Cluster analysis of BK and GluN2B. Graphs and analysis are distributed in three groups corresponding to three different settings where the cluster radius is fixed to 60 nm (top), 40 nm (middle), or 20 nm (bottom). (A, B, I, J, Q, and R) Histograms representing the distribution of clusters in HEK293T cells co-expressing either BK and GluN1/2BWT or BK and GluN2B-V618G, containing BKα alone (green bars), GluN2BWT alone (red bars), or both proteins (yellow bars). Colored curves outline the histograms to facilitate visualization. In all cases, the clustering properties of the samples were quantified by adjusting the density filtering with a count of 10 molecules and radius cutoff set to (A and B) 60 nm (r = 60 nm), (I and J) 40 nm (r = 40 nm), and (Q and R) 20 nm (r = 20 nm). (C, K, and S) Distributions of BK homoclusters in HEK293T cells co-expressing either BK and GluN1/2BWT (gray bars) or BK and GluN1/2B-V618G (orange bars) obtained with the different radius values (C, r = 60 nm; K, r = 40 nm; S, r = 20 nm). (D, L, and T) Distributions of GluN2B homoclusters in HEK293T cells co-expressing either BK and GluN1/2BWT (gray bars) or BK and GluN1/2B-V618G (orange bars) obtained with the different radius values (D, r = 60 nm; L, r = 40 nm; T, r = 20 nm). (E, M, and U) Distributions of NMDAR–BK heteroclusters in HEK293T cells co-expressing either BK and GluN1/2BWT (gray bars) or BK and GluN1/2BV618G (orange bars) obtained with the different radius values (E, r = 60 nm; M, r = 40 nm; U, r = 20 nm). (F, N, and V) Cumulative probability analysis of heterocluster distribution in cells co-expressing BK and either GluN2BWT or GluN2B-V618G at the different values of radius (F, r = 60 nm, [Kolmogorov–Smirnov test {K–S} ****P < 0.0001, D = 0.06232]; N, r = 40 nm, [K–S test ****P < 0.0001, D = 0.09336]; V, r = 20 nm [K–S test ****P < 0.0001, D = 0.1183]). (G, H, O, P, W, and X) Pie charts illustrating the proportion (%) of cluster distribution in cells co-expressing BK and either GluN1/2BWT or GluN1/2BV618G at radius values (G and H) r = 60 nm, (O and P) r = 40 nm, and (W and X) r = 20 nm.
Figure S1.

Cluster analysis of BK and GluN2B. Graphs and analysis are distributed in three groups corresponding to three different settings where the cluster radius is fixed to 60 nm (top), 40 nm (middle), or 20 nm (bottom). (A, B, I, J, Q, and R) Histograms representing the distribution of clusters in HEK293T cells co-expressing either BK and GluN1/2BWT or BK and GluN2B-V618G, containing BKα alone (green bars), GluN2BWT alone (red bars), or both proteins (yellow bars). Colored curves outline the histograms to facilitate visualization. In all cases, the clustering properties of the samples were quantified by adjusting the density filtering with a count of 10 molecules and radius cutoff set to (A and B) 60 nm (r = 60 nm), (I and J) 40 nm (r = 40 nm), and (Q and R) 20 nm (r = 20 nm). (C, K, and S) Distributions of BK homoclusters in HEK293T cells co-expressing either BK and GluN1/2BWT (gray bars) or BK and GluN1/2B-V618G (orange bars) obtained with the different radius values (C, r = 60 nm; K, r = 40 nm; S, r = 20 nm). (D, L, and T) Distributions of GluN2B homoclusters in HEK293T cells co-expressing either BK and GluN1/2BWT (gray bars) or BK and GluN1/2B-V618G (orange bars) obtained with the different radius values (D, r = 60 nm; L, r = 40 nm; T, r = 20 nm). (E, M, and U) Distributions of NMDAR–BK heteroclusters in HEK293T cells co-expressing either BK and GluN1/2BWT (gray bars) or BK and GluN1/2BV618G (orange bars) obtained with the different radius values (E, r = 60 nm; M, r = 40 nm; U, r = 20 nm). (F, N, and V) Cumulative probability analysis of heterocluster distribution in cells co-expressing BK and either GluN2BWT or GluN2B-V618G at the different values of radius (F, r = 60 nm, [Kolmogorov–Smirnov test {K–S} ****P < 0.0001, D = 0.06232]; N, r = 40 nm, [K–S test ****P < 0.0001, D = 0.09336]; V, r = 20 nm [K–S test ****P < 0.0001, D = 0.1183]). (G, H, O, P, W, and X) Pie charts illustrating the proportion (%) of cluster distribution in cells co-expressing BK and either GluN1/2BWT or GluN1/2BV618G at radius values (G and H) r = 60 nm, (O and P) r = 40 nm, and (W and X) r = 20 nm.

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