This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract: The phospholipid bilayer plays a central role in the lifecycle of the endogenous cannabinoid, N- arachidonoyl-ethanolamine (anandamide, AEA). AEA has been shown to be synthesized from lipid, to interact with the membrane embedded cannabinoid CB1 receptor, to be transported to intracellular compartments possibly via caveolae related endocytosis and finally to be degraded by fatty acid amide hydrolase (FAAH), an integral membrane protein whose active site is accessed by AEA possibly via the bilayer. The long term goal of our NIH/NIDA (DA03934 and DA00489) sponsored research is to elucidate the basis for the actions of the cannabinoids (CBs) at the molecular level. In previous AAB supported work, we have explored the properties of AEA alone in a dioleoylphosphatidylcholine (DOPC) phospholipid bilayer via multi-nanosecond molecular dynamics simulations. We have also tested the hypothesis that AEA approaches the CB1 receptor not from the extracellular milieu, but from lipid; interacts with a specific recognition element (the V6.43/I6/46 groove on the lipid face of CB1) and, then enters CB1 by passing between TMHs 6 and 7. Our recent collaborative mutation studies of the V6.43/I6.46 groove lend strong support to this hypothesis. The work proposed here focuses on the fate of AEA after it leaves the CB1 receptor. In this proposal, we request 184,000 SUs on the TCS Lemieux machine at the Pittsburgh Computing Center for lipid-protein simulation work. These resources are proposed to be used for molecular dynamics simulations using the NAMD program, including an atomic level description of a phospholipid bilayer model of the cellular membrane with the FAAH protein inserted into one leaflet of this bilayer. The Steered Molecular Dynamics (SMD) method will be used to test the hypothesis that the entry path for AEA into FAAH is from the lipid bilayer. To this end, we will study the unbinding of AEA from FAAH and assess the importance of Ile491, a residue near the FAAH lipid entry portal to this process.
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