kca3.1 k+ channel  (Matos labs)

Journal: Scientific Reports

Article Title: K ca 3.1 Activation Via P2y 2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration

doi: 10.1038/s41598-017-04292-6

Figure Lengend Snippet: Ionic basis of the outward current elicited by ATP and effect of K + channel modulators. ( A ) Current responses obtained by applying voltage pulses, from −120 to +60 mV in 20 mV steps, to a SKOV-3 cell held at −40 mV. Currents during superfusion in control conditions (upper traces) and at the outward component peak elicited either by ATP (middle traces) or UTP (lower traces). Control currents were subtracted from those obtained at the peak response, and the amplitude values were plotted against the respective membrane voltage; the linear I/V relationship obtained (23 cells) showed a reversal membrane potential close to −100 mV. ( B ) Traces illustrate an example of current responses elicited by ATP while a cell was held at different values as indicated, confirming a linear I/V relationship and the E rev estimation for the outward component. ( C ) Relationship between E rev (estimated as in ( A ) and external K + concentration; line is the Nernst relationship considering internal and external concentrations in each case. Data represent means ± SEM of 10–15 cells. ( D ) A battery for distinct K + -channel inhibitors was tested on the response, including non-specific blockers of K + channels (TEA + and 4-AP), those specific for two-pore K + channels, and blockers apamin and TRAM-34 specific for K Ca 2 and K Ca 3.1, respectively; bars indicate the proportion of current inhibited by the different antagonists (*p < 0.05). ( E ) Traces show membrane current recorded during the outward peak response to ATP (upper traces) and that obtained in the same cell by co-applying ATP together with TRAM-34 (10 µM), a specific blocker of K Ca 3.1 channels (lower traces). The graph shows data (means ± SEM) obtained in 30 cells under the same protocol in which the basal currents were subtracted for each case, averaged and plotted. ( F ) Currents elicited by 1-EBIO (100 or 300 µM), a K + channel opener specific for K Ca 3.1 channels, applied at two different intracellular Ca 2+ concentrations: low and high (estimated concentration of 10 and 300 nM, respectively). The bar graph shows the averaged current density obtained in each condition monitored in 14 cells. ( G ) Traces illustrate a case in which initially, 1-EBIO was unable to generate any current (upper trace); however, after a response elicited by ATP, a second application of 1-EBIO (lower trace) at the same concentration generated a meaningful outward response.

Article Snippet: Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated overnight at 4 °C with the appropriate mouse monoclonal antibody (1:1000) directed against the K Ca 3.1 channel protein (ALM-051, Alomone; Jerusalen, Israel).

Techniques: Concentration Assay, Generated

Journal: Scientific Reports

Article Title: K ca 3.1 Activation Via P2y 2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration

doi: 10.1038/s41598-017-04292-6

Figure Lengend Snippet: P2Y 2 and K Ca 3.1 protein expression and electric response to agonists in esiRNA transfected SKOV-3 cells. ( A ) Analysis by immunocytochemistry after 48 h of esiRNA treatment in control (CNT) and P2Y 2 -esiRNA-treated groups. Panels show the fluorescence signal for DAPI (blue) in the first column, the signal obtained with a specific antibody against P2Y 2 receptor protein (in green) in the second column, and the corresponding merged image. ( B ) Similar analysis was made in K Ca 3.1-esiRNA-treated cells using an antibody against K Ca 3.1 channel protein (in A and B bar = 50 µm). ( C ) Traces illustrate current response elicited by agonists (all 3 µM) or 1-EBIO (300 µM) in SKOV-3 cells 24 h after esiRNA transfection. In ( D and E ) traces illustrate current responses in SKOV-3 cells 24 h and 72 h after esiRNA transfection, respectively. Currents were normalized against the ATP-elicited response in the CNT group (18–19 cells in each condition). Every set of bars corresponds to either CNT, P2Y 2 -esiRNA, or K Ca 3.1-esiRNA as indicated in the group for ATP application (*p < 0.05, experimental condition vs. respective CNT).

Article Snippet: Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated overnight at 4 °C with the appropriate mouse monoclonal antibody (1:1000) directed against the K Ca 3.1 channel protein (ALM-051, Alomone; Jerusalen, Israel).

Techniques: Expressing, esiRNA, Transfection, Immunocytochemistry, Fluorescence

Journal: Scientific Reports

Article Title: K ca 3.1 Activation Via P2y 2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration

doi: 10.1038/s41598-017-04292-6

Figure Lengend Snippet: 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 K Ca 3.1 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. and indicated in the set stimulated with ATP (*p < 0.05, experimental condition vs. respective CNT).

Article Snippet: Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated overnight at 4 °C with the appropriate mouse monoclonal antibody (1:1000) directed against the K Ca 3.1 channel protein (ALM-051, Alomone; Jerusalen, Israel).

Techniques: Migration, esiRNA, Incubation, Transfection

Journal: Scientific Reports

Article Title: K ca 3.1 Activation Via P2y 2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration

doi: 10.1038/s41598-017-04292-6

Figure Lengend Snippet: Co-localization of P2Y 2 receptor and K Ca 3.1 channel in human ovarian carcinoma. Expression of P2Y 2 receptors and K Ca 3.1 channels in slices from human ovarian carcinoma evaluated immunohistochemically. Biopsies from ovarian carcinoma tissue were collected, processed, and stained with hematoxylin-eosin and/or labeled with K Ca 3.1- and P2Y 2 -specific antibodies that were revealed by second antibodies coupled to green and red fluorescent dyes, respectively. Nuclei in blue labeled with DAPI. Positive co-expression was detected in ovarian neoplastic cells (see merged image), whereas no signal was observed in control assays in which the primary antibody was omitted. Expression of K Ca 3.1 was also observed in stromal cells that did not show neoplastic morphology (*); however, P2Y 2 did not co-localize with K Ca 3.1 in these cells. Patient 1, IC16-532-6.

Article Snippet: Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated overnight at 4 °C with the appropriate mouse monoclonal antibody (1:1000) directed against the K Ca 3.1 channel protein (ALM-051, Alomone; Jerusalen, Israel).

Techniques: Expressing, Staining, Labeling