Molecular Determinants of Acidic pH-Dependent Transport of Human Equilibrative Nucleoside Transporter-3 [Membrane Biology]

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 Asp219 and Glu447, 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 Asp219 or Glu447 with any other residues resulted in robust activity that was pH independent. Dual substitution of Asp219 and Glu447 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 Asp219 and Glu447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2vpbYIh
via IFTTT