Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport.
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.
Abuladze
Natalia
N
Division of Nephrology, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Room 7-155 Factor Building, Los Angeles, CA 90095, USA.
Azimov
Rustam
R
Newman
Debra
D
Sassani
Pakan
P
Liu
Weixin
W
Tatishchev
Sergei
S
Pushkin
Alexander
A
Kurtz
Ira
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D004563:Electrochemistry; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics* / Q000378:metabolism*
D001639:Bicarbonates; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D012964:Sodium