GluN2B disease-linked mutants are permeable to Ca ²⁺ . (A) Representative microscopy images showing HEK cells co-transfected with BK plus NMDARs containing the GFP-tagged GluN2B mutants used in this study, as well as the red fluorescent, membrane-linked Lyn-R-GECO1 Ca²⁺ indicator. Images were obtained in bright field (BF, left panel), with 488-nm excitation light (middle) and with 540-nm excitation light (right panel). Scale bar = 10 µM. (B) Simultaneous whole-cell current (black traces) and normalized fluorescence recordings (∆F/F₀, red traces) from cells co-expressing BK channels, the membrane calcium sensor Lyn-R-GECO1 and GluN1-GluN2B^WT (left), GluN1-GluN2B^V15M (middle), or GluN1-GluN2B^V618G (right). Recordings were obtained at the indicated holding potentials after application of 1 mM glutamate for 1 s. (C) Graphs represent the averaged maximal peak from Lyn-R-GECO1 recordings (top) or the normalized area under the curve (AUC) (bottom) versus voltage relationships corresponding to the experiments shown in B, in the absence of BK; Data points represent mean ± SEM; n = 5–7.