Fig 2. Tungstate-mediated activation of BKαβ₁ channels is independent of G proteins.

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 Methods). (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 versus 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) versus 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) [14] and 147 ± 2 mV (n = 7) [26] (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) [14]. 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).