title: Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis.
abstract: Two PDZ domain-containing proteins, NHERF and E3KARP are necessary for cAMP-dependent inhibition of Na(+)/H(+) exchanger 3 (NHE3). In this study, we demonstrate a specific role of E3KARP, which is not duplicated by NHERF, in Ca(2+)-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca(2+) ([Ca(2+)](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca(2+)-dependent inhibition requires Ca(2+)-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca(2+)-dependent interaction between alpha-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of alpha-actinin-4. Elevation of [Ca(2+)](i) results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of alpha-actinin-4 potentiates the inhibitory effect of ionomycin on NHE3 activity by accelerating the oligomerization and endocytosis of NHE3. In contrast, overexpression of the actin-binding domain plus spectrin repeat domain acts as a dominant-negative mutant and prevents the inhibitory effect of ionomycin on NHE3 activity as well as the oligomerization and internalization of NHE3. From these results, we propose that elevated Ca(2+) inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NHE3-E3KARP-alpha-actinin-4 complex.
authors.affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Jae Ho
authors.initials: JH
authors.lastname: Kim
authors.forename: Whaseon
authors.initials: W
authors.lastname: Lee-Kwon
authors.forename: Jong Bae
authors.initials: JB
authors.lastname: Park
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Ryu
authors.forename: C H Chris
authors.initials: CH
authors.lastname: Yun
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D000185:Actinin; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000502:physiology*; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000502:physiology*; D010750:Phosphoproteins; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: C111453:ACTN4 protein, human; D000199:Actins; C491752:Actn4 protein, mouse; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D000185:Actinin; D002118:Calcium