Abstract The activity of the CFTR Cl – channel is de- pendent on its phosphorylation status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regulators of the cystic fibrosis conductance regulator (CFTR). Treating CFTR-expressing 3T3 cells with ei- ther of the two specific PP2B blockers cyclosporin A (CsA, 1 μM) or deltamethrin (DM, 30 nM) caused rapid activation of CFTR in cell-attached patches. As deter- mined by noise analysis of multi channel patches, DM- or CsA-activated CFTR displayed gating kinetics com- parable to those of forskolin-activated CFTR. After acti- vation of CFTR by blocking PP2B, CFTR still inactivat- ed. CFTR-mediated currents were, on average, 6.1 times larger when cells were stimulated by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphorylation site of CFTR is regulated by cellular PKA, PP2B and another phosphatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, CsA and DM had no effect on CFTR activity in both cell-attached patch-clamp and transepithelial experiments. In con- trast, when exogenous PP2B was added to patches ex- cised from 3T3 or Calu-3 cells, PKA-activated CFTR currents were quickly inactivated. This indicates that free exogenous PP2B can inactivate CFTR in patches from both cell types. We propose that in order to reg- ulate CFTR in an intact cell, PP2B may require a se- lective subcellular localization to become active. When excised patches were PKC-phosphorylated, the gating kinetics of CFTR were significantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indis- criminate dephosphorylation of different phosphoryla- tion sites by PP2B. Key words CFTR gating · Chloride transport · Cyclosporin A · Deltamethrin · Noise analysis · Patch clamp Introduction The cystic fibrosis transmembrane conductance regulator (CFTR) is the major epithelial apical membrane Cl – channel regulated by phosphorylation of its regulatory domain (R domain). In vivo phosphorylation studies have shown that at least five different serines are phosphorylat- ed by protein kinase A (PKA) and two others by protein kinase C (PKC; [9]). There is recent evidence for an or- dered phosphorylation of R domain serines during stimu- lation [4, 5]. CFTR dephosphorylation and inactivation by phosphatases is probably similarly organized, but nei- ther biochemical nor functional data are available that distinguish between effects of phosphatases on specific sites of CFTR. Serine/threonine-specific phosphatases are present in all cell types [18], including epithelial cells [23, 24]. Phosphatases have been classified into two groups ac- cording to their dependence on intracellular factors. Type 1 protein phosphatase (PP1) is inhibited by two intracel- lular proteins, termed inhibitors 1 and 2, while type 2 phosphatases are not. The latter were further subdivided by their dependence on divalent cations. PP2A is active in the absence of divalents, while PP2B and PP2C are active only in the presence of Ca 2+ and Mg 2+ , respective- ly [18]. In in vitro assays phosphatases show a very broad and overlapping substrate specificity, though tar- geted localization of phosphatases to certain cell do- mains might result in spatial specificity. Whether CFTR is regulated by a certain phosphatase might therefore depend on the cellular localization and en- vironment. CFTR is expressed in many different cell types, which makes cell-type-specific regulation of CFTR H. Fischer ( ✉ ) · B. Illek · T.E. Machen University of California, Department of Molecular and Cell Biology, Division of Cell and Developmental Biology, 231 Life Science Addition, Berkeley, CA 94720, USA Present address: H. Fischer · B. Illek Children’s Hospital Oakland, Research Institute, 747 Fifty Second Street, Oakland, CA 94609, USA, Pflügers Arch – Eur J Physiol (1998) 436:175–181 © Springer-Verlag 1998 ORIGINAL ARTICLE Horst Fischer · Beate Illek Terry E. Machen Regulation of CFTR by protein phosphatase 2B and protein kinase C Received: 29 October 1997 / Received after revision: 13 February 1998 / Accepted: 2 March 1998