The auto-recessive mutation R298S of the sodium bicarbonate cotransporter NBCe1-A leads to a trafficking defect that underlies proximal renal tubule acidosis and myriad ocular pathologies
BACKGROUND: Proximal renal tubule acidosis (pRTA) is a devastating disease in which afflicted individuals fail to reabsorb bicarbonate in the proximal segment of the nephron and thus are unable to maintain blood pH. The electrogenic NBCe1-A is an integral membrane co- transporter that normally reabsorbs Na+ and HCO3− across the basolateral membrane. A naturally occurring mutation, R298S, in the cytoplasmic, N-terminal domain (Nt) of NBCe1-A leads to pRTA.
OBJECTIVE: We hypothesize that the R298S genetic mutation results in a fragile monomer- dimer equilibrium of NBCe1-A, leading to intracellular aggregation and the inability to incorporate into the plasma membrane.
METHODS: To investigate the basis of the R298S, we expressed NBCe1-A and NBCe1-A-R298S in cultured human proximal tubular (HK-2) cells to monitor intracellular trafficking and localization by confocal microscopy. The molecular basis of the disease was investigated by in-depth biophysical studies of the Nt and Nt-R298S (including Nt crystal structure, homodimer dissociation constant analyses, molecular mass measurements by Rayleigh-scattering, and hydrodynamic radii temperature dependence).
RESULTS: Confocal studies show that, unlike NBCe1-A, NBCe1-A-R298S is largely retained in the endoplasmic reticulum and post-Golgi vesicles of proximal tubule cells. The Nt crystal structure reveals a dimer with putative conduits in the interior responsible for substrate transport. But biophysical experiments reveal a significantly higher KD of the NBCe1-A-R298S dimer compared to wild-type, with a propensity to fall out of solution that is dependent on pH, temperature, and concentration.
CONCLUSION: The R298S mutation is predominantly a trafficking defect, where cellular localization at the plasma membrane is dependent on growth temperature of the cell line and expression levels.
IMPLICATIONS: Patients afflicted with this mutation can be characterized with a failure to recover bicarbonate in the proximal tubule due to low expression levels of the cotransporter at the plasma membrane.