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. 2019 Sep 3;9:12706. doi: 10.1038/s41598-019-48971-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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CFTR activation by CSC depends on adenylyl cyclase activity and PKA phosphorylation but does not increase intracellular cAMP. (a) Representative I_sc measurements depict the effect of 100 μM ACs inhibitors (KH7 and SQ22536) or DMSO on CFTR -mediated basal chloride current in WT-CFTR expressing CFBE. (b) Summary of ACs inhibitors (KH7 and SQ22536) effect on basal CFTR-mediated I_sc calculated as the percentage of maximal current reached after maximal forskolin stimulation compared to the baseline after blocking CFTR with Inh₁₇₂. Data are means ± SEM, n = 3–16 *p < 0.05 (c) CFTR-mediated I_sc activation with the successive addition of phosphodiesterase inhibitor IBMX (200 μM), cell permeable cAMP agonist (CPT-cAMP; 500 μM) and forskolin (10 μM) on SQ22536 (100 μM), KH7 or DMSO pre-treated CFBE. (d) Summary of (c) Percentage of maximal current after CPT-cAMP and IBMX activation calculated as the percentage of maximal current reached after maximal forskolin stimulation compared to the baseline after Inh₁₇₂. Data are means ± SEM, n = 3 *p < 0.05 (e) Representative I_sc current of CSC titration on DMSO (black) or SQ22536 (100 μM) pre-treated (blue) in CFBE. (f) Summary of SQ22536 (50 and 100 μM) pre-treatment on CSC-triggered CFTR activation in CFBE. Data are means ± SEM, n = 3 with technical duplicates (g) cAMP level and Inh₁₇₂-sensitive I_sc in CFBE upon inhibition by SQ22536 (100 μM SQ, green) or stimulation by forskolin (0.01, 0.05, 0.1, 2 and 10 μM, black) or CSC (30, 50, 100 and 200 μg/ml red) measured with cAMP ELISA kit. Data are means ± SEM. n = 3(h) Immunoblot analysis of WT and 15SA PKA-phosphorylation deficient CFTR-3HA expression in CFBE. Complex- and core-glycosylated CFTR are indicated by an empty and filled arrowhead, respectively. Na/K ATPase was probed as a loading control. (i) 15SA mutations largely prevent the forskolin-induced CFTR activation. The WT and 15SA CFTR mediated I_sc (left panel) and PM density (right panel) were measured in CFBE. I_sc was measured in the presence of 20 μM forskolin. Data are means ± SEM, n = 3. Right panel: representative 15SA CFTR mediated I_sc upon CSC and forskolin stimulation. (j) Summary of WT- and 15SA-mediated I_sc current normalised for CFTR PM density and expressed as the percentage of forskolin-stimulated WT I_sc after 10 μM forskolin stimulation. Note that proportionally reduced activation of the 15SA CFTR was detectable by CSC (200 μg/ml) Data are means ± SEM, n = 4.