The structural insight into the functional modulation of human anion exchanger 3

Article https://doi.org/10.1038/s41467-024-50572-x The structural insight into the functional modulation of human anion exchanger 3 Received: 20 February 2024 Accepted: 15 July 2024 1234567890():,; 1234567890():,; Check for updates Liyan Jian 1,2,8, Qing Zhang2,3,8, Deqiang Yao 2,4,8, Qian Wang2, Moxin Chen5,6, Ying Xia2, Shaobai Li2, Yafeng Shen2, Mi Cao2, An Qin 1,7 , Lin Li5,6 & Yu Cao 1,2 Anion exchanger 3 (AE3) is pivotal in regulating intracellular pH across excitable tissues, yet its structural intricacies and functional dynamics remain underexplored compared to other anion exchangers. This study unveils the structural insights into human AE3, including the cryo-electron microscopy structures for AE3 transmembrane domains (TMD) and a chimera combining AE3 N-terminal domain (NTD) with AE2 TMD (hAE3NTD2TMD). Our analyzes reveal a substrate binding site, an NTD-TMD interlock mechanism, and a preference for an outward-facing conformation. Unlike AE2, which has more robust acid-loading capabilities, AE3’s structure, including a less stable inwardfacing conformation due to missing key NTD-TMD interactions, contributes to its moderated pH-modulating activity and increased sensitivity to the inhibitor DIDS. These structural differences underline AE3’s distinct functional roles in specific tissues and underscore the complex interplay between structural dynamics and functional specificity within the anion exchanger family, enhancing our understanding of the physiological and pathological roles of the anion exchanger family. Cells meticulously regulate their intracellular pH within a narrow range to sustain an optimal environment for biochemical reactions and the functional integrity of organelles. Various channels and transporters are instrumental in cellular pH control, including proton pumps, sodium-potassium pumps, lactic acid transporters, and ammonium transporters1–4. To counteract the pH shifts caused by the accumulation of CO2 and bicarbonate from energy metabolism and biosynthesis, bicarbonate transporters evolved to export the HCO3− anions, accompanied by either chloride influx (Cl−/HCO3− exchangers) or sodium influx (Na+/HCO3− cotransporter)3,5. Anion exchangers (AEs), belonging to the solute carrier superfamily SLC4, encompass three members: AE1-3, encoded by the SLC4A1-3 genes. AE1 predominantly acts as the Cl−/HCO3− exchanger in erythrocytes6, while AE2 is crucial for pH homeostasis in acid-secreting cells such as osteoclasts and parietal cells7,8. Notably, AE3 is primarily expressed in excitable tissues, including the retina, heart, and brain9–11, where it serves to counteract alkalosis and maintain cellular and organ functions. Disruption of AE3 function can lead to disturbances in pH homeostasis, potentially resulting in pathological conditions. In humans, loss-of-function mutations in AE3 have been linked with short QT syndrome, which may be caused by elevated intracellular pH when AE3 are deactivated12,13. Additionally, the Asp867 variant of AE3 is associated 1 Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2Institute of Precision Medicine, the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 115 Jinzun Road, Shanghai, China. 3Structural Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, USA. 4Institute of Aging & Tissue Regeneration, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 5Department of Ophthalmology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. 6Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China. 7Department of Orthopaedics, Shanghai Frontiers Science Center of Degeneration and Regeneration in Skeletal System, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 8These authors contributed equally: Liyan Jian, Qing Zhang, Deqiang e-mail: ; ; Yao. Nature Communications | (2024)15:6134 1 Article with common subtypes of idiopathic generalized epilepsy. Disruption of AE3 reduces the seizure threshold and decreases susceptibility to epileptic seizures14–16. Furthermore, animal models demonstrate that knockout or mutations of Slc4a3 can induce retinal degeneration, progressing to retinitis pigmentosa17,18, thereby positioning SLC4A3 as a potential candidate gene for human retinal diseases19. In summary, tissues with high metabolic rates, such as the heart and retina, rely on normal AE3 function to manage metabolic alkalosis and keep pH homeostasis. As the anion transporter family, SLC4 genes could be categorized based on the mechanism driving carbonate/bicarbonate transport, three sodium-independent anion exchangers (AE1-3), five sodiumdependent co-transporters (NBCe1, NBCe2, NBCn1, NDCBE and NBCn2) and two unusual members (AE4 and BTR1)3,20,21. Within the SLC4 family, members exhibit high similarity in their transmembrane domains (TMDs) but display significant diversity in their N-terminal soluble domains (NTDs). For instance, human AE1-3 shares ~40–51% protein sequence identity in their NTDs, while the identities in their TMDs range from 67–71%. Furthermore, alternative mRNA splicing is a common occurrence across the NTDs of SLC4A members. A notable example is human AE1, which exists in two isoforms differing in NTD lengths: the kidney-type AE1 (kAE1) lacks the first 65 residues found in the erythrocyte-type AE1 (eAE1)6,22. In the case of human AE3, two major forms have been identified cardiac AE3 (cAE3) and brain AE3 (bAE3), with cAE3 missing residues 1-296 present in bAE33,23,24. Previous structural studies on AE1 and AE2 have elucidated their inward- and outward-facing conformations, as well as the substrate-binding pockets in these different states25–28. Additionally, the full-length structures of human AE2 revealed TMD-NTD interplay mediating the conformational change during the anion exchange27. As one of the major pH and carbonate mediators in excitable tissues, AE3 demonstrates exchange activities that are distinctly regulated compared to AE1 and AE2. While its deficiency could cause a severe pH disorder in the heart and brain, intriguingly, AE3’s transport activity is about 60–70% lower than that of AE1 and AE2 in recombinant expression systems29,30. Unlike AE2, which is highly pH-sensitive, AE3 shows relative pH-independence, functioning stably across a range of pH levels29. Moreover, AE3 is more sensitive to the anion transporter inhibitor DIDS (4,4’-diisothiocyanatostilbene-2,2’-disulfonate) than AE2, with an IC50 of about 300-fold less than that of AE230. These functional differences within the AE subfamily suggest subtle yet critical structural variations in AE1-3, underpinning their specialized adaptations to meet the distinct demands of various cells and tissues. Here (...truncated)