NBCe1A dimer assemble visualized by bimolecular fluorescence complementation.
Mutations in the electrogenic Na(+)/HCO3(-) cotransporter (NBCe1) that cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic although their tissues should make some 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 fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An enhanced yellow fluorescent protein (EYFP) fragment (1-158, EYFP(N)) or (159-238, EYFP(C)) was fused to the NH2 or COOH terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the NH2 terminus of NBCe1A (EYFP(N)-N-NBCe1A w/ EYFP(C)-N-NBCe1A), and the electrophysiology data demonstrated this EYFP-NBCe1A coexpressed pair have wild-type transport function. These data suggest NBCe1A forms dimers and that NH2 termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% wild-type function individually): S427L (naturally occurring) and E91R (for NH2-terminal structure studies). When we coexpressed S427L and E91R, we measured 50% wild-type function, which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.
Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. chang.minhwang@mayo.edu.
Min-Hwang
MH
Chang
An-Ping
AP
Chen
Michael F
MF
Romero
D000141:Acidosis, Renal Tubular / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D055503:Protein Multimerization; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics*; D014981:Xenopus
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C054562:yellow fluorescent protein, Bacteria
Na+/HCO3- cotransporter; SLC4; Xenopus oocyte; acid base; electrophysiology; membrane current; protein structure