Fig 2: Decreased association between BKCa a1 and BKCa ß1 subunits in vascular smooth muscle cells from diabetic patients. (A–C) Differential interference contrast (DIC) and confocal fluorescent PLA puncta (red) and DAPI (blue) images of freshly dissociated human vascular smooth muscle labeled with mouse-anti BKCa a1 (A), rabbit-anti BKCa a1 (B), and rabbit-anti BKCa ß1 (C) antibodies. (D,E) DIC (left) and fluorescence PLA (red)/DAPI (blue) (right) images of dissociated vascular smooth muscle from non-diabetic (D) and diabetic (E) patients co-labeled with two distinct antibodies for the BKCa a1 subunit. (F,G) DIC (left) and fluorescence PLA (red)/DAPI (blue) (right) images of dissociated human vascular smooth muscle from non-diabetic (F) and diabetic (G) patients co-labeled for BKCa a1 and BKCa ß1 subunits. Scale bar = 10 µm. (H) Quantification of PLA fluorescent puncta per µm2 cell area for non-diabetic and diabetic vascular smooth muscle cells labeled for mouse-anti BKCa a1 (n = 19 cells), rabbit-anti BKCa a1 (n = 27 cells), rabbit-anti BKCa ß1 (n = 12 cells), mouse-anti BKCa a1 + rabbit-anti BKCa a1 (n = 24 non-diabetic, 14 diabetic cells); mouse-anti BKCa a1 + rabbit-anti BKCa ß1 (n = 27 non-diabetic, 24 diabetic cells). *P < 0.05, Mann-Whitney test. Significance for columns 1, 2, 3 and 5 was compared to column 4, and column 7 was compared to column 6.

Fig 3: Decreased BKCa channel activity in vascular smooth muscle from diabetic patients. (A) Representative single BKCa channel traces at -40 mV from excised membrane patches of isolated human non-diabetic and diabetic vascular smooth muscle cells in the presence of 1 µM and 10 µM free Ca2+ bath solution (C: closed; O: open). (B) Scatter/bar plot summarizing BKCa channel open probability (Po) at the indicated free Ca2+ concentration from human non-diabetic (n = 18 cells, 9 subjects) and diabetic (n = 27 cells, 15 subjects) cells. *P < 0.05, Mann-Whitney test. (C) Open dwell time histograms of BKCa channels in non-diabetic and diabetic vascular smooth muscle. The goodness of the fit was assessed with R2 and the F test was used for comparison between open time fits. Significance was compared between non-diabetic and diabetic datasets.

Fig 4: Fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA-FLS) express proteins of multiple calcium-activated potassium channel KCa1.1 ß subunits. a Representative Western blot from a gel loaded with proteins from one RA-FLS donor with different lanes probed with antibodies against different subunits of KCa1.1 (top) and actin (bottom). b Intensity of KCa1.1 a and ß subunit protein bands normalized to actin expression levels in RA-FLS. Each symbol on the scatterplot represents results from a different donor. The horizontal bar represents the mean for each subunit

Fig 5: No change in BKCa subunit total protein levels in arterial lysates from non-diabetic and diabetic patients. (A) Representative immunoreactive bands corresponding to BKCa a1 and BKCa ß1 subunits in lysates from untransfected HEK293 cells (HEK), HEK293 cells transfected with either BKCa a1 or BKCa ß1 subunit and human arteries from non-diabetic patients (n = 3 lysates per condition). Full-length blots are shown in Supplementary Fig. S2A. (B) Representative immunoreactive bands corresponding to BKCa a1 and BKCa ß1 subunits, and ß-actin as a normalization control. Full-length blots are shown in Supplementary Fig. S2B. (C) Corresponding densitometric summary data for each subunit obtained using arterial lysates from non-diabetic (n = 10 lysates) and diabetic (n = 10 lysates) patients. *P < 0.05, Mann-Whitney test. Significance was compared between non-diabetic and diabetic datasets.