Entry to "formula tunnel" revealed by SLC4A4 human mutation and structural model

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Point mutations in the electrogenic sodium bicarbonate cotransporter (NBCe1/SLC4A4) are linked to serious diseases like glaucoma, cataracts, and proximal renal tubular acidosis.
• The R298S mutation in NBCe1, located in the N-terminal domain, results in moderately reduced transporter function.

Purpose of the Study:

• To investigate the structural and functional roles of specific residues in the cytoplasmic N-terminal domain of NBCe1.
• To test a homology model of NBCe1 based on the Band 3 (AE1) crystal structure.

Main Methods:

• Homology modeling of NBCe1 using Band 3 (AE1) crystal structure coordinates.
• Site-directed mutagenesis to perturb interactions between Arg-298 and Glu-91/Glu-295.
• Functional analysis of mutated NBCe1 transporters expressed in Xenopus oocytes.

Main Results:

• Arg-298 is predicted to be in a subsurface pocket, interacting with Glu-91 or Glu-295 via hydrogen bonding and charge-charge interactions.
• Mutagenesis of either Arg-298 or Glu-91 significantly reduced NBCe1 transport function.
• Reversing the charges of these residues (E91R/R298E) restored transporter function, indicating their interdependence.

Conclusions:

• The cytoplasmic N termini of NBCe1 and potentially other SLC4 transporters play a direct role in controlling bicarbonate permeation.
• These findings challenge the established view of N-terminal domains in related transporters (like AE1) solely as protein-binding sites.