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.
Highlights
Bicarbonate, known as baking soda, plays an important metabolic role in acid-base balances
Similar to other human Solute carrier family 4 (SLC4), NBCe1-A contains a large (~400 aa) cytoplasmic, Nterminal domain (NtNBCe1-A), of which little is known about its role in transport or regulation [9,10,11]
Measurements using dynamic-light scattering Dynamic Light Scattering (DLS) (Fig. 1, solid curve) indicate that the observed RH average (r H) of 4.6 nm at slightly acidic pH is in close agreement with theoretical calculations [28] of 4.2
Summary
Bicarbonate, known as baking soda, plays an important metabolic role in acid-base balances. Solute carrier family 4 (SLC4) transporters are integral membrane proteins that transport HCO3– ions across epithelial cells throughout the body [1,2,3,4,5,6]. Each group contains many splice variants that differ in their cytoplasmic domains [9]. The sodium bicarbonate cotransporter, NBCe1-A, is one major variant of the electrophoretic group, which without ATP transports Na+ and HCO3– ions out of the cell at a ratio that yields a net negative charge across the membrane. Similar to other human SLC4s, NBCe1-A contains a large (~400 aa) cytoplasmic, Nterminal domain (NtNBCe1-A), of which little is known about its role in transport or regulation [9,10,11]. Defects in NtNBCe1-A result in severe autosomal recessive disorders that are associated with acidic blood pH, vision loss, dental abnormalities, and mental retardation [12,13,14,15,16,17,18]
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