Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

Abstract

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.

Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

“…For example, we showed that the interaction between the C3′ ribose hydroxyl group and Ser 371 on TM7b is important in vcCNT (Figures 1C and 4A). Consistent with our observation, the hCNT1S546P variant is non-functional (Ser 546 in hCNT1 is equivalent to Ser 371 in vcCNT) (Cano-Soldado et al, 2012). Another important finding of our studies is that interactions with the nucleobase and ribose can be additive, and thus the loss of binding energy from modification of part of the nucleoside can be compensated for by the gain of energy from modification of another part of the nucleoside.…”

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner

“…This variant lacks nucleoside transport function, but apparently retains the ability to fold normally and insert into the plasma membrane. 18 Forty-eight hours after infecting cells with an hCNT1S546P adenoviral vector (AdhCNT1SP) at an MOI of 10, no increase in cytidine uptake was observed; in contrast, upregulation of hCNT1-related activity was evident under the same experimental conditions in AdhCNT1-infected cells (Figure 6a). Interestingly, like AdhCNT1, AdhCNT1SP induced cell-cycle arrest at S phase in both NP-9 and NP-29 cells (Figure 6b).…”

Functional disruption of pyrimidine nucleoside transporter CNT1 results in a novel inborn error of metabolism with high excretion of uridine and cytidine

“…The variant affects a highly conserved residue in the CNT1 protein (Figure ). This specific variant has previously been shown to abolish the nucleoside transporter function of CNT1 . Disruption of this function would result in a biochemical phenotype with unusually high excretion of uridine and cytidine as CNT1 is known to be a high affinity transporter of both pyrimidine nucleosides.…”

“…The Ser546 residue of CNT1 is highly conserved in evolution and an analogous serine residue also occurs in the other CNT‐type transporters CNT2 and CNT3 . Based on data from Leabman et al, Gray et al described a high degree of haplotype diversity in SLC28A1 and functionally analyzed coding region variants of the gene.…”

“…The allele frequency of the variant in ExAc amounts to 0.001055% . The variant has been extensively studied by independent groups . Thymidine uptake in Xenopus laevis oocytes expressing the mutant SLC28A1 gene showed that the mutant CNT1 transporter is nonfunctional .…”