Urate transport function of rat sodium-dependent nucleobase transporter 1

Abstract

Sodium‐dependent nucleobase transporter 1 (SNBT1) is a nucleobase‐specific transporter identified in our recent study. In an attempt to search for its potential substrates other than nucleobases in this study, we could successfully find urate, a metabolic derivative of purine nucleobases, as a novel substrate, as indicated by its specific Na+‐dependent and saturable transport, with a Michaelis constant of 433 μmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin‐Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co‐introduction of rat breast cancer resistance protein (rBCRP), a urate efflux transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.

Distinct specific interactions of the UapA transporter with membrane lipids are critical for dimerization, ER-exit and function

“…hypoxanthine, uracil or thymine) (Fig 7B). Importantly, rSNBT1-dependent 5-FU sensitivity was Na + -dependent (Fig 7C), compatible with the physiological mechanism of functioning of rSNBT1 in rat [22,23].…”

“…To test this idea, we used a characterized NAT homologue from rat, rSNBT1, which similarly to UapA acts as nucleobase transporter [22,23]. Noticeably, UapA and rSNBT1 have different specificity and cation dependence, as rSNBT1 is a rather promiscuous Na + symporter specific for pyrimidines (uracil, thymine) and most purines (hypoxanthine, guanine, xanthine and uric acid), while UapA is quite specific for xanthine and uric acid and utilizes H + for co-transport.…”

Specific Residues in a Purine Transporter Are Critical for Dimerization, ER Exit, and Function

“…To test this idea, we used a characterized NAT homolog from rat, rSNBT1, which similarly to UapA acts as nucleobase transporter (Yamamoto et al 2010;Yasujima et al 2018). Noticeably, UapA and rSNBT1 have different specificities and cation dependence, as rSNBT1 is a rather promiscuous Na + symporter specific for pyrimidines (uracil and thymine) and most purines (hypoxanthine, guanine, xanthine, and uric acid), while UapA is quite specific for xanthine and uric acid, and utilizes H + for cotransport.…”

“…Importantly, using information on a specific core residue that proved to have a key role in stabilizing UapA, Thr401, we genetically manipulate and achieve the functional expression, for the first time, of a mammalian homolog of UapA in A. nidulans. This opens the way for the functional expression of mammalian transporters, that belong to the nucleobase ascorbate transporter (NAT) family, including those essential for vitamin C transport in humans (Yamamoto et al 2010;Yasujima et al 2018), in the model Aspergillus system.…”

Structure-function relationships in transmembrane transporters

“…In order to understand more about the evolution of substrate specificity in the NAT family, we tried to functionally express characterized NAT homologues in A. nidulans and study structurefunction relationships. At first, we decided to express the well characterized NAT homolog from rat, rSNBT1 that is a rather promiscuous Na + symporter specific for pyrimidines (uracil and thymine) and purines (hypoxanthine, guanine, xanthine, and uric acid) [4,173]. Previous attempts to express rSNBT1 or rSNBT1/UapA chimeric transporters in A. nidulans or S. cerevisiae have failed, always due to total ER retention (C. Gournas, S. Amillis, B. Byrne, and G. Diallinas, unpublished data).…”