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Item Open Access Underlying Mechanisms Which Regulate Equilibrative Nucleoside Transporter 1 (ENT1): From Fundamental Forms of Regulation to Unifying Signalling Pathways(2016-09-20) Bicket, Alex Christopher; Coe, Imogen; Benchimol, SamuelEquilibrative nucleoside transporter 1 (ENT1) is a membrane protein critical for the movement of nucleosides across biological membranes. ENT1 plays a role in nucleoside salvage, cell signalling, cardioprotection, and the uptake of nucleoside analog drugs involved in the treatment of cancer, viruses, and parasites. Although ENT1 is clinically relevant as a drug transporter, relatively little is known about how it is regulated. I have identified two gaps in our knowledge of ENT1 how calcium regulates ENT1-dependent nucleoside flux, and the role of N-linked glycosylation on ENT1. I have defined a novel mode of ENT1 regulation, whereby direct binding of CaM to ENT1 modulates nucleoside flux in a calcium-dependent manner. Moreover, I have shown that in a physiologically relevant model (U-87 MG, human glioblastoma cell line) that activation of NMDA receptors led to increased intracellular calcium, which increased chloroadenosine flux that can be blocked with the NMDA receptor antagonist MK-801 (50 M) or the CaM antagonist W7 (50 M). Calcium-dependent regulation of ENT1 is relevant in many cell types, since both ENT1 and CaM are ubiquitously expressed. I also examined N-linked glycosylation of ENT1. Previous work performed in yeast and oocytes have established that ENT1 is glycosylated at Asn48, but expression of a glycosylation deficient hENT1 mutant in a human cell line has not been published. I predicted that glycosylation was necessary for proper ENT1 function, and would alter protein abundance at the plasma membrane. Using chloroadenosine transport assays, I showed that N48Q mutant ENT1 protein is non-functional, and using NBTI binding analysis and immunofluorescence I identified that there was less glycosylation aberrant ENT1 at the plasma membrane compared to wild type protein. Furthermore, we have previously established in the lab that ENT1 has a variety of protein-protein interactions with other ENTs. I established here that removing N-glycosylation from ENT1 blocked the ability for ENT1 to have protein interactions with wild type ENT1. This work provides an understanding of novel mechanisms of ENT1 regulation which can be used as the basis of future clinically relevant research to enhance nucleoside drug uptake.