Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII.

doi: 10.1073/pnas.2108967118

Figure Lengend Snippet: Fig. 1. The activation of Panx1 channels induced by MS in HeLa rPanx1 cells depends on the Ca2+–calmodulin complex, CaMKII kinase activity, and residue S394. (A) DAPI uptake rate normalized with respect to baseline uptake in HeLa rPanx1-EGFP cells pretreated or not for 30 min with 100 μM W7, 10 μM KN62, or 10 μM PP2, subjected to MS. The DAPI uptake assays were performed in the presence of the above-mentioned compounds. Each value corresponds to the mean ± SE of a total of three to four independent experiments. (B) To test the involvement of putative phosphorylation site by CaMKII, DAPI uptake rate was normalized with respect to baseline uptake of nontransfected HeLa-P cells subjected to MS, or transfected with the pRK5 vector with or without the following inserts: rPanx1-EGFP, rPanx1S384A-EGFP, or rPanx1S394A-EGFP. The dye uptake assay was performed 24 h posttransfection. Each value corresponds to the mean ± SE of a total of three to eight independent experiments. **P < 0.005; ***P < 0.001; n.s., nonsignificant.

Article Snippet: In order to detect submembrane localized Ca2+ signals, HeLa cells grown on coverslips were transfected with a genetically encoded Ca2+ indicator protein pN-Lck-GCAMP3 plasmid (a generous gift from Baljit Khakh, Los Angeles, CA, Addgene plasmid no. 26974) as previously described (47).

Techniques: Activation Assay, Activity Assay, Residue, Phospho-proteomics, Transfection, Plasmid Preparation