Ca2+-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-α-actinin-4 complex for oligomerization and endocytosis

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 Ca2+-dependent inhibition of NHE3 activity. NHE3 activity is inhibited by elevation of intracellular Ca2+ ([Ca2+](i)) in PS120 fibroblasts stably expressing E3KARP but not those expressing NHERF. In addition, this Ca2+-dependent inhibition requires Ca2+-dependent association between alpha-actinin-4 and E3KARP. NHE3 is indirectly connected to alpha-actinin-4 in a protein complex through Ca2+-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 [Ca2+](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 Ca2+ inhibits NHE3 activity through oligomerization and endocytosis of NHE3, which occurs via formation of an NRE3-E3KARP-alpha-actinin-4 complex.