title: 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.
abstract: 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 coliCNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression inXenopusoocytes and the engineered cysteine-less hCNT3 protein hCNT3(C-), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagentp-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and 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.
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Ras
authors.initials: R
authors.lastname: Mulinta
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Sylvia Y M
authors.initials: SYM
authors.lastname: Yao
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and.
authors.forename: Amy M L
authors.initials: AML
authors.lastname: Ng
authors.affiliation: Oncology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and.|the Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Carol E
authors.initials: CE
authors.lastname: Cass
authors.affiliation: From the Membrane Protein Disease Research Group, Departments of Physiology and james.young@ualberta.ca.
authors.forename: James D
authors.initials: JD
authors.lastname: Young
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C490231:NupC protein, E coli; C065171:cif nucleoside transporter
keywords: human concentrative nucleoside transporter 3 (hCNT3); membrane protein; membrane transport; nucleoside transport; nucleoside transporter; nucleoside/nucleotide transport; recombinant protein expression; substituted cysteine accessibility (SCAM) analysis; transporter