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The cystic fibrosis conductance regulator (CFTR) is a cAMP-regulated Cl(-) channel expressed predominantly at the apical membrane of secreting epithelial cells. Mutations in the CFTR gene lead to cystic fibrosis, the most frequent genetic disease in the Caucasian population. The most common mutation, a deletion of phenylalanine at position 508 (F508del), impairs CFTR folding and chloride channel function. Although an intense effort is under way to identify compounds that target the F508del CFTR structural defect and promote its expression and stability at the plasma membrane, so far their clinical efficacy has proven to be poor, highlighting the necessity to better understand the molecular mechanism of CFTR regulation and of the pathogenesis of the disease. Accumulating evidence suggests that the inclusion of the CFTR in macromolecular complexes and its interaction with the cortical cytoskeleton may play a key role in fine-tuning the regulation of channel function. Here we review some recent findings that support a critical role for protein-protein interactions involving CFTR and for the cytoskeleton in promoting local control of channel activity. These findings indicate that compounds that rescue and stabilize CFTR at the apical membrane may not be sufficient to restore its function unless the appropriate intracellular milieu is also reconstituted., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na(+)/H(+) exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1-ezrin-actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-MeSADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity.

The effect of noradrenaline on intracellular levels of cAMP in epithelial cells isolated from frog skin, has been studied both in the presence and in the absence of alpha (phentolamine) or beta (propranolol) adrenergic blocking agents. Such levels are increased by hormonal treatment and are linked to the stimulation of beta-adrenergic receptors, while alpha-stimulation has an inhibitory effect. Higher levels of cAMP are reached in the presence of both Ca2+ and Mg2+, confirming the existence of a Ca2+ and Mg2+-dependent adenyl-cyclase, stimulated by noradrenaline. The experimental conditions necessary to induce the highest intracellular cAMP levels, are those in which the permeabilizing effect reaches its maximum.