Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Abstract

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na؉ and H ؉ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na ؉ /nucleoside and H ؉ /nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H ؉ dependence (all mutants), shift in Na ؉ :nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na ؉ -specific hCNT1, uncoupled Na ؉ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

“…In TM 13, three mutants (A601C(CϪ), A609C(CϪ), and L612C(CϪ)) were PCMBS-inhibitable in both cation conditions. Inhibition of transport by PCMBS was previously described for E519C in hCNT3 TM 11A (30). Scanning of the loop between TMs 11 and 12, which includes TM 11A, revealed additional PCMBS-inhibitable residues.…”

“…In hCNT3, this motif corresponds to residues GYKT-FFNEFVAY, which span the region from Gly 512 to Tyr 523 . Although the transport activity of E519C(CϪ) was Ͻ0.1 pmol/ oocyte⅐min Ϫ1 and therefore not analyzed in the present study, we have previously characterized the transport activities of two Glu 519 mutants (E519D and E519C) in an hCNT3 wild-type background (30). Both of these mutants displayed robust transport of uridine in the presence of Na ϩ but only low (E519D) or not detectable (E519C) levels of transport activity in Na ϩ -free, acidified medium (30).…”

“…Studies from our laboratory have previously used PCMBS to investigate individual topological features and residues of hCNT1 and hCNT3 (26,29,30,34). Incorporating results from TM 12 (26), the present investigation reports a SCAM analysis of the entire C-terminal one-third of the latter protein (hCNT3CϪ TMs 11-13, inclusive) (Fig.…”

“…Within the conserved CNT family (G/A)XKX 3 NEFVA(Y/M/F) motif of TM 11A, a sequence of eight consecutive residues, extending from Phe 516 to Tyr 523 , were PCMBS-sensitive upon conversion to cysteine under both cation conditions, the only exceptions being Glu 519 , which showed low levels of uridine transport activity, and Ala 522 , which showed PCMBS inhibition only in Na ϩ -containing, H ϩ -reduced medium. In the wild-type hCNT3 background, mutant E519C exhibited Na ϩ -specific nucleoside transport activity and was PCMBSsensitive in the presence of Na ϩ (30). Within this region of eight PCMBS-inhibitable residues, three (Phe 516 , Ala 522 , and Tyr 523 ) were uridine-protectable.…”

“…Located deeper within the plane of the membrane, other uridine-protectable residue positions in TM 12 were PCMBS-sensitive in both H ϩ -and Na ϩ -containing media (26). hCNT3 Glu 519 and the corresponding residue in hCNT1 (Glu 498 ) in region TM 11A were also identified as having key roles in permeant and cation binding and translocation (29,30), and hCNT3 E519C showed inhibition of uridine uptake by PCMBS (30). Centrally positioned within the highly conserved CNT family motif (G/A)XKX 3 NEFVA(Y/M/F), residue 519 is proposed to be a direct participant in cation coupling via the common hCNT3 Na ϩ /H ϩ -binding site that, in other CNTs, is either Na ϩ -specific (e.g.…”