Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes

A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis

Phosphorylation of CFTR by PKA promotes binding of the regulatory domain

The unphosphorylated regulatory (R) domain of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is often viewed as an inhibitor that is released by phosphorylation. To test this notion, we studied domain interactions using CFTR channels assembled from three polypeptides. Nucleotides encoding the R domain (aa 635–836) were replaced with an internal ribosome entry sequence so that amino- and carboxyl-terminal half-molecules would be translated from the same mRNA transcript. Although only core glycosylation was detected on SplitΔR, biotinylation, immunostaining, and functional studies clearly demonstrated its trafficking to the plasma membrane. SplitΔR generated a constitutive halide permeability, which became responsive to cAMP when the missing R domain was coexpressed. Each half-molecule was co-precipitated by antibody against the other half. Contrary to expectations, GST-R domain was pulled down only if prephosphorylated by protein kinase A, and coexpressed R domain was precipitated with SplitΔR much more efficiently when cells were stimulated with cAMP. These results indicate that phosphorylation regulates CFTR by promoting association of the R domain with other domains rather than by causing its dissociation from an inhibitory site

Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA

Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the ‘9CA’ mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5–10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA