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. 2021 Jan 13;11:1009. doi: 10.1038/s41598-020-80520-w

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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d,l-Methadone evokes Ca²⁺ release from internal stores, causing increased [Ca²⁺]_i that is inhibited by loss of OPRM1. (A) Resting [Ca²⁺]_i levels in *+pRS and *+pRS-shOPRM1 cells were measured as described in Materials and Methods. Values are means ± SEM from three independent experiments (n = 3). **p < 0.018. (B) Cells loaded with Fura-2 AM and treated with d,l-methadone were analyzed for Ca²⁺ transients in Ca²⁺-free buffer by ratiometric single-cell Ca²⁺ imaging using a Shimadzu RF 5301PC spectrofluorometer as described in Materials and Methods. Values represent means of Ca²⁺ signal traces in 10 cells from one of three independent experiments showing similar results (n = 3). Ca²⁺ response following TBHQ addition indicates that cells were viable during the assay. Analysis of the Ca²⁺ signal traces (left panel) indicates that d,l-methadone-induced increase in [Ca²⁺]_i is greater in *+pRS cells compared to *+pRS-shOPRM1 cells. Values are means ± SEM from three independent experiments (n = 3). **p < 0.025.