Abstract
Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.
Highlights
Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling
HENT1 and hENT2 function at pH 7.4, hENT3 functions at pH 5.5, and hENT4 functions at pH 5.5– 6.5 [10, 18]. Consistent with their apparent transport maxima, hENT1 and hENT2 are primarily localized to the cell surface [1, 3], whereas hENT3 is localized to vesicular and tubular intracellular organelles including the late endosome/lysosome and mitochondria [10, 19]. hENT4 is found in the plasma membrane of ventricular myocytes and vascular endothelial cells and transports adenosine in an acidic pH-dependent manner
The estimated R values for hENTs demonstrated a rank order of hENT3 (0.07) Ͼ hENT4 (0.63) Ͼ hENT2 (1.02) Ͼ hENT1 (1.02). This indicates that hENT3 is the strongest pH-dependent member of the family followed by hENT4, and hENT1 and hENT2 are pH-independent members (Fig. 1B)
Summary
Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Site-directed mutagenesis studies and chimeric ENT analysis are beginning to identify specific transmembrane regions and residues involved in the substrate selectivity (endogenous nucleosides and therapeutic nucleoside analogs) and inhibitor (e.g. cardiovascular drugs)-binding characteristics of ENTs [15,16,17], no crystal structures are available for any member of this family. -4) have a very similar membrane topology as well as substrate and inhibitor-binding characteristics [1, 3], yet individual hENTs drastically differ in their functional activity at various physiological pH as well as their cellular and subcellular distributions. HENT4 has been renamed as the plasma membrane monoamine transporter due to its relatively higher affinity to monoamines (e.g. serotonin) than adenosine [18]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
