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. 2019 Feb 20;9:2405. doi: 10.1038/s41598-019-39555-x

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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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Effects of convallatoxin on morphine-mediated membrane potential hyperpolarization in MOR-expressing AtT-20 cells. (A) Flowchart showing experiments testing the effects of convallatoxin on morphine activation of GIRK channels. AtT-20 Cells were transfected with myc-MOR expression plasmid for 24 h prior to all membrane potential assays. (B) Acute effects of each treatment in MOR-expressing AtT-20 cells were measured using membrane potential assay. AUC: F_4,15 = 23.97, p < 0.0001 (1-way ANOVA). (C) After 2 h of incubation, chronic effects of each treatment on MOR desensitization were determined by rechallenging cells with 10 μM morphine. AUC: F_4,15 = 84.32, p < 0.0001 (1-way ANOVA). (D,F,H) AtT-20 Cells were co-transfected with myc-MOR and sh-control (D), sh-clathrin (F), or sh-AP2 (H) for 24 h prior to membrane potential assay. Silencing of clathrin (F) and AP2 (H) did not attenuate the acute effect of convallatoxin. Immunoblot showing expression of clathrin (F) or AP2 (H) in clathrin- or AP2-knockdown AtT-20 cells (upper panel). AUC: (D) F_2,12 = 88.35; (F) F_2,12 = 323.2; (H) F_2,15 = 75.45; all p < 0.001 (1-way ANOVA). (E, G, I) Both clathrin (I) and AP2 (G) were involved in the chronic convallatoxin effect. AUC: (E) F_2,12 = 458.8; (G) F_2,12 = 287.2; (I) F_2,15 = 42.45; all p < 0.001 (1-way ANOVA). ***p < 0.001 versus vehicle group. ^##p < 0.01 versus morphine + convallatoxin group. ^$$$p < 0.001 versus morphine-alone group (Newman-Keuls post hoc tests). All values indicate the mean ± SD.