Fig 2: SKOV-3 migration promoted by ATP was inhibited by K+ channel blockers or esiRNA treatment. (A) Bar graph shows SKOV-3 cell migration quantified after a 16-h incubation with 3 different ATP concentrations (0.3, 0.6, or 3 µM). (B) ATP-promoted migration was evaluated in different conditions. Images illustrate cell migration in control condition without added ATP, with 3 µM ATP, and 3 µM ATP co-applied with 10 µM TRAM-34. (C) Bar graph shows cell migration normalized against control. Cells were incubated in different conditions as indicated by + signs in experiments like those shown in (B) (*p < 0.05, experimental condition vs. control without treatment). Note that drugs that activate the K+ response promoted migration, while KCa3.1 blockers (last 3 bars) in the presence of ATP significantly inhibited migration to basal level, or even below, as in TRAM-34 application (*p < 0.05, compared vs. control without treatment). Migration in the presence of K+ blockers was also statistically different with respect to ATP-promoted migration (#p < 0.05). The data represent means ± S.E.M. of 3 different culture preparations. (D) SKOV-3 cells after 48 h of treatment with esiRNA were assayed for migration promoted either by 3 µM ATP, 3 µM UTP or 300 µM 1-EBIO. For each drug treatment the distinct esiRNA transfection conditions were evaluated as in Fig. 5 and indicated in the set stimulated with ATP (*p < 0.05, experimental condition vs. respective CNT).

Fig 3: K+ channel expression and hyperpolarization to bradykinin in human and porcine IMCAs. Confocal micrographs of immunolabelling for KCa3.1 and KCa2.3 in isolated, cannulated and pressurized human (A) and porcine (B) RA-IMCAs with myogenic tone and full dilation to BK. Punctate and diffuse KCa3.1 label was evident in the ECs of human and porcine arteries, whereas KCa2.3 was less clear in human IMCAs, and highly expressed at EC borders of porcine IMCAs (yellow arrowheads). The elastin was dense in human arteries, with the internal elastic lamina (IEL) seen as longitudinal strings in the porcine arteries. Representative of at least 3 arteries for each label; asterisks indicate corresponding nuclei in upper and lower panels. The pink dashed line in the schematic represents the focal plane. (C) The schematic indicates a sharp microelectrode impaled into a SMC of a porcine RA-IMCA mounted for isometric tension recording. Under control conditions the thromboxane mimetic U46619 (0.6 µM) depolarized and contracted arteries, and BK (10 nM) caused hyperpolarization and relaxation (C), summarized in (D). (E) Addition of L-NAME depolarized and contracted porcine left ventricular (LV)-IMCAs. Under these conditions BK (0.1 nM to 100 nM) repolarized and relaxed the artery. Addition of 100 nM acetylcholine (ACh) depolarized and contracted the artery. The asterisk indicates when the electrode came out of the cell. The RMP and tension prior to the addition of L-NAME (100 µM) are indicated by dashed lines. Drugs were added to a static bath at the arrows. RMP, resting membrane potential.

Fig 4: Brain morphology and immunohistochemistry.(A) No overt differences between genotypes in morphology and amounts of glial fibrillary acidic protein (GFAP)+ astrocytes and (B) of CD11b+ microglia in the molecular layer of the hippocampal CA1 region. (C) Immune histochemistry of KCa3.1 in brain tissues from KCa3.1+/+ and KCa3.1-/- mice and (D) in human brain tissues, meningeal artery, and glioblastoma multiforme together with a negative control (-AB).

Fig 5: Immunocytochemistry fluorescence images showing results of staining for ion channels in mouse kidney cortical sections at low magnification.Results show staining for the ion channel of interest and for AQP2 (Red, 594 nM), a marker of CNT and CCD in the cortex. A bifurcation of AQP2-positive tubules was used to identify CNT segments (above the bifurcation) and CCD segments (single tubule below the bifurcation). A. SK1 staining of tubule segments (green, 488 nm) shows strong staining of many cells along both the CNT and CCD (SK1, labels CNT and CCD). Merged images with AQP2 staining verify the CNT and CCD sites of localization. B. SK3 staining of tubule segments (green, 488 nm) shows prominent staining of most cells along both the CNT and CCD (SK3, labels CNT and CCD). Merged images with AQP2 verify the CNT and CCD sites of localization. C. IK1 staining (red, 594 nm) is strong in most cells of both the CNT and CCD (IK1, labels CNT and CCD) as verified in the Merged image. D. BKa staining (green, 488 nm) is prominent in most cells of both the CCD (BKa, CCD) and along the CNT (data not shown) as verified in the Merged images. E. TRPV4 staining (green, 488 nm) is apparent in most cells of the CCD (TRPV4, CCD) and in the CNT (data not shown) as verified in the Merged images. All fluorescence images were obtained at 40x (oil) with a Zeiss Axioskop 40 microscope using a AxioCam MRm CCD camera.