NBCe1 (SLC4A4) functions as a dimer and N‐termini are required for unifying the transporter

Proximal renal tubular acidosis, glaucoma and cataracts are caused by recessive mutations in NBCe1 (SLC4A4). Expression of S427L or E91R results in deficient NBCe1 transport activity (<10% wt NBCe1 function), yet, we observe ~55% wt NBCe1 function when these mismatched mutations are co‐expressed. Only a functional dimer can explain these results. To directly examine dimerization, we engineered two concatemeric human NBCe1‐A (wt‐wt) constructs varying by different linkers. The hkNBCe1 wt‐wt tandem dimer has the same biophysical fingerprint as a wt hkNBCe1. We use the concatemeric approach forcing a wild‐type NBCe1 and a “transport dead” NBCe1 (hkNBCe1 with N‐ or C‐terminus deletion) together to determine if a monomer or a dimer is the NBCe1 functional unit. These results provide insight into how NBCe1‐transport entities are configured and whether two subunits are necessary for normal transport function. We made hkNBCe1 constructs with N‐terminal deletion (ΔN‐wt) and C‐terminal deletion (wt‐ΔC) and dimer constructs (ΔN‐wt)–wt, wt–(wt‐ΔC), and (ΔN‐wt)–(wt‐ΔC) using tandem linkers. Three more truncated hkNBCe1 tandem dimer constructs were also engineered (wt–(ΔN‐wt), (wt‐ΔC)–wt, and (wt‐ΔC)–(ΔN‐wt)) with altered order of the subunit arrangement. The cRNA from these truncated hkNBCe1 tandem concatemeric constructs were expressed in Xenopus oocytes for electrophysiology functional assay. These experiments provide further validation that NBCe1 forms a functional dimer. The data also reveal that N‐termini from two subunits are necessary for normal transport function and N‐termini coming from opposite subunits are most likely responsible for unifying the NBCe1‐transport entity. EY017732 & AHA SDG2640146.