Ca(2+)-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis

• Published in: The Journal of biological chemistry Vol. 277; no. 26; pp. 23714 - 23724
• Main Authors: Kim, Jae Ho, Lee-Kwon, Whaseon, Park, Jong Bae, Ryu, Sung Ho, Yun, C H Chris, Donowitz, Mark
• Format: Journal Article
• Language: English
• Published: United States 28.06.2002
• Subjects: Actinin; Actins - metabolism; Animals; Binding Sites; Calcium - pharmacology; Cell Line; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - physiology; Endocytosis; Fibroblasts - metabolism; Humans; Mice; Microfilament Proteins - chemistry; Microfilament Proteins - physiology; Phosphoproteins; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers - antagonists & inhibitors; Sodium-Hydrogen Exchangers - chemistry; Sodium-Hydrogen Exchangers - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M200835200

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.