(732.18) Insights Into the Na+-Dependent Anion Exchange in the SLC4 Family From the Cryo-EM Structure of the Sodium-Driven Chloride/Bicarbonate Exchanger SLC4A8

Poster Board Number: E263

Hristina Zhekova (University of Calgary), Weiguang Wang (UCLA), Kirill Tsirulnikov (UCLA), Gülru Kayık (University of Calgary), Hanif Khan (University of Calgary), Rustam Azimov (UCLA), Natalia Abuladze (UCLA), Liyo Kao (UCLA), Debbie Newman (UCLA), Sergei Noskov (University of Calgary), D. Tieleman (University of Calgary), Z. Zhou (UCLA, UCLA), Alexander Pushkin (UCLA), Ira Kurtz (UCLA, UCLA)

SLC4 transporters are involved in the transport of HCO3‾, CO32‾, Na+, Cl‾ and H+ required for regulation of pH and ion homeostasis in several important physiological processes. Dysfunction of these transporters can lead to acid-base abnormalities, pH dysregulation, and various disease processes. Understanding of their transport mechanism is therefore critical for development of pharmacological strategies for SLC4-related diseases. The studies of the SLC4 transport mechanisms have been hindered by lack of information on the specific substrate binding sites within the protein matrix. No binding sites have been resolved in the two previously published structures of SLC4 proteins, the chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1) and the electrogenic sodium carbonate cotransporter 1 (NBCe1, SLC4A4), although putative binding sites have been identified on the basis of these structures from computational modelling and functional mutagenesis experiments. Here we present a new cryo-EM structure at 3.4Å of the outward facing open (OF) conformation of the sodium dependent chloride/bicarbonate exchanger (NDCBE, SLC4A8) [1], which exchanges Na+-CO32‾ for Cl‾, combining the feature functionalities of both AE1 and NBCe1. The structure resolves for the first time a Na+ and a CO32‾ ion bound at the center of the protein in the area of the previously identified putative binding site S1 in NBCe1 and AE1. Mutation of the residues involved in ion binding leads to significant impairment of NDCBE transport. Computational modelling with the new NDCBE structure outlines two separate ion pathways for cations and anions in the OF cavity and demonstrates that a Na+ ion bound at D800 is necessary for anion binding in site S1 and that a Na+-CO32‾ ion pair in this site is the most likely ion load. A second anion binding site, S2, is also identified in the permeation cavity in proximity to site S1. We conclude that Na+-dependent transport in the SLC4 family is influenced both by thermodynamic effects at the S1 binding site and by permeation effects governed by the residues lining the cation and anion permeation pathways in the OF cavity and site S2.

  [1] Wang W, Tsirulnikov K, Zhekova HR, et al. Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE. Nat Commun. 2020;12:5690

IK was supported by NIH R01 DK077162, Allan Smidt Charitable Fund, Ralph Block Family foundation, and Factor Family Foundation. SYN lab was supported by the NIH R01 DK077162 and the NSERC grant RGPIN-2021-02439. HZ was supported by NIH R01 DK077162. MD simulations were performed on the CFI supported GladOS cluster, West-Grid/Compute Canada clusters, and Anton 2 computer provided by the PSC through NIH Grant R01GM116961. We acknowledge the Electron Imaging Center for NanoMachines for use of their instruments supported by the UCLA and grants from NIH (1S10RR23057; 1U24GM116792) and NSF (DBI-1338135; DMR-1548924).