Entry to "formula tunnel" revealed by SLC4A4 human mutation and structural model.
Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na(+) bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge-charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was "repaired" by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling HCO(3)(-) permeation.
Chang
Min-Hwang
MH
Department Physiology & Biophysics and Biology, Case Western Reserve University, Cleveland, OH 44106, USA.
DiPiero
Jennifer
J
Sönnichsen
Frank D
FD
Romero
Michael F
MF
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics*; D012997:Solvents / Q000737:chemistry; D014981:Xenopus
C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D012997:Solvents