Governing effect of regulatory proteins for Cl⁻/HCO₃⁻ exchanger 2 activity

Abstract

Anion exchanger 2 (AE2) has a critical role in epithelial cells and is involved in the ionic homeostasis such as Cl ¡ uptake and HCO 3 ¡ secretion. However, little is known about the regulatory mechanism of AE2. The main goal of the present study was to investigate potential regulators, such as spinophilin (SPL), inositol-1,4,5-trisphosphate [IP 3 ] receptors binding protein released with IP 3 (IRBIT), STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase, and carbonic anhydrase XII (CA XII). We found that SPL binds to AE2 and markedly increased the Cl ¡ /HCO 3 ¡ exchange activity of AE2. Especially SPL 1-480 domain is required for enhancing AE2 activity. For other regulatory components that affect the fidelity of fluid and HCO 3 ¡ secretion, IRBIT and SPAK had no effect on the activity of AE2 and no protein-protein interaction with AE2. It has been proposed that CA activity is closely associated with AE activity. In this study, we provide evidence that the basolateral membrane-associated CA isoform CA XII significantly increased the activity of AE2 and co-localized with AE2 to the plasma membrane. Collectively, SPL and CA XII enhanced the Cl ¡ /HCO 3 ¡ exchange activity of AE2. The modulating action of these regulatory proteins could serve as potential therapeutic targets for secretory diseases mediated by AE2.

Exploring the autoinhibitory domain of the electrogenic Na+/HCO3− transporter NBCe1‐B, from residues 28 to 62

“…The weak cationic cluster is consistent with the idea that AE2 neither exhibits autoinhibition, nor is rescued by IRBIT. Indeed, IRBIT does not stimulate AE2 (Jeong & Hong, ). SLC4A3 (Fig. B ).…”

Chloride Influx of Anion Exchanger 2 Was Modulated by Calcium-Dependent Spinophilin in Submandibular Glands

“…Intracellular Cl − was evaluated from N -(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE, Thermo, E-3101) fluorescence as previously described ( Park et al, 2013 ; Jeong and Hong, 2016 ). HEK293T and primary isolated SMG cells seeded on coverslips were treated with 5 mM MQAE for 30 min at room temperature and perfused with NaCl-based solution until signal stabilization.…”

“…In this study, we found that AE2 localized in the plasma membrane of submandibular ducts. Recently, our previous study revealed the regulatory role of SPL on the AE2, wherein SPL enhances the chloride bicarbonate exchanger (CBE) activity in vitro ( Jeong and Hong, 2016 ). However, the role of SPL in the regulation of basolateral AE2 and Cl − /HCO 3 − balance in exocrine glands remains unknown.…”

The Fundamental Role of Bicarbonate Transporters and Associated Carbonic Anhydrase Enzymes in Maintaining Ion and pH Homeostasis in Non-Secretory Organs

“…The SPAK phosphorylation site of NBCe1-B is S65 and T49 in the first 85 N-terminus residues, which acts as an autoinhibitory domain (AID) of NBCe1-B [39]. More recently, Lee et al and Jeong et al suggested that scaffolding protein SPL, as an actin cytoskeletal modulator, binds to AE2 and induces the enhanced AE2 activity [41,42]. SPL consists of a binding motif for F-actin, receptor, and canonical PP1 (R-K-I-H-F motif); PDZ domain; and three coiled-coil (C-C) domains [55].…”

“…SPL consists of a binding motif for F-actin, receptor, and canonical PP1 (R-K-I-H-F motif); PDZ domain; and three coiled-coil (C-C) domains [55]. Actually, the SPL can enhance AE2 activity through the 1-480 amino acid residues containing F-actin binding motif, receptor binding motif, and PP1 binding motif [42]. In addition, SPL activity is regulated by Ca 2+ signaling with kinases including SPAK and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [41].…”