Tungstate-induced increase in the open probability of BK channels endogenously expressed in freshly isolated vascular myocytes requires the BK channel β₁ subunit.

<p>(A) Representative recordings obtained from inside-out patches clamped at + 60mV from wild-type (WT) and β₁- knockout freshly isolated mouse vascular myocytes, before (control) and after exposure to 1 mM tungstate (1 mM WO₄^2-), as stated. Arrows indicates the closed state level. (B) Average changes (in %) of BK channel open probability (NP_o) induced by 1 mM tungstate on WT and β₁- knockout mouse vascular myocytes at the indicated voltage membrane. Significant differences were found among WT (n = 7) and β₁- knockout (n = 9), P < 0.001 (Student’s t-test). Further depolarization to +80 mV of β₁- knockout inside-out patches did not change the response to tungstate found at +60 mV.</p

Tungstate-mediated activation of BKαβ₁ channels is independent of G proteins.

<p>Representative currents recorded from excised inside-out macropatches obtained from HEK293 cells expressing the BKαβ₁ channels in the presence of 500 μM GDPβS (added to the bath solution) (A) or from transfected HEK293 cells pre-incubated with PTX (500 ng/ml, 24 hours) (C). Currents were recorded at cytosolic 0 Ca²⁺ and 0.7 mM Mg²⁺ before (control, top panels) and 5–10 minutes after cytosolic application of 1 mM tungstate (WO₄^2-, bottom panel). The voltage protocol was as described in the Methods. (B), (D) Average G-V curves for BKαβ₁ channels under the experimental conditions above mentioned. Solid curves were obtained by fitting the normalized conductance to the Boltzmann equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118148#sec002" target="_blank">Methods</a>). (E) Voltage for half maximal activation (V_{1/2 act}) of BKαβ₁ channels before (control, filled circles) and after addition of tungstate (1 mM WO₄^2-, open circles) obtained for the indicated experimental conditions (+GDPβS, n = 4; +PTX, n = 6). No significant difference was found in the decrease on V_{1/2 act} induced by tungstate when comparing both treatments: 24 ± 4 mV (n = 4) for GDPβS treatment <i>versus</i> 16 ± 3 mV (n = 6) for PTX treatment (P = 0.11, Mann-Whitney U-test). Besides the substantial difference in the duration of both treatments to inhibit G proteins (24–28 hours for PTX treatment and minutes for GDPβS treatment), no significant difference was found among them regarding V_{1/2 act} before the addition of tungstate (control situation for GDPβS treatment: V_{1/2 act} = 144 ± 4 mV (n = 4) <i>versus</i> control situation for PTX treatment: V_{1/2 act} = 145 ± 2 mV (n = 6); P > 0.99, Mann-Whitney U-test). Furthermore, these V_{1/2 act} control values (before tungstate application) were similar to the ones previously reported by us under identical experimental conditions but without interfering with the activation of G proteins: 139 ± 2 mV (n = 7) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118148#pone.0118148.ref014" target="_blank">14</a>] and 147 ± 2 mV (n = 7) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118148#pone.0118148.ref026" target="_blank">26</a>] (P = 0.1, ANOVA). Note that, as previously reported, 1 mM tungstate also reduced substantially the K⁺ current amplitude in the absence of cytosolic Ca²⁺ (A, C), an effect that, contrary to the tungstate-induced reduction of V_{1/2 act}, has been shown to occur either in the absence or presence of Mg²⁺ and in the absence or presence of the different regulatory β subunits (β₁-β₄) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118148#pone.0118148.ref014" target="_blank">14</a>]. For each experimental condition V_{1/2 act} and k_act values were (in mV): 144 ± 4 and 20 ± 0.3 (control situation for GDPβS treatment, n = 4); 120 ± 1 and 22 ± 1 (after WO₄^2- addition for GDPβS treatment, n = 4); 145 ± 2 and 19 ± 0.3 (control situation for PTX, n = 6); 129 ± 2 and 21 ± 1 (after WO₄^2- addition for PTX treatment, n = 6). No significant differences were found among k_act values (P = 0.07, ANOVA).</p

BK channels mediate both ERK1/2 phosphorylation and reduction of PDGF-stimulated proliferation induced by tungstate in human vascular smooth muscle cells.

<p>(A) Representative Western-blot showing phosphorylation of ERK1/2 using phospho-ERK-specific antibodies after 10 minutes incubation in control conditions (0% FBS), with PDGF (20 ng/ml), and with tungstate (1 mM) alone or in combination with iberiotoxin (IbTX 100 nM), as indicated. Beta-actin was used as loading control. (B) Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal and normalized to beta-actin. The ratio p-ERK/beta-actin in control conditions was taken as 1, so that fold-changes relative to control are shown. (C) VSMCs were serum starved for 48 hours and then incubated 30 hours with 20 ng/ml PDGF alone or in combination with the indicated compounds. During the last 6 hours of incubation, EdU was added to the media to detect the number of cells entering S-phase. Mean ± S.E.M. of 6–9 determinations from at least four different cultures. ***P < 0.001 (ANOVA followed by Tukey <i>post hoc</i> test).</p

BKαβ₁ channels potentiate tungstate-induced ERK1/2 phosphorylation in a G_i/o protein-dependent manner.

<p>Phosphorylation of ERK1/2 was analyzed by Western blot using phospho-ERK-specific antibodies. Total ERK was used as loading control (data not shown). Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal. Relative density (phosphorylated versus total ERK) was normalized to the inner control (EGF/IGF for each condition, which was considered as 100%). (A) Representative Western blots obtained from HEK293, HEKα and HEKαβ₁ cells for ERK1/2 phosphorylation levels, without treatment (-) or after treatment with 1 mM tungstate (WO₄^2-) (during 5 and 10 minutes, as indicated) in the absence of toxins. (B) Average normalized relative density (phosphorylated versus total ERK) in the absence of toxins. (C) Representative Western blots obtained from HEK293, HEKα and HEKαβ₁ cells for ERK1/2 phosphorylation levels, without treatment (-), after treatment with 100 ng/ml EGF (during 10 minutes) (EGF) or after treatment with 1 mM tungstate (WO₄^2-) (during 5 and 10 minutes, as indicated) in the absence (left) or presence of PTX (right). (D) Average normalized relative density (phosphorylated versus total ERK) in the presence of PTX. (E) Representative Western blots obtained from HEK293, HEKα and HEKαβ₁ cells for ERK1/2 phosphorylation levels, without treatment (-), after treatment with 100 ng/ml IGF (during 10 minutes) (IGF) or after treatment with 1 mM tungstate (WO₄^2-) (during 5 and 10 minutes, as indicated) in the absence (left) or presence of IbTX (right). (F) Average normalized relative density (phosphorylated versus total ERK) in the presence of IbTX. n = 4–12 in each experimental group. *P < 0.05 when compared to the other HEK cell lines (Kruskal-Wallis test followed by Dunn <i>post hoc</i> test). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118148#sec002" target="_blank">Methods</a> for further details.</p

Tungstate-induced activation of heterologously expressed Gi/o proteins is mediated by BKαβ₁ channels.

<p>(A) Example of the reinforced membrane fluorescence pattern and emission levels from CFP channel (up-left), YFP (FRET channel) (up-right) and the merge channels (bottom-left) from HEK293 heterologously expressing Gα_i-YFP and CFP-Gβ. Fluorescence microscopy images were recorded by using confocal microscopy (for more details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118148#sec002" target="_blank">Methods</a>). FRET signal was determined by using donor ratiometric parameters (458/514) after excitation in the CFP frequency and registering in the YFP emission frequency. (B) Representative FRET changes from HEK293, HEKα or HEKαβ₁ cells transfected with the cDNAs of G protein subunits, in response to 1 mM tungstate (either in the absence or presence of IbTX), as indicated. (C), Average FRET changes for the different experimental conditions illustrated in B (n = 5–9). *P < 0.05 (Kruskal-Wallis test followed by Dunn <i>post hoc</i> test).</p