A novel mutant Na⁺/HCO3⁻ cotransporter NBCe1 in a case of compound‐heterozygous inheritance of proximal renal tubular acidosis

Abstract

Proximal renal tubular acidosis (pRTA) is a rare, recessively-inherited disease characterized by abnormally acidic blood, blindness, as well as below average height and weight. pRTA is typically associated with homozygous mutation of the solute carrier 4 family gene SLC4A4. SLC4A4 encodes the electrogenic sodium bicarbonate cotransporter NBCe1, a membrane protein that acts to maintain intracellular and plasma pH. We present the first description of a case of compound-heterozygous inheritance of pRTA. The individual has inherited two mutations in NBCe1: p.Arg510His (R510H) and p.Gln913Arg (Q913R), one from each parent. In addition to the usual features of pRTA, the patient exhibits unusual signs, such as muscle spasms and fever. We have recreated these mutant transporters for expression in model systems. We find that both of the mutant proteins exhibit substantial intracellular retention when expressed in mammalian renal cell lines. When expressed in Xenopus oocytes, we find that the R510H and Q913R-mutant NBCe1 molecules exhibit apparently normal Na /HCO cotransport activity but that Q913R is associated with an unusual HCO independent anion-leak. We conclude that a reduced accumulation of NBCe1 protein in the basolateral membrane of proximal-tubule epithelia is the most probable cause of pRTA in this case. We further note that the Q913R-associated anion-leak could itself be pathogenic if expressed in the plasma membrane of mammalian cells, compromising the benefit of strategies aiming to enhance mutant NBCe1 accumulation in the plasma membrane.

The role of disease-linked residue glutamine-913 in support of the structure and function of the human electrogenic sodium/bicarbonate cotransporter NBCe1-A

“…Figure 9A shows that WT (disclosed by EGFP immunoreactivity, green) exhibits a peripheral distribution that substantially overlaps (yellow) with that of the basolateral marker Na + /K + -ATPase (red), consistent with lateral expression of WT. Figure 9B shows that the distribution of Q913R does not substantially overlap with the location of Na + /K + -ATPase, consistent with the previous report of enhanced intracellular retention for Q913R 14 . Of the de novo mutants (Fig.…”

“…On a technical note, a failure of NBCe1 mutants to accumulate in the oocyte plasma membrane typically presages intracellular retention of those mutants in polarised MDCK cells, as has been previously described for R510H, A799I, A799V, R881C, and Q913R 14 , 17 , 21 and is presently described for Q913C and Q913K. However in the present study Q913L and Q913S are intracellularly retained in MDCK cells yet accumulate normally in the oocyte plasma membrane.…”

“…The construction of the plasmids for expression of wild-type NBCe1-A with a carboxy-terminal enhanced green fluorescent protein (EGFP) tag in mammalian cells (NBCe1-A-EGFP.pcDNA3.1) and EGFP-tagged NBCe1-A in Xenopus oocytes (NBCe1-A-EGFP.pGH19), and the introduction to each of the Q913R mutation, has been described previously 14 . Other codon substitutions at position 913 (underlined below, collectively referred to as Q913X, where X is any amino acid:) 25 were introduced using a QuikChange site-directed mutagenesis kit (Agilent Technologies Inc., Santa Clara, CA) using the following primers with their reverse complements: 5′-GTTCACTTTCCTG TGC GTGTTGTGTCTGGC-3′ (p.Gln913Cys aka Q913C: chosen because this de novo mutant had been previously included in a cysteine scanning study and was shown to exhibit defective plasma membrane targeting:) 26 , 5′-GTTCACTTTCCTG GAG GTGTTGTGTCTGGC-3′ (pGln913Glu aka Q913E: chosen as an example of a ‘negatively charged’ residue), 5′-GTTCACTTTCCTG AAG GTGTTGTGTCTGGC-3′ (p.Gln913Lys aka Q913K: chosen as a second example of a ‘positively charged’ residue), 5′-GTTCACTTTCCTG CTG GTGTTGTGTCTGGC-3′ (pGln913Leu aka Q913L: chosen as an example of a ‘nonpolar, aliphatic’ residue), and 5′-GTTCACTTTCCTG AGC GTGTTGTGTCTGGC-3′ (p.Gln913Ser aka Q913S: chosen as an example of a ‘polar, uncharged’ residue).…”

“… 6 ) and missense (p.Arg298Ser 7 , 8 , p.Ser427Leu 9 , p.Thr485Ser 10 , p.Gly486Arg 11 , p.Arg510His 5 , 7 , 12 , p.Leu522Pro 13 , p.Ala799Val 10 , and p.Arg881Cys 5 , 10 ). The fourteenth and most recently described mutation p.Gln913Arg is inherited in compound heterozygous form with p.Arg510His 14 . All mutations result in the loss of functional NBCe1 expression by decreasing NBCe1 abundance in the plasma membrane and/or Na + /2HCO 3 − cotransport activity.…”

Molecular Pathophysiology of Acid-Base Disorders

“…Mutations identified are mostly missense mutations that affect trafficking of mutant transporters to the membrane and/or alter function of the transporter, e.g. reduced activity or inducing anion leaks 55,58,59 .…”