title: Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.
abstract: NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.
authors.affiliation: Division of Nephrology, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689, USA.
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Zhu
authors.forename: Liyo
authors.initials: L
authors.lastname: Kao
authors.forename: Rustam
authors.initials: R
authors.lastname: Azimov
authors.forename: Natalia
authors.initials: N
authors.lastname: Abuladze
authors.forename: Debra
authors.initials: D
authors.lastname: Newman
authors.forename: Alexander
authors.initials: A
authors.lastname: Pushkin
authors.forename: Weixin
authors.initials: W
authors.lastname: Liu
authors.forename: Connie
authors.initials: C
authors.lastname: Chang
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters