Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

Abstract

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent proteintagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

“…The LL/AA sorting mutant provided favorable conditions for testing this hypothesis because it allows replacing the poorly tractable lysosomal activity by a classic, whole-cell influx equivalent to lysosomal efflux. Several lysosomal transporters have been successfully characterized using this whole-cell approach (6,12,18,37,38). In preliminary experiments, we expressed PQLC2-LL/AA-EGFP in HEK-293 cells and examined their ability to take up…”

Adenosine Signaling in Striatal Circuits and Alcohol Use Disorders

“…ENT1 is sensitive to nanomolar concentrations of nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside; NBMPR), whereas ENT2 is resistant to NBMPR up to 1 mM (Baldwin et al, 1999;Yao et al, 1997). Furthermore, ENT1 and ENT2 are widely expressed throughout the central nervous system (Jennings et al, 1998;, while ENT3 appears to be expressed outside the CNS (Baldwin et al, 2005). The expression and pharmacological properties of ENT4 have yet to be fully characterized.…”

Regulation of adenosine levels during cerebral ischemia

“…ENT3 and ENT4 are less studied members of the ENT family and their importance in regulating adenosine levels and adenosine receptor activity is poorly characterized. ENT3 has an intracellular localization and is, thus, unlikely to affect adenosine levels and adenosine receptor activity [14] . ENT4 is also known as plasma membrane monoamine transporter (PMAT).…”