Inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hCNT3) predicts an elevator-type transport mechanism

The human SLC28 family of concentrative (Na⁺-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNT_NW to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter’s outward-facing conformation. In addition, both the Na⁺ and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.