Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter

Yuma Iwai, Setsuko Kamatani, Sawako Moriyama, Hiroshi Omote

Research output: Contribution to journalArticle

Abstract

Vesicular nucleotide transporter (VNUT) plays a key role in purinergic signalling through its ability to transport nucleotides. VNUT belongs to the SLC17 family, which includes vesicular glutamate transporters (VGLUTs) and Type I Na+/phosphate cotransporters. All of these transporters exhibit membrane potential and Cl--dependent organic anion transport activity and have essential arginine in the transmembrane region. Previously, we reported that ketoacids inhibit these transporters through modulation of Cl- activation. Although this regulation is important to control signal transmission, the mechanisms underlying Cl--dependent regulation are unclear. Here, we examined the functional roles of Cl- and essential arginine residue on ATP binding to VNUT using the fluorescent ATP analogue trinitrophenyl-ATP (TNP-ATP). The fluorescence of TNP-ATP was enhanced by VNUT, whereas no enhancement was observed by VGLUT. Concentration-dependence curves showed that TNP-ATP was a high-affinity fluorescent probe for VNUT, with a Kd of 4.8 μM. TNP-ATP binding was competitive to ATP and showed similar specificity to transport activity. Addition of Cl- and ketoacids did not affect the apparent affinity for TNP-ATP. The Arg119 to Ala mutant retained TNP-ATP binding ability with slightly reduced affinity. Overall, these results indicated that Cl- and essential arginine were not important for ATP binding.

Original languageEnglish
Pages (from-to)479-486
Number of pages8
JournalJournal of biochemistry
Volume165
Issue number6
DOIs
Publication statusPublished - 2019

Keywords

  • SLC17
  • chloride
  • ketone body
  • trinitrophenyl-ATP
  • vesicular nucleotide transporter

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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