Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport

Abstract

We have previously reported a topological model of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na(+)-dependent base flux of the mutants was determined using intracellular pH measurements with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na(+)-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented.

Figure 1. Amino acid sequence of human kNBC1

A, alignment of human kNBC1 and AE1. B, location of kNBC1 amino acids mutated in this study (filled circles).

Figure 2. Functional characterization of wt-kNBC1 and mutant-kNBC1

Constructs were expressed in HEK-293T cells.

Figure 3. Determination of wt-kNBC1 and mutant-kNBC1 construct plasma membrane expression in HEK-293T cells

Cell surface biotinylation and immunoblotting with kNBC1-specific antibody was used. Shown are the constructs that were studied functionally.

Figure 4. Critical amino acid residues in kNBC1 that determine its functional properties

Grey rectangles represent transmembrane segments (TMs). Extracellular loops are designated as EL1–EL5, and intracellular loops are designated as IL1–IL4. Acidic, basic and neutral amino acid residues are shown in red, blue and grey, respectively.

Figure 5. Amino acid mutations, which had a severe (< 25% of wt-kNBC1 function, ), moderate (25–60% of wt-kNBC1 function, ) and a small (> 60% of wt-kNBC1 function, ) effect on kNBC1 function

Designation of TMs, ELs and ILs is same as in Fig. 4. The residues that are required for plasma membrane expression are shown in brown.

Figure 6. Isoelectric point (pI) of ELs and ILs of human kNBC1 and AE1

–, pI below 7; +, pI above 7; 0, pI ∼7.

Figure 7. Amino acids in ILs and in ELs involved in ISF of kNBC1

A, amino acids in ILs involved in ISF in the inward-facing conformation of kNBC1. B, amino acids in ELs involved in ISF in the outward-facing conformation of kNBC1. Amino acids involved in the ITS are shown with a smaller font in A and B.