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Abstract #25367 Published in IGR 12-1
NBCe1 (Slc4A4) functional dimer assembly
Chang M; Holmes H L; Ranatunga W K; Chen A; Romero M FFASEB Journal 2009; 23: S1
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 NBCe 1 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 fully explored. For our studies we chose two severe NBCe1 mutations (<10% WT function individually): S427L (naturally occurring) and E91R (for N-terminal structure studies). When we co-expressed S427L and E91R, we measured ~55% wild-type function which can only occur if the S427L-E91R heterodimer is the functional unit. To further investigate this functional dimerization, we have engineered concatemeric hkNBCe1 constructs. When the hkNBCe1 tandem-dimer (WT-WT) cRNA was expressed in Xenopus oocytes, the electrophysiology data demonstrate the WT-WT homodimer functions as a WT hkNBCe1. The electrophysiology data also showed that the WT-S427L or WT-E91R heterodimers functioned very similarly to WT hkNBCe1. Intriguingly, we observed ~50% WT function in both the S427L-E91R and E91R-S427L heterodimers constructs. These data illuminate why the nullNBCe1 phenotypenull only occurs recessively. An eYFP fragment (1-158, YFP(N)) was fused to the N- or C-terminus of hkNBCe1 and another eYFP fragment (159-238, YFP(C)) was fused to N- or C-terminus of hkNBCe1. The electrophysiology data demonstrate the eYFP-hkNBCe1 fusion proteins have WT hkNBCe1 like function. The hkNBCe1 dimerization can also be visualized by biomolecular fluorescence complementation (BiFC) method. We plan to further use these molecular tools to probe the nature of the hkNBCe 1 dimer interactions.
M. Chang. Physiology and Biomedical Engineering, Mayo Clinic, RochesterUnited States.
Classification:
11.14 Investigational drugs; pharmacological experiments (Part of: 11 Medical treatment)