Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.
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.
Zhu
Quansheng
Q
Division of Nephrology, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1689, USA.
Kao
Liyo
L
Azimov
Rustam
R
Abuladze
Natalia
N
Newman
Debra
D
Pushkin
Alexander
A
Liu
Weixin
W
Chang
Connie
C
Kurtz
Ira
I
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*
D001457:Anion Exchange Protein 1, Erythrocyte; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters