Structural Characterization of the Kidney Specific Form of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐A

The kidney specific form of the electrogenic sodium bicarbonate cotransporter NBCe1‐A (SLC4A4 gene) plays a key role in bicarbonate reabsorption in the kidney proximal tubule. Mutations in the SLC4A4 gene cause severe proximal renal tubular acidosis (pRTA). The current therapy of pRTA is limited to bicarbonate administration. We have recently reported an approach to target pRTA induced by premature stop codons using compounds that induce ribosomal read‐through of mutant NBCe1‐A. To develop specific therapeutic small molecules for the known NBC1‐A missense mutations requires that the high‐resolution structure of NBCe1‐A be characterized. Towards this goal, we have cloned and expressed tagged‐human NBCe1‐A in insect Sf9 and yeast (Pichia) cells. The protein was predominantly tetrameric in Sf9 and dimeric in Pichia cells using size‐exclusion chromatography, non‐denaturing polyacryl amide gel electrophoresis, and transmission electron microscopy (EM). Given the greater stability of the dimer than the tetramer (that aggregated easily), we used Pichia as an expression system. Purified NBCe1‐A dimers were negatively stained with uranyl acetate using the double carbon method, given the recent success of this method in the structural characterization of other macromolecules at medium resolution. The structural models of the NBCe1‐A dimer generated using single particle reconstruction and electron tomography, were very similar. A model of the NBCe1‐A dimer was generated that will be used for high‐resolution structural characterization of NBCe1‐A using cryoEM.