pH-sensitive self-associations of the N-terminal domain of NBCe1-A suggest a compact conformation under acidic intracellular conditions.
NBCe1-A is an integral membrane protein that cotransports Na+ and HCO3 - ions across the basolateral membrane of the proximal tubule. It is essential for maintaining a homeostatic balance of cellular and blood pH. In X-ray diffraction studies, we reported that the cytoplasmic, N-terminal domain of NBCe1-A (NtNBCe1-A) is a dimer. Here, biophysical measurements show that the dimer is in a concentration-dependent dynamic equilibrium among three additional states in solution that are characterized by its hydrodynamic properties, molar masses, emission spectra, binding properties, and stabilities as a function of pH. Under physiological conditions, dimers are in equilibrium with monomers that are pronounced at low concentration and clusters of molecular masses up to 3-5 times that of a dimer that are pronounced at high concentration. The equilibrium can be influenced so that individual dimers predominate in a taut conformation by lowering the pH. Conversely, dimers begin to relax and disassociate into an increasing population of monomers by elevating the pH. A mechanistic diagram for the inter-conversion of these states is given. The self-associations are further supported by surface plasmon resonance (SPR-Biacore) techniques that illustrate NtNBCe1-A molecules transiently bind with one another. Bicarbonate and bicarbonate-analog bisulfite appear to enhance dimerization and induce a small amount of tetramers. A model is proposed, where the Nt responds to pH or bicarbonate fluctuations inside the cell and plays a role in self-association of entire NBCe1-A molecules in the membrane.
Case Western Reserve University, Department of Physiology & Biophysics, 10900 Euclid Avenue, Robbins Building E549, Cleveland, OH 44106-4970, USA. hgill@gwu.edu
Harindarpal S
HS
Gill
D001639:Bicarbonates / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D001639:Bicarbonates; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D012964:Sodium