NHE3 regulatory complexes

Abstract

The epithelial brush border Na/H exchanger NHE3 is active under basal conditions and functions as part of neutral NaCl absorption in the intestine and renal proximal tubule, where it accounts for the majority of total Na absorbed. NHE3 is highly regulated. Both stimulation and inhibition occur post-prandially. This digestion related regulation of NHE3 is mimicked by multiple extracellular agonists and intracellular second messengers. The regulation of NHE3 depends on its C-terminal cytoplasmic domain, which acts as a scaffold to bind multiple regulatory proteins and links NHE3 to the cytoskeleton. The cytoskeletal association occurs by both direct binding to ezrin and by indirect binding via ezrin binding to the C-terminus of the multi-PDZ domain containing proteins NHERF1 and NHERF2. This is a review of the domain structure of NHE3 and of the scaffolding function and role in the regulation of NHE3 of the NHE3 C-terminal domain.

Fig. 1.

Cartoon of mammalian small intestinal Na absorptive processes. Neutral NaCl absorption is made up of NHE3 linked to either DRA or PAT-1.

Fig. 2.

(A) Model of the transport domain of NHE1 showing the area of exchange as two opposing funnel structures facing the intracellular and extracellular surfaces with membrane spanning domains indicated by roman numerals with critical amino acids shown. With permission from Landau et al. (Landau et al., 2007). (B) Comparison of NhaA and NHE1 transport domains, modeled from the crystal structure of NhaA. The homologus amino acids crucial to exchange of NhaA and NHE1 are shown. With permission from Landau et al. (Landau et al., 2007). (C) Amino acid alignment of human NHE1 (SLC9A1) and human NHE3 (SLC9A3) with arrows designating the amino acids shown in Fig. 2B, demonstrating identity. ^*, identical amino acids;:, conserved amino acids; ·, semi-conserved amino acids.

Fig. 3.

Secondary structure prediction for human NHE3 C-terminus (amino acids 457–834) based on Chou-Fasman modeling (Chou and Fasman, 1974).

Fig. 4.

Cartoon of domain structure of rabbit NHE3. The N-terminus includes amino acids 1–454. The C-terminus is the regulatory domain and in blue are some of the extracellular and second messenger regulators with the parts of the C-terminus necessary for their effects shown. In green is the area of attachment of NHE3 to the cytoskeleton via ezrin/radixin/moesin (ERM) proteins which associate with NHE3. In yellow are shown the proteins binding to and regulating the NHE3 C-terminus.

Fig. 5.

(A) Sucrose density gradient centrifugation demonstrating NHE3 exists in multiple large complexes in all cells in which it is expressed. Lysates from the cells shown were Triton X-100 solubilized, separated by centrifugation and identified by immunoblotting with sizes shown in comparison with proteins of known size studied simultaneously on parallel gradients (E3HA, HA-NHE3). (B) Carbachol (Carb) exposure changed the NHE3 complex size. With permission from Li et al. (Li et al., 2004).

Fig. 6.

Cartoon of `switch domain' of NHE3 which has been shown to dynamically associate with at least seven proteins in a short α-helical domain (amino acids 586–605). We draw this as an interacting dimer although evidence that this is the organization structure of the C-terminus is lacking. Drawing by Virginia Ferrante (Ferrante Medical Media).