CFTR chloride channel in the apical compartments: spatiotemporal coupling to its interacting partners

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel located primarily at the apical or luminal surfaces of epithelial cells in the airway, intestine, pancreas, kidney, sweat gland, as well as male reproductive tract, where it plays a crucial role in transepithelial fluid homeostasis. CFTR dysfunction can be detrimental and may result in life-threatening disorders. CFTR hypofunctioning because of genetic defects leads to cystic fibrosis, the most common lethal genetic disease in Caucasians, whereas CFTR hyperfunctioning resulting from various infections evokes secretory diarrhea, the leading cause of mortality in early childhood. Therefore, maintaining a dynamic balance between CFTR up-regulating processes and CFTR down-regulating processes is essential for maintaining fluid and body homeostasis. Accumulating evidence suggests that protein-protein interactions play a critical role in the fine-tuned regulation of CFTR function. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might be coupled spatially and temporally to a wide variety of interacting partners including ion channels, receptors, transporters, scaffolding proteins, enzyme molecules, signaling molecules, and effectors. Most interactions occur primarily between the opposing terminal tails (amino or carboxyl) of CFTR protein and its binding partners, either directly or mediated through various PDZ scaffolding proteins. These dynamic interactions impact the channel function, as well as localization and processing of CFTR protein within cells. This article reviews the most recent progress and findings about the interactions between CFTR and its binding partners through PDZ scaffolding proteins, as well as the spatiotemporal regulation of CFTR-containing macromolecular signaling complexes in the apical compartments of polarized cells lining the secretory epithelia.

Fig. 1. Putative CFTR topology and its interactions with various binding proteins

CFTR is a member of the ABC transporter superfamily and consists of two repeated motifs, each composed of a membrane-spanning domain (MSD) containing six helices and a cytosolic nucleotide binding domain (NBD), which can bind and hydrolyze ATP. These two identical motifs are linked by a cytoplasmic regulatory (R) domain that contains a number of charged residues and multiple consensus phosphorylation sites (substrates for PKA, PKC, cGMP-dependent protein kinase II, etc.). The CFTR chloride channel can be activated through phosphorylation of the R domain by various protein kinases and by ATP binding to, and hydrolysis by, the NBD domain. Both the amino (NH2) and carboxyl (COOH) terminal tails of this membrane protein are cytoplasmically oriented and mediate the interaction between CFTR and a wide variety of binding proteins.

Fig. 2. A model of secretory epithelial cell and secretory diarrhea

Cholera toxin or heat-stable enterotoxin can increase the intracellular cAMP or cGMP levels by activating the membrane-localized adenylate cyclase (AC) or guanylate cyclase (GC). Increase in the intracellular cAMP or cGMP leads to the phosphorylation of the R domain of CFTR by PKA or cGMP-dependent protein kinase II (cGK II), which in turn activates the CFTR chloride channel, resulting in Cl⁻ secretion into the lumen. As a consequence, Na⁺ and water are effluxed into the lumen through the paracellular transport mechanism. Therefore, the net result is the secretion of fluid and electrolytes across the apical surface into the gut lumen. Cl⁻ is taken up from the basolateral (blood) side by the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC). K⁺ recycles through basolateral K⁺ channels, and Na⁺ is pumped out of the cell by Na⁺-K⁺-ATPase (adapted from ref. 4).

Fig. 3. PDZ domain-containing scaffolding proteins that can bind CFTR protein

Six different PDZ domain-containing scaffolding proteins have been reported to bind to the C-terminal tail of the CFTR protein mediated through their PDZ domains: NHERF1/EBP50, NHERF2/E3KARP, PDZK1/CAP70, PDZK2/IKEPP, CAL, and Shank2. CAL is primarily localized to Golgi apparatus, whereas the rest are localized to the apical membranes of epithelial cells. NHERF1 and NHERF2 are closely related and share ~50% sequence identity. NHERF1 and NHERF2 contain two PDZ domains, both of which can bind CFTR, as well as a C-terminal domain (ERM domain) that mediates association with MERM proteins to link CFTR to the actin cytoskeleton. Both PDZK1 and PDZK2 contain four tandem PDZ domains, and PDZ3 and PDZ4 of PDZK1 are reported to bind two CFTR molecules simultaneously. CAL possesses only one PDZ domain and two coiled-coil (CC) domains that associate CAL to the membrane. In addition to one PDZ domain, Shank2 also contains other sites for protein-protein interaction, including an SH3 domain, a long proline-rich region, and a sterile alpha motif (SAM) domain. Shank2 is expressed abundantly in brain, as well as in kidney, liver, intestine, and pancreas, and is localized to the luminal pole in pancreatic duct cells and luminal area of colonic epithelia. The tissue distributions of these CFTR interacting proteins are not identical, suggesting that CFTR might interact with different PDZ proteins in different tissues (adapted from ref. 4).

Fig. 4. The spatiotemporal coupling of β₂-adrenergic receptor signaling to CFTR channel function in the airway

CFTR, NHERF1, and β₂AR form a macromolecular complex together with signaling molecules at the apical surfaces of airway epithelia. G proteins can be associated with β₂AR and protein kinase A (PKA) anchored to AKAP (ezrin) and is likely to be in the complex. Upon agonist activation of the receptor, adenylate cyclase is stimulated through the Gs pathway, leading to an increase in highly compartmentalized cAMP. This increased local concentration of cAMP leads to the activation of PKA, which is in close proximity to CFTR, resulting in a compartmentalized and specific signaling from β₂AR to the CFTR channel. Phosphorylation disrupts the complex, leading to the receptor-based activation of CFTR.

Fig. 5. The spatiotemporal coupling of LPA inhibition to CFTR-dependent Cl⁻ transport in the gut

LPA₂ and CFTR are physically associated with NHERF2, which clusters LPA₂ and CFTR into a macromolecular signaling complex at the apical plasma membranes of gut epithelia. This macromolecular complex is the foundation of functional coupling between LPA signaling and CFTR-mediated Cl⁻ transport. Upon LPA stimulation of the receptor, adenylate cyclase is inhibited through the G_i pathway, leading to a decrease in cAMP level. This decreased local or compartmentalized accumulation of cAMP results in the reduced activation of Cl⁻ channel in the vicinity by CFTR agonists (e.g., adenosine) (adapted from ref. 73).

Fig. 6. The spatiotemporal coupling of MRP4 cAMP transporter to CFTR Cl⁻ channel function in the gut

Underneath the apical plasma membrane in the gut epithelia, there exist highly localized compartments that are composed of a series of signaling molecules such as adenosine receptor (AR); G protein (Gs); AC; PKA and its anchoring proteins, AKAPs (not shown in this figure); CFTR; cAMP transporter (MRP4); and PDZ scaffolding protein (PDZK1), which functions to physically connect CFTR to MRP4. This macromolecular signaling complex provides an anatomical basis for the generating and modulating local cAMP compartments. When an agonist (such as adenosine) binds AR, a series of G-protein-mediated reactions leads to activation of AC present in the apical membrane. Sufficient cAMP is locally generated in a diffusionally restricted apical microdomain (but not in other cellular compartments). cAMP activates PKA, which is anchored also to the apical membrane by AKAP (i.e., ezrin), and phosphorylates CFTR Cl⁻ channel in close vicinity, resulting in an increase of Cl⁻ currents. The CFTR-mediated Cl⁻ currents can be further increased (potentiated) through the additional increase of local cAMP resulting from the reduced or blocked efflux via a neighboring apical membrane cAMP transporter (MRP4) in the same subcellular compartment. The interaction between CFTR and MRP4 provides an additional layer of mechanism to regulate CFTR function, which is important in maintaining epithelial and body homeostasis (adapted from ref. 84).