Rescue of the temperature-sensitive, autosomal-recessive mutation R298S in the sodium-bicarbonate cotransporter NBCe1-A characterized by a weakened dimer and abnormal aggregation.

backgroundBand 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.

methodsWe 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.

resultsWe 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.

conclusionsThe R298S is a temperature-sensitive mutation in Nt that results in instability of the colloidal system leading to abnormal aggregation. GENERAL SIGNIFICANCE: Our findings provide new perspectives to the aberrant mechanism of certain ocular pathologies and type-II RTA associated with the R298S mutation.