Evidence of functional NBCe1 (SLC4A4) dimer assembly

Mutations in the electrogenic Na + /HCO 3 − cotransporter (NBCe1) result in proximal renal tubular acidosis, glaucoma and cataracts in patients and are only recessive. Parents and siblings of these affected individuals are asymptomatic though their tissues should make some of the mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been explored. As an initial test of functional dimerization, we co‐expressed mismatched NBCe1 mutations pairs in Xenopus oocytes. When we co‐expressed two severe NBCe1 mutations, such as S427L (naturally occurring), and E91R (synthetic for N‐terminal structure studies), we obtained intriguing results. Each mutations individually results in quite deficient NBCe1 transport activity (<10% wild‐type function). However, when these mutations are co‐expressed in Xenopus oocytes, we observe ~55% wild‐type function which can only occur if heterodimers are the functional NBCe1 unit. While informative, the coexpression experiments only imply that a dimer is formed and functional. A more direct method of examining dimerization is to make tandem NBCe1 constructs. We have engineered two concatemeric hkNBCe1 cDNA constructs varying only by linkers length. When the hkNBCe1 tandem‐dimer cRNA was expressed in Xenopus oocytes, the electrophysiology data demonstrate that both hkNBCe1 tandem‐dimers function very similar to wild‐type hkNBCe1. Currently we have functional complementation data illustrating that NBCe1 forms dimers. As we learn more about this dimer and its interaction, we will make use of biochemical or binding studies to further probe dimer interfaces. Supported by EY017732.