NBCe1‐A residues mutated in patients with pRTA: A Topological Analysis

Abstract

Eight missense mutations in Na+ coupled HCO3− cotransporter 1 (NBCe1‐A) (R298S, S427L, T485S, G486R, R510H, L522P, A799V and R881C) are reported to cause proximal renal tubular acidosis (pRTA). In this study, we explored location of the pRTA causing mutations and their structural importance in NBCe1‐A by substituted cysteine accessibility method (SCAM). The 8 residues were individually substituted with cysteine (cys) in a modified NBCe1‐A backbone with no free reactive cys (NBCe1‐A‐5C−). Locations of the introduced cys were determined by whole cell labeling with two cysteine specific reagents: membrane permeant biotin maleimide and a membrane impermeant MTS‐TAMRA. Results showed none of the 8 residues is on the surface of NBCe1‐A. Selective mutation of T485 revealed it resides in a unique position that is important for normal function. Further topologic analysis of NBCe1‐A by SCAM on 56 individually introduced cys in the NBCe1‐A‐5C− revealed that the first half of the transmembrane region of NBCe1‐A contains 8 transmembrane segments (TM) highly homologous to anion exchanger 1 (AE1), whereas the remainder of the cotransporter folds tightly unlike AE1. On the basis of these results, we propose a new topologic model of NBCe1‐A which allows the assignment of pRTA causing mutations to the following regions: S427L, TM1; T485S and G486R, TM2; R510H and L522P, TM3; A799V, TM10; R881C, TM12.Source of Research Support: NIH