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pH-sensitive Self-associations of the N-terminal Domain of NBCe1-A Suggest a Compact Conformation under Acidic Intracellular Conditions

[ Vol. 19 , Issue. 10 ]

Author(s):

Harindarpal S. Gill   Pages 1054 - 1063 ( 10 )

Abstract:


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.

Keywords:

NBCe1, SLC4, bicarbonate, cotransporter, light scattering, surface plasmon resonance, basolateral membrane, homeostatic balance, X-ray diffraction studies, hydrodynamic properties

Affiliation:

The George Washington University & The GW Medical Faculty Associates, Department of Medicine & Division of Renal Disease & Hypertension, 2300 I St. NW, Ross Hall 436B, Washington DC, DC 20052-0011.



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