Structural Biology of SoLute Carriers: NBCe1 NBCn1 AE1 in Acidemia
The Atomic Basis for Enzymatic Mechanisms and Diseases


 



























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Welcome to the Gill-Lab website at The George Washington University and The George Washington Medical Faculty Associates. We integrate basic research with clinical and translational research by fostering an interdisplinary approach expertise that covers a wide range of levels of reductionism from X-ray crystallography to Molecular Biology to Confocal Imaging to Human Pathology and Treatment. The tabs to the right are links to discussions on various diseases ranging from Acidemia to Eye Disorders to Tuberculosis. Our main focus on this first tab is on how integral-membrane proteins in the kidney and eye function at the atomic level and to use this knowledge to implement diagnostics and therapuetics for human diseases and conditions such as type-II RTA and a myriad of eye disorders (glaucoma, band kerathapy, cataracts).


  Carbon dioxide interconverts with bicarbonate in the body

Everyday our kidneys filter 0.5 pound of bicarbonate (HCO3-), commonly known as baking soda. Bicarbonate plays an important metabolic role in acid-base balances. Without this buffering molecule, our blood would become too acidic. In every part of our body, bicarbonate is interconverted with carbon dioxide (CO2) by the following reaction:

CO2 + H2O ↔ H2CO3 (Carbonic acid or carbonated drinking-water) ⇆ HCO3- (Bicarbonate)

Unlike the equilbrium between carbon dioxide and carbonic acid that occurs on its own, the reaction between carbonic acid and bicarbonate is catalyzed by an enzyme called carbonic anhydrase that is ubiquitous and comes in many varieties in the body. This allows for transport of HCO3- molecules into cells from the apical-side that cannot freely do so because of their charged nature and a lack of an appropriate membrane-transporter. Acetylzolmide is a common drug used to combat Acute Mountain Sickness (e.g. pilots or hikers ascending too rapidly to high altitudes). The drug inhibits carbonic anhydrase and ultimately induces blood acidosis to compensate for the respiratory alkalosis caused by the rapid removal of carbon dioxide due to hyperventilation.

  Metabolic Acidosis linked to Defects in Sodium-Bicarbonate
  Transporters at the Basolateral Membrane of the Nephron

Solute carrier family 4 (SLC4) transporters are integral membrane proteins that are located on the basolateral-side of the cell and move HCO3- ions across the epithelium throughout the body. The sodium bicarbonate cotransporter, NBCe1, is one major member 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. The electrophoretic nature allows both substrates to move uphill against their chemical concentration gradients. Defects in NBCe1 result in severe autosomal recessive disorders such as proximl renal tubule acidosis (pRTA), occular abnormailities (i.e. band keratherapy, glaucoma, cataracts), dental defects, short stature, and mental retardation. Patients are characterized by labworks having normal serum gaps, hypokalemia, and hyperchloremic metabolic acidosis. Below we diagnose and illuminate the molecular pathology of the devastating R298S mutation, which is an autosomal-recessive mutation that has been described in the literature to be prelavant among Jewish and Japanese populations.

copyright of Gill

The figure shows live proximal tubule (kidney) cells, where we monitored the expression of NBCe1 trafficking to the plasma membrane using a small green-fluorescent protein attached to the N-terminus of the protein. Protein movement in live cells was monitored in real-time by green-fluorescent signals using a confocal microscope equipped with a 5% CO2 stage-incubator. The upper right panel shows NBCe1-R298S mutatant is unable to make it from the Golgi apparatus to the plasma membrane, preventing bicarbonate from being recycled back into blood.

copyright of Gill

The figure shows live human corneal (eye) cells, where we can monitor the expression of NBCe1 trafficking to the plasma membrane using a small green-fluorescent protein attached to the N-terminus of the protein. Protein movement in live cells was monitored in real-time by green-fluorescent signals using a confocal microscope equipped with a 5% CO2 stage-incubator. The lower left panel shows NBCe1-R298S mutant reveals an aggregated state, having formed string-like clusters. Likely, these were in the plasma membrane and now endocytosed, suggesting a maladly state within the membrane.

The figure is a cartoon presentation illustrating how the cytoplasmic domain regulates blood pH. The NBCe1-A splice variant is located at the basolateral membrane of the proximal tubule and normally reabsorbs 80% of the filtered HCO3- from the lumen to blood, thereby playing a major role in regulating blood pH. NBCe1-A contains a large (~400 aa) cytoplasmic, N-terminal domain (NtNBCe1-A) illustrated in the figure to sense both intracellular pH and bicarbonate levels. The crystal structure of NtNBCe1-A is shown to the right.

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