Topological Location and Structural Importance of the NBCe1-A Residues Mutated in Proximal Renal Tubular Acidosis

Abstract

NBCe1-A electrogenically cotransports Na

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis

“…Forty-eight hours after transfection, HEK 293 cell membranes were prepared as described (46). [ 14 C]NEM labeling was performed as described previously (13) -free solution and monitoring the pHi changes.…”

“…The COOH-terminal cytoplasmic tail is critically involved in NBCe1-A plasma membrane processing (22,26,38). The transmembrane region of NBCe1-A contains 14 transmembrane segments (TM), and the last five TMs may participate in forming a scaffold to accommodate NBCe1-A substrate ion interaction sites (45,46). Three amino acids in NBCe1-A-TM1 (Ala428, Ala435, and Thr442) (44) and one amino acid in TM8 (Leu750) (25) have been determined to line the ion permeation pathway and are potentially involved in ion translocation.…”

“…Based on our most recent NBCe1-A topological model (46), all the residues mutated in pRTA (pRTA residues) are located in the transmembrane region (TM1, 3, 4, 10, and 12, respectively) of the transporter except R298S, which resides in the NH 2 -terminal cytoplasmic domain. The transmembrane region-localized pRTA residues are not exposed to the intra-or extracellular surface of NBCe1-A (46), indicating that they may reside in a critical region involved in either protein folding, helices packing, or ion translocation. Specifically, R298S was predicted to affect ion entry into the NBCe1-A ion translocation pathway (4).…”

“…Unlike the aforementioned missense mutations, NBCe1-A-T485S processes to the plasma membrane normally and retains ϳ50% of wild-type NBCe1-A (wt-NBCe1-A) base transport function when expressed in ECV 304 or HEK 293 cells (15,38,46). Given the structural and chemical similarities between threonine and serine, it would not be predicted that this substitution impairs NBCe1-A base transport causing pRTA.…”

Pathophysiological Roles of Mutations in the Electrogenic Na+-HCO - Cotransporter NBCe1

“…Topological analysis using the substituted cysteine accessibility method suggests that most of these mutations are buried in the protein complex/lipid bilayer where they perform important structural roles [38]. In particular, the amino acid substitution analysis revealed that Thr 485 might reside in a special position, which seems to require the OH group side chain to maintain a normal conformation of NBCe1A.…”