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

摘要

Mutations in SLC4A4, the gene encoding the electrogenic Na⁺-HCO3⁻ cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ∼50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3⁻ as a surrogate ion for CO3²⁻, our result indicated that NBCe1-A mediates electrogenic Na⁺-CO3²⁻ cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3⁻, compatible with the hypothesis that it mediates Na⁺-HCO3⁻ cotransport. In patients, NBCe1-A-T485S is predicted to transport Na⁺-HCO3⁻ in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3⁻ absorption, possibly representing a new pathogenic mechanism for generating human pRTA.