Anne Vankeerberghen

The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions

Cystic fibrosis is a frequent autosomal recessive disorder that is caused by the malfunctioning of a small chloride channel, the cystic fibrosis transmembrane conductance regulator. The protein is found in the apical membrane of epithelial cells lining exocrine glands. Absence of this channel results in imbalance of ion concentrations across the cell membrane. As a result, fluids secreted through these glands become more viscous and, in the end, ducts become plugged and atrophic. Little is known about the pathways that link the malfunctioning of the CFTR protein with the observed clinical phenotype. Moreover, there is no strict correlation between specific CFTR mutations and the CF phenotype. This might be explained by the fact that environmental and additional genetic factors may influence the phenotype. The CFTR protein itself is regulated at the maturational level by chaperones and SNARE proteins and at the functional level by several protein kinases. Moreover, CFTR functions also as a regulator of other ion channels and of intracellular membrane transport processes. In order to be able to function as a protein with pleiotropic actions, CFTR seems to be linked with other proteins and with the cytoskeleton through interaction with PDZ-domain-containing proteins at the apical pole of the cell. Progress in cystic fibrosis research is substantial, but still leaves many questions unanswered.

Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator.

Abstract. We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl– channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant ΔF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when ΔF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel–channel interaction.

Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator

1 To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume‐activated chloride current (ICl,swell) using the whole‐cell patch‐clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild‐type (WT) CFTR and the deletion mutant ΔF508 CFTR. 2 I Cl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 ± 8.8% (n= 13; mean ±s.e.m.) of the control value (n= 11). This reduction was independent of activation of the CFTR channel. 3 Expression of ΔF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl− current. In these cells ICl,swell was reduced to 52.7 ± 18.8% (n= 5) of the control value (n= 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 ± 13.7%, n= 11; 21 control cells). 4 Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 ± 11.9% (n= 14) of the control value (n= 12) without any changes in the kinetics of the current. Non‐stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non‐expressing COS cells. 5 We conclude that expression of WT CFTR down‐regulates ICl,swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1). It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR. Determination of the three‐dimensional structure of NBF1 would help to understand this defect. We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1‐ATPase protein. This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.

The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels.

Abstract. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca2+-activated Cl– channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5′-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-1-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-ΔPDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-ΔR780–830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-ΔR-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Characterization of mutations located in exon 18 of the CFTR gene

In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I1139V and ΔM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild‐type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for ΔM1140, indicating that these mutations interfere with the proper gating of the chloride channels.

Isolation and characterization of Locusta migratoria accessory gland myotropin I (Lom-AG-MT-I) from the brain of the Colorado potato beetle, Leptinotarsa decemlineata

A novel myotropic Colorado potato beetle peptide, active in the Locusta oviduct motility assay, was isolated from a methanolic extract of 9,000 brain complexes of adult Leptinotarsa decemlineata by means of HPLC. Its sequence is Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This peptide is identical to Lom-AG-MT-I, a myotropin previously isolated from the male accessory glands of Locusta migratoria, using the L. migratoria oviduct motility bioassay as a monitoring system. It strongly stimulated the frequency, amplitude, and tonus of the myogenic oviduct contractions, even at low concentrations.

Suppressive interactions between mutations located in the two nucleotide binding domains of CFTR

The S1235R locus in CFTR was studied in combination with alleles found at the M470V and G628R loci. While R628 caused a maturational defect, R1235 did not. The impact of R1235 was found to be influenced by the alleles present at the G628R and M470V loci. At the single channel level, R1235‐V (R1235 on a V470 background) was characterized by an open probability significantly higher than V470‐wildtype CFTR. M470, which on its own increases CFTR chloride transport activity when compared to V470‐wildtype CFTR, suppressed the activity of R1235 in such a way that a protein with an open probability not significantly different from V470‐wildtype CFTR was obtained. While R628‐V CFTR had similar current densities as V470‐wildtype CFTR in Xenopus laevis oocytes, R1235‐V resulted in current densities that were more than twofold higher than those of V470‐wildtype CFTR. However, the current densities generated by R1235/R628‐V (R1235 and R628 on a V470 background) CFTR were significant lower than R1235‐V or R628‐V CFTR.

Isolation of Ala1-proctolin, the first natural analogue of proctolin, from the brain of the Colorado potato beetle

Methanolic head and brain extracts of the Colorado potato beetle contain several myotropins, active in the Locusta oviduct motility assay. Reversed phase high performance liquid chromatography (RP HPLC) gave evidence for the presence of three myotropic factors, with retention times close to that of proctolin. Both strongly stimulated the frequency, amplitude and tonus of the myogenic oviduct contractions. Gas phase sequencing and FAB-MS revealed that, besides proctolin (Arg-Tyr-Leu-Pro-Thr), two natural proctolin analogues were present. The first one is Ala-Tyr-Leu-Pro-Thr and is designed as Ala1-proctolin. The threshold concentration for biological activity of Ala1-proctolin was 10(-7) M, compared to 10(-10) M for proctolin itself. Ala1-proctolin is the first identified biological analogue of proctolin. The full nature of the first amino acid of a third proctolin-analogue (x-Tyr-Leu-Pro-Thr) is probably a modified amino acid of which the identity could as yet not be revealed. Our results suggest the existence of a family of proctolin-like peptides.

Functional integrity of the vesicle transporting machinery is required for complete activation of cFTR expressed in xenopus laevis oocytes.

Abstract. We expressed the human cystic fibrosis transmembrane conductance regulator (CFTR) in oocytes of the South African clawed frog Xenopus laevis. We performed simultaneous and continuous recording of membrane current (Im), conductance (Gm) and capacitance (Cm), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxanthine (IBMX) increased all parameters, demonstrating that CFTR activation was partly achieved by exocytotic delivery and insertion of preformed CFTR molecules into the plasma membrane. CFTR currents after cAMP-cocktail were correlated with the capacitance of the oocytes: oocytes with larger Cm exhibited larger currents. Expression of CFTR itself did not change the Cm of the oocytes. However, activation of CFTR with cAMP-cocktail increased Im and Gm 15- and 20-fold, respectively while membrane surface area increased by about 7%, indicating the functional insertion of preformed CFTR into the plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, IBMX alone, but not caffeine or theophylline, was sufficient to stimulate more than half of the increases in Im and Gm as observed with cAMP-cocktail. Since Cm was not significantly stimulated by IBMX, we conclude that IBMX alone activated the CFTR channels already present in the oocyte membrane. CFTR stimulation by cAMP-cocktail was independent of external Ca2+ and ATP had no additional activating potency. The role of protein trafficking in the activation of CFTR evoked by increases of cytoplasmic cAMP was assessed by measuring the effects of brefeldin A (BFA), nocodazole and primaquine on the bioelectric parameters and membrane surface area. All these compounds that interfere with the protein trafficking machinery at different stages prevented the translocation of CFTR from intracellular pools to the plasma membrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways including direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we show that complete activation of CFTR requires an intact protein trafficking machinery.