The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia

• Published in: American Journal of Physiology: Cell Physiology 2006-10, Vol.291 (4), p.C788-C801
• Main Authors: Toye, Ashley M, Parker, Mark D, Daly, Christopher M, Lu, Jing, Virkki, Leila V, Pelletier, Marc F, Boron, Walter F
• Format: Article
• Language: English
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Acidosis, Renal Tubular - genetics; Acidosis, Renal Tubular - metabolism; Acidosis, Renal Tubular - physiopathology; Animals; Arginine; Cell Line; Cell Membrane - metabolism; Cell Polarity; Cellular biology; Cysteine; Dogs; Electric Conductivity; Electrophysiology; Epithelium - metabolism; Epithelium - physiopathology; Fluorescent Dyes - pharmacokinetics; Gene expression; Genotype & phenotype; Green Fluorescent Proteins - antagonists & inhibitors; Green Fluorescent Proteins - pharmacokinetics; Humans; Hydrogen-Ion Concentration; Immunologic Techniques; Kidney - metabolism; Kidney - physiopathology; Membranes; Mutation, Missense; Oocytes - metabolism; Pathology; Proteins; Sodium-Bicarbonate Symporters - genetics; Sodium-Bicarbonate Symporters - metabolism; Studies; Tissue Distribution; Transfection; Xenopus
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00094.2006

1 Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom; and 2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut Submitted 28 February 2006 ; accepted in final form 9 May 2006 The human electrogenic renal Na-HCO 3 cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO 3 – from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na + affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4 ; Na + -HCO 3 cotransporter 1 Address for reprint requests and other correspondence: W. F. Boron, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520-8026 (e-mail: walter.boron{at}yale.edu )