Rescue of the temperature-sensitive, autosomal-recessive mutation R298S in the sodium-bicarbonate cotransporter NBCe1-A characterized by a weakened dimer and abnormal aggregation.
BACKGROUND
Band keratopathy, an ocular disease that is characterized by hypercalcemia and opaque bands across the cornea, has been associated with kidney disease. Type-II renal tubular acidosis (RTA), a condition in which the kidneys fail to recover bicarbonate (HCO3-) in the proximal tubule of the nephron, results in HCO3- wastage in the urine and low blood pH. The development of these diseases is associated with autosomal-recessive mutations in the Na+-coupled HCO3- cotransporter NBCe1-A located at the basolateral membranes of either cell type.


METHODS
We provide insight into the devastating R298S mutation found in type-II RTA-afflicted individuals using confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in human corneal endothelial and proximal tubule cells and from in-depth biophysical studies of their cytoplasmic N-terminal domains (Nt and Nt-R298S), including Nt crystal structure, melting-temperature, and homodimer dissociation constant (KD) analyses.


RESULTS
We illuminate and rescue trafficking defects of the R298S mutation of NBCe1-A. The KD for Nt monomer-dimer equilibrium is established. The KD for Nt-R298S is significantly higher, but immeasurable due to environmental factors (pH, temperature, concentration) that result in dimer instability leading to precipitation. The crystal structure of Nt-dimer shows that R298 is part of a putative substrate conduit and resides near the dimer interface held together by hydrogen-bond networks.


CONCLUSIONS
The R298S is a temperature-sensitive mutation in Nt that results in instability of the colloidal system leading to abnormal aggregation.


CONCLUSIONS
Our findings provide new perspectives to the aberrant mechanism of certain ocular pathologies and type-II RTA associated with the R298S mutation.
Department of Medicine, The George Washington University; Division of Renal & Hypertension, The GW Medical Faculty Associates, 2300 I (eye) Street NW, Ross Hall Room 436B, Washington D.C. 20052, United States. Electronic address: hgill@gwu.edu.
Harindarpal S
HS
Gill
Department of Medicine, The George Washington University; Division of Renal & Hypertension, The GW Medical Faculty Associates, 2300 I (eye) Street NW, Ross Hall Room 436B, Washington D.C. 20052, United States.
Kun-Young
KY
Choi
Department of Medicine, The George Washington University; Division of Renal & Hypertension, The GW Medical Faculty Associates, 2300 I (eye) Street NW, Ross Hall Room 436B, Washington D.C. 20052, United States.
Lakshmi
L
Kammili
Department of Medicine, The George Washington University; Division of Renal & Hypertension, The GW Medical Faculty Associates, 2300 I (eye) Street NW, Ross Hall Room 436B, Washington D.C. 20052, United States.
Anastas
A
Popratiloff
D002460:Cell Line; D003102:Colloids; D003198:Computer Simulation; D004728:Endothelium, Corneal / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D006860:Hydrogen Bonding; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D066329:Protein Aggregates; D011487:Protein Conformation; D011489:Protein Denaturation; D055503:Protein Multimerization; D055550:Protein Stability; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D044366:Transition Temperature
D003102:Colloids; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters
Composition-gradient light scattering; Confocal microscopy; Protein aggregation; X-ray crystallography