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Abstract: SA-PO297

Molecular Dynamics Simulations Reveal the Residues Involved in NBCe1 Ion Coordination

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Zhekova, Hristina R., University of Calgary, Calgary, Alberta, Canada
  • Pushkin, Alexander, UCLA, Los Angeles, California, United States
  • Azimov, Rustam, UCLA, Los Angeles, California, United States
  • Abuladze, Natalia, UCLA, Los Angeles, California, United States
  • Kao, Liyo, UCLA, Los Angeles, California, United States
  • Newman, Debra, UCLA, Los Angeles, California, United States
  • Noskov, Sergei, University of Calgary, Calgary, Alberta, Canada
  • Kurtz, Ira, UCLA, Los Angeles, California, United States
Background


We have recently solved by CryoEM the near atomic (3.9 Å) structure of NBCe1-A, an electrogenic Na+-CO32- cotransporter expressed on the basolateral membrane of the proximal tubule, which plays a key role in tubular bicarbonate absorption. Although our structure and functional mutagenesis data suggest that a set of residues from TMs 3, 8, 10 and their vicinity are likely involved in ion coordination, their exact roles cannot be determined given that 3.9 Å resolution was not sufficient to detect coordinated ions.

Methods

The NBCe1 membrane domain was placed in a cubic periodic box in a POPC bilayer using the CHARMM-GUI online server. MD simulations were performed with the CHARMM36 force field and the NAMD program, and ~600 ns long MD trajectories with 2-fs time steps were collected and used for analysis. Ion interactions with the NBCe1 residues were quantified as contact frequency (i.e. percentage of the MD trajectory steps, in which a given ion was found at 3.5 Å from a specific protein residue). Residues exhibiting high contact frequencies with respect to Na+ and CO32- were used for identification of the ion coordination sites.

Results

In the wild type NBCe1 protein, the Na+ contact frequency was highest for residues D754 and T758 (TM8) and A799 (loop prior to TM10). The CO32- contact frequency was highest for K924 (TM13), A800 (loop prior to TM10), T801 (TM10), with less interaction at T485 (loop prior to TM3), and G486 and P487 (TM3). Mutational data supported these results. The A799V mutation found in patients with proximal RTA affecting the Na+ coordination site leads to significantly impaired protein-ion interactions for both Na+ and CO32-. Another proximal RTA causing mutation,G486R, affecting the CO32- coordination site, demonstrates impaired Na+ protein contacts and strong interaction between R486 and CO32- with the arginine residue positioned away from the coordination site.

Conclusion

All-atom molecular dynamics simulations of the membrane domain of NBCe1 revealed that it binds stably Na+ and CO32-. Na+ is coordinated by D754, T758, and A799. CO32- is coordinated by K924, A800, A801, T485, G486, and P487. The proximal RTA patient mutations A779V and G486R drastically alter the coordination of Na+ and CO32-.

Funding

  • NIDDK Support