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.
Highlights
Of the known SLC4 transporters, the structural and functional importance of the C-terminal transmembrane region of the anion exchanger AE1 has been extensively analyzed
Given the biological importance of addressing this question for understanding the transport mechanism(s) of SLC4 HCO3Ϫ transporters, we extensively examined the properties of the C-terminal region of NBCe1-A
Construction of 168 Introduced Cysteines in the C-terminal Region of NBCe1-A—In our previous random cysteine mutagenesis study of NBCe1-A, we were unable to detect the two reentrant loops that were predicted based on the topology of AE1 (Fig. 1) [10, 14]
Summary
MTS-TAMRA Labeling Assay—HEK 293 cells expressing mutant NBCe1-A proteins were washed three times with PBS and incubated with 100 M MTS-TAMRA (1:1,000 dilution in PBS, pH 7.4) for 3 min on ice. The cells were washed twice with PBS and subsequently imaged (Olympus BH2 microscope) using an excitation wavelength of 545 nm and an emission wavelength of 590 nm. The membrane pellet was rinsed twice and resuspended in 500 l of PBS, pH 7.5, followed by adding 1 l of MTS-TAMRA (50 mM stock in dimethyl sulfoxide), and incubated for 20 min at room. For KCl treatment, the EDTA-treated membranes were incubated with 1 M KCl at room temperature for 30 min, and the samples were BM labeled as described above. Dunnett’s t test was used to assess statistical significance with p Ͻ 0.05 considered significant
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