Ca2+-dependent Inhibition of Na+/H+ Exchanger 3 (NHE3) Requires an NHE3-E3KARP-α-Actinin-4 Complex for Oligomerization and Endocytosis

• Published in: The Journal of biological chemistry Vol. 277; no. 26; p. 23714
• Main Authors: Jae Ho Kim, Whaseon Lee-Kwon, Jong Bae Park, Sung Ho Ryu, C. H. Chris Yun, Mark Donowitz
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 28.06.2002
• ISSN: 0021-9258; 1083-351X
• 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 α-actinin-4 and E3KARP. NHE3 is indirectly connected to α-actinin-4 in a protein complex through Ca 2+ -dependent interaction between α-actinin-4 and E3KARP, which occurs through the actin-binding domain plus spectrin repeat domain of α-actinin-4. Elevation of [Ca 2+ ] i results in oligomerization and endocytosis of NHE3 as well as in inhibition of NHE3 activity. Overexpression of α-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-α-actinin-4 complex.