP 4转运蛋白似乎抑制cAMP水平的<$2肾上腺素能受体（<$2AR）刺激。我们假设MRP 4在神经元中具有这样的功能，其与<$2AR的关联可能增强cAMP对高度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）与Cav1.2复合，用于局部和有效调节该L型通道。Cav1.2占大脑中L型通道的约80%，这种Ca 2+通道是研究突触可塑性基础的分子途径的主要目标。在2AR激活后，L型Cav1.2通过PKA在丝氨酸1700处的磷酸化而增强。虽然很明显，通过cAMP的β-肾上腺素能信号传导在空间上被磷酸二酯酶（PDE）所包含，MRP 4正在成为该经典的β 2 AR纳米结构域内的另一种潜在调节剂。拟议的研究将确定MRP 4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池进入这些复合物。在有和没有MRP 4抑制或shRNA敲低（KD）的情况下，通过荧光共振能量转移在Δ 2AR刺激后监测cAMP水平。将确定蛋白质相互作用位点，并将使用取代MRP 4的肽来评价MRP 4与Δ 2AR和Cav1.2相关的功能作用。将通过监测在有和没有MRP 4抑制或KD的情况下，Cav1.2通道对Δ 2AR的磷酸化来测试MRP 4对Cav1.2的调节。将通过监测突触后位点Cav1.2介导的Ca 2+瞬变来进一步测试MRP 4功能。检查MRP 4与<$2AR/Cav1.2纳米结构域的组装将促进对局部cAMP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，抑郁症，焦虑症，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.