Missense mutations and proximal RTA. Have we reached a new threshold? Focus on “Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis”

Snezana Petrovic, Thomas D. DuBose Jr.

the reabsorption of bicarbonate by the kidney requires parallel operation of apical transporters that secrete H+ and corresponding basolateral transporters that absorb bicarbonate. Approximately 80% to 90% of the filtered bicarbonate is reabsorbed in the proximal tubule, where secretion of H+ into HCO3-rich glomerular filtrate by the Na+/H+ exchanger, NHE-3, results in the formation of H2CO3 that is rapidly converted, within the tubule lumen, by membrane-bound carbonic anhydrase (type IV), to H2O and CO2, which can freely diffuse into the proximal tubule and, via intracellular carbonic anhydrase (type II), is converted to intracellular H2CO3 and HCO3. Bicarbonate exits the cell via the electrogenic Na+-3HCO3 symporter (NBCe1-A), with the negative cell potential serving as the primary driving force. This process is summarized in the proximal tubule cell model displayed in Fig. 1. Renal tubular acidosis (RTA) is a rare group of disorders whether inherited or acquired, although the incidence and prevalence are not well defined. The phenotype, when fully expressed, is recognized clinically as a chronic non-gap metabolic acidosis in the face of evidence of an inability of the kidney to excrete net acid appropriately, and is often associated with abnormalities of growth and development. RTAs involving the proximal tubule (PRTA) are the least common and are typically divided into two major categories: generalized disorders of proximal tubule reabsorption and isolated abnormalities in renal acidification. Several examples of the latter have been documented to date and include genetic defects of the basolateral Na+-3HCO3 symporter or NBCe1-A, the enzyme carbonic anhydrase type II, and NHE-3 (2).

Fig. 1.

Apical and basolateral transporters involved in bicarbonate absorption in the proximal convoluted tubule. See text for explanation.

Some of the mutations in genes that encode acid-base transport proteins completely abrogate transporter function or cause intracellular retention of the transporter, so that the disease-causing phenotype is the result of impaired proton or bicarbonate transport (7). Of particular interest are the functional consequences of mutations encoding transporters that retain a significant percentage of their transport function and are targeted to the cell membrane. In some of these instances, the alterations in the mutated proteins amount to the loss of proper targeting of a transporter to the luminal or basolateral membrane (1, 6), which illustrates the remarkable importance of the polar orientation of epithelial cells for preservation of transepithelial ion transport.

The issue of the directionality of transepithelial ion transport, via a novel mechanism, is highlighted in the study by Zhu et al. (8), published in this issue of American Journal of Physiology-Cell Physiology. In a series of originally conceived and elegantly designed studies, Zhu et al. probed the basis of the electrogenicity and stoichiometry of one of the key transporters in the proximal tubule, the sodium/bicarbonate symporter 1 (NBCe1-A), encoded by the solute-linked carrier 4 A4 (SLC4A4) gene. NBCe1-A sustains robust absorptive transepithelial flux of bicarbonate in the proximal tubule. Missense, nonsense, and deletion mutations of SLC4A4 cause proximal renal tubular acidosis, characterized by severe metabolic acidosis, often associated with ocular abnormalities such as cataracts and glaucoma, mental retardation, and hemiplegic migraines (7), as NBCe1 is also expressed in these tissues. Since the first report by Igarashi et al. (5) in 1999, SLC4A4 mutations have been the focus of detailed analysis, indicating that the phenotype of pure bicarbonate wasting in this form of PRTA results from loss of function, intracellular retention or mistargeting of NBCe1-A (7). Zhu et al. hypothesized that PRTA caused by the substitution of threonine by serine at position 485 in NBCe1-A (T485S) is mediated by the loss of NBCe1-A electrogenicity, which reverses basolateral bicarbonate flux and therefore impairs transepithelial bicarbonate reabsorption (8). The electrogenic properties of NCBe1-A are essential for its transport function because the reversal potential of NBCe1-A is near the membrane potential of the proximal tubule cell, rendering the magnitude and polarity of bicarbonate transport, mediated by NBCe1-A, very sensitive to alterations in membrane potential (7).

The T485S mutation was originally described by Horita et al. (4), who analyzed homozygous mutations in the common coding regions of NBCe1-A (T485S, A799V, and R881C) to correlate the degree of NBCe1-A impairment with the severity of PRTA. These studies indicated that a reduction of NBCe1-A activity of about 50% was sufficient to cause severe PRTA. While Horita et al. reported that T485S exhibited approximately half the activity of the wild-type NBCe1-A in ECV304 cells (4), the molecular mechanism by which this substitution altered transporter activity remained unclear. A technically challenging aspect of defining the molecular mechanism of NBCe1-A impairment produced by T485S substitution is inherent in the structural similarity of threonine and serine. Zhu and associates approached this prospective difficulty by labeling cysteine-substituted T485 with a small thiol-reactive reagent, [14C]NEM, which labels −SH groups of the cysteine residues only when they are exposed to an aqueous environment of a presumed ion permeation pathway, or labeling with membrane-permeable versus impermeable methanethiosulfonate (MTS) reagents, which can indicate whether a specific residue is located at the ion interaction site. These experiments lend strong support to the notion that T485 is, in fact, positioned at the NBCe1-A ion interaction site (8). Another interesting aspect of the study design was the use of NO3 ion instead of CO3−2 to examine NBCe1-A substrate specificity, that is, whether it carries HCO3 or CO3−2, since neither ion could be measured directly. NO3 is a logical substitute for CO3−2 because the two ions have a similar molecular radius and electrostatic properties, and transport of NO3 can be measured as a rate of H+/NO3 exchange expressed endogenously in HEK 293 cells. The difference in valence of NO3 and CO3−2 makes some of the conclusions extrapolated from these experiments somewhat speculative. Nonetheless, the data suggest that NBCe1-A preferentially transports CO3−2, which led the authors to propose that the T485S mutation alters the affinity of wild-type NBCe1-A for CO3−2, such that the mutant form transports Na+ and HCO3 preferentially at 1:1 stoichiometry, rendering the transporter electroneutral (8). In other words, Zhu et al. propose that the electrogenicity of NBCe1-A depends on the affinity of the transporter for the substrate accompanying the sodium ion (carbonate vs. bicarbonate ion) and that this affinity ratio is altered with T485S substitution. The high expression of several cytosolic and membrane-bound isoforms of carbonic anhydrase in proximal tubule cells in association with NBCe1-A predicts a low concentration of carbonate relative to a high concentration of bicarbonate in vivo. Whether there is an advantage of preferential uptake of carbonate over bicarbonate in wild-type NBCe1-A in vivo is not entirely clear.

This uncertainty notwithstanding, the most important contribution of the current study is the demonstration that a single amino acid substitution may cause human disease solely by altering the electrogenicity of a membrane transporter. Future studies will need to address whether the observation in the expression system used in this study holds true for suitable proximal tubule cell lines or isolated proximal tubules. This approach assumes importance since the stoichiometry of NBCe1-A is known to be cell-type specific (3). Therefore, the issue of whether this substitution results in a reversal of basolateral bicarbonate flux, as predicted here, will require additional study. Since cell models that faithfully recapitulate the transporters involved in bicarbonate transport by the human proximal tubule are not yet available, and given the technical challenges, which Zhu et al. overcame to the extent currently possible, addressing this issue with greater specificity may take some time.


No conflicts of interest, financial or otherwise, are declared by the author(s).


S.P. and T.D.D. prepared the figure; S.P. drafted the manuscript; T.D.D. edited and revised the manuscript; T.D.D. approved the final version of the manuscript.


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