Substituted Cysteine Accessibility Method (SCAM) Analysis of the Transport Domain of Human Concentrative Nucleoside Transporter 3 (hCNT3) and Other Family Members Reveals Features of Structural and Functional Importance [Membrane Biology]

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members, hCNT1, hCNT2 and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C-), substituted cysteine accessibility method (SCAM) analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS) was performed on the transport domain (IH2, HP1, TM7, TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C-) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C-) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance and provided evidence of an emerging novel elevator-type mechanism of transporter function.

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