Calcium inhibits NHE3 by an E3KARP-dependent mechanism which involves a signaling complex alpha-actinin-4, E3KARP, and NHE3

X11sciescopu

Ca2+-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP-alpha-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.X11101sciescopu

Leu143 in the Putative Fourth Membrane Spanning Domain Is Critical for Amiloride Inhibition of an Epithelial Na+/H+ Exchanger Isoform (NHE-2)

A family of Na+/H+ exchanger isoforms (called NHE1, NHE2, and NHE3) which exhibits a wide range of amiloride sensitivity has recently been cloned and characterized. A part of the domain, which determines amiloride sensitivity in the epithelial Na+/H+ exchanger isoform, NHE2, was identified by site-directed mutagenesis and functional studies using cDNAs stably expressed in a fibroblast cell line. It has previously been reported that AR300, an amiloride resistant mutant of the ubiquitous Na+/H+ exchanger isoform, NHE1, is 30-fold more resistant to methylpropyl amiloride (MPA) compared to NHE1 and contains a single amino acid substitution of L167F in the fourth putative transmembrane helix, which corresponds to L143 in NHE2. Therefore, in the present study point mutational substitutions were introduced into the equivalent of this fourth transmembrane helix of rabbit NHE2 (including Y144F; L143F; L143F and Y144F) to mimic the corresponding amino acids in NHE1, NHE3 (another epithelial isoform) and AR300, respectively. NHE2/L143F (mimicking NHE3) increased the IC50 for amiloride by 5-fold and for ethylisopropyl amiloride (EIPA) by 20-fold. Similarly, NHE2/L143F and Y144F (mimicking AR300) increased the resistance to both amiloride and EIPA by 10-fold. On the other hand, NHE2/Y144F (mimicking NHE1) did not affect the sensitivity to amiloride or EIPA, and this mutant, like wild type NHE2, is partially resistant to EIPA. Thus, amino acid 143 of NHE2 is critical for, but is not the only amino acid responsible for, amiloride and EIPA inhibition of Na+/H+ exchange. That none of the mutations studied altered the Na+ affinity of these Na+/H+ exchangers further suggests that amiloride binding and Na+ transport sites are not identical

Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+ exchanger NHE3 by glucocorticoids

SGK1 plays an important role in regulation of Na+/H+ exchanger (NHE) 3 in vivo. We show that SGK3 colocalizes with NHE3 in recycling endosomes. These studies identify SGK3 as the effector of the PI3K pathway that activates NHE3 and show that endosomal localization of SGK3 is essential for acute activation of NHE3