title: IRBIT plays an important role in NHE3-mediated pHi regulation in HSG cells.
abstract: Expression of inositol-1,4,5-trisphosphate (IP3) receptor-binding protein (IRBIT) has been reported in epithelial cells. However, its role in pHi regulation is not well understood. In this study, we investigated the role of IRBIT in pHi regulation, mediated by Na(+)/H(+) exchangers (NHEs), in salivary glands. We measured pHi recovery from cell acidification in BCECF-loaded salivary HSG cells. Western blot and co-immunoprecipitation (CO-IP) assays were also performed, showing that NHE1, 2 and 3 are expressed, and IRBIT binds to NHE3. HOE642, a specific NHE1 blocker, inhibited pHi recovery, but 40% pH(i) recovery was still observed even at the highest concentration of HOE642. Furthermore, pretreatment of the cells with siIRBIT significantly inhibited pHi recovery, indicating that NHE3 potentially plays a role in pHi recovery as well. The amount of membrane-localized NHE3 and its interaction with IRBIT are also significantly increased by cell acidification. In addition, we found that Ste20p-related proline alanine-rich kinase (SPAK) reverses the effect of IRBIT on membrane NHE3 translocation. Taken together, we conclude that IRBIT plays an important role in pHi regulation, mediated by NHE3, and further regulated by SPAK.
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Yeongeondong 28, Chongnoku, Seoul 110-749, Republic of Korea.
authors.forename: Tien Manh
authors.initials: TM
authors.lastname: Tran
authors.forename: Moon-Yong
authors.initials: MY
authors.lastname: Park
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Lee
authors.forename: Jun-Seok
authors.initials: JS
authors.lastname: Bae
authors.forename: Sung-Min
authors.initials: SM
authors.lastname: Hwang
authors.forename: Se-Young
authors.initials: SY
authors.lastname: Choi
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
authors.forename: Kyungpyo
authors.initials: K
authors.lastname: Park
mesh_terms: D027682:Cation Transport Proteins / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism*; D037181:Lectins, C-Type / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D012469:Salivary Glands / Q000166:cytology*; D000074082:Sodium-Hydrogen Exchanger 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: C470184:CLECL1 protein, human; D027682:Cation Transport Proteins; D037181:Lectins, C-Type; D008565:Membrane Proteins; C490998:SLC9A1 protein, human; C081517:SLC9A2 protein, human; C000613385:SLC9A3 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human
keywords: HOE642; IRBIT; Na(+)/H(+) exchangers; SPAK; pH(i) Recovery