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.

Public Health Relevance

Increased activation of the ?-adrenergic receptor (?-AR) is clinically linked to Alzheimer's disease and stroke by up-regulation of activity of the L-type channel Cav1.2. Downstream cAMP signaling is a critical element in propagating the deleterious effects of improper ?-AR activity. This proposed research aims to uncover whether Multidrug Resistant Protein 4 selectively regulates ?2AR-stimulated cAMP, and thereby Cav1.2, in brain, with the aim of advancing current perspectives on neurological disorders.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F03B-A (20))
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Stewart, Randall R
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University of California Davis
Schools of Medicine
United States
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