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A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.

The most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), DeltaF508-CFTR, is misprocessed and subsequently degraded in the endoplasmic reticulum. Using the patch-clamp technique, we showed that co-expressions of DeltaF508-CFTR with the N-terminal CFTR truncates containing bi-arginine (RXR) retention/retrieval motifs result in a functional rescue of the DeltaF508-CFTR mutant channel in COS-1 cells. This DeltaF508-CFTR rescue process was strongly impaired when truncated CFTR constructs possessed either the DeltaF508 mutation or arginine-to-lysine mutations in RXRs. In conclusions, our data demonstrated that expression of truncated CFTR constructs could be a novel promising approach to improve maturation of DeltaF508-CFTR channels.

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, I11139V and deltaM1140), 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 deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.

Possible heteromultimer formation between Kv- and Kir-type K+ channels was investigated, in connection with the known functional diversity of K+ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K+ currents could be approximated by the algebraic sum of the 2 K+ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K+ channels.

It has been proposed that the pICln protein forms a nucleotide-sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide-binding site. To evaluate this hypothesis, we have performed voltage-clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pICln in which the three glycines of the putative nucleotide-binding site have been changed into alanines (G54A; G56A; G58A). The injected oocytes displayed outwardly rectifying anion currents, which were voltage-dependently blocked by extracellular cAMP, but which were not affected by removal of extracellular Ca2+. Furthermore, the mutation did not affect the voltage-dependent inactivation. We therefore conclude that there is no evidence in favour of an extracellular nucleotide-binding site in pICln.

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.

The Rho/Rho-associated kinase (ROK) pathway has been shown to modulate volume-regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (MLCP) on VRAC in CPAE. Application of ML-9, an MLCK inhibitor, inhibited VRAC, both when applied extracellularly or when dialyzed into the cell. A similar inhibitory effect was obtained by dialyzing the cells with AV25, a specific MLCK inhibitory peptide. Conversely, NIPP1191–210, an MLCP inhibitory peptide, potentiated the activation of VRAC by a 25% hypotonic stimulus. These data indicate that activation of VRAC is modulated by MLC phosphorylation.

We investigated CFTR channel activity of mature R-domain mutants showing single alterations at sites other than the predicted phosphorylation sites. All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. The anion permeability sequence for all three mutants was the same as that of wild type CFTR. Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Single channel conductances were unchanged in all mutants. Our results suggest that additional sites in the R-domain other than phosphorylation sites influence gating of CFTR channels.

The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.