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Figure 1. Macroscopic currents recorded after cbv1+β1–4 subunit expression in Xenopus oocytes show characteristic features of BK currents. (A) Representative current records from I/O macropatches expressing different BK channel subunit combinations (cbv1±β1/wt β2/β2-IR/β3/β4; 10 µM Ca2+i). Currents were evoked by 100 ms-long (except for cbv1+wt β2; 600 ms-long; 3 µM Ca2+i), 10-mV depolarizing steps from −150 to 150 mV, with a holding potential set to −80 mV. cbv1+wt β2 channels are characterized by fast inactivation. (B) V0.5 versus [Ca2+]i plot underscores that β subunit types differentially modulate the apparent Ca2+i sensitivity of the channel complex. (C and D) Bar graphs show averaged activation (τact) and deactivation (τdeact) time constants, respectively, obtained at Vmax with Ca2+i = 10 µM. Mean Vmax (mV) values for different constructs were (mean ± SEM) cbv1 = 117 ± 8.2; cbv1+β1 = 55 ± 14; cbv1+β2-IR = 40 ± 3.3; cbv1+β3 = 80 ± 17; cbv1+β4 = 130 ± 8. τdeact was estimated from tail currents after stepping down to −80 mV from Vmax. n = 4–8; *, different from cbv1 (P < 0.05); #, different from cbv1+β1 (P < 0.05). Data are expressed as mean ± SEM.
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Figure 2. Ethanol effect on BK macroscopic currents is Ca2+i dependent. 50 mM ethanol activates homomeric cbv1 channels at submicromolar (0.3 µM) Ca2+i while causing mild inhibition at higher Ca2+i (100 µM). (A) Macroscopic current recordings evoked from I/O patches at 0.3 and 100 µM Ca2+i in the absence or presence of 50 mM ethanol after cbv1 expression in Xenopus oocytes. (B) At 0.3 µM Ca2+i, ethanol shifts the G/Gmax-V plot to the left, indicating channel activation. (C) At 100 µM Ca2+i, ethanol shifts the G/Gmax-V plot to the right, indicating inhibition of channel activity. (D) At nominal zero Ca2+i, ethanol does not shift the G/Gmax-V plot, indicating ethanol fails to modulate cbv1 channel activity at nominal zero Ca2+i. (E) V0.5 versus [Ca2+]i plot showing that the activation to inhibition crossover for ethanol effect on cbv1 currents occurs at ≈20 µM Ca2+i. (F) Bar graph showing ethanol-induced change in V0.5 from control obtained at nominal zero, 0.3, and 100 µM Ca2+i. n = 5–8; each patch was excised from a different cell. *, different from control (P < 0.05); **, different from control (P < 0.01). Data are expressed as mean ± SEM.
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Figure 3. Ethanol activates β1-containing BK channels at submicromolar (0.3 µM) Ca2+i, while causing strong inhibition at higher (100 µM) Ca2+i. (A) Macroscopic current recordings from I/O patches obtained at 0.3 and 100 µM Ca2+i in the absence or presence of 50 mM ethanol after cbv1+β1 expression in Xenopus oocytes. (B and C) Ethanol shifts the G/Gmax-V plot to the left at 0.3 µM Ca2+i, indicating BK current potentiation (B), while shifting the plot to the right at 100 µM Ca2+i, indicating inhibition (C). (D) β1 subunits set the activation to inhibition crossover of ethanol responses at ≈3 µM Ca2+i. (E) Bar graph representing ethanol-induced change in V0.5 values from pre-ethanol application obtained at 0.3 µM and 100 µM Ca2+i. n = 5–8; each patch was excised from a different cell. *, different from control (P < 0.05); **, different from control (P < 0.01). Data are expressed as mean ± SEM.
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Figure 4. The ethanol response of wt β2–containing BK channels is similar to the ethanol response of cbv1+β1 channels. (A and B) Single-channel recordings of cbv1+wt β2 channels from I/O patches at 0.3 (A) and 100 µM Ca2+i (B); Vm = −40 mV. Records were obtained before (top traces), during (middle traces), and immediately after (bottom traces) patch exposure to 50 mM ethanol. Arrows indicate the baseline (all channels in nonpermeant states). (C) Averaged NPo ratios in the presence and absence of ethanol from cbv1+wt β2 at 0.3, 3, 10, 30, and 100 µM Ca2+i. Data demonstrate that the activation to inhibition crossover for ethanol effect on cbv1+wt β2 channels occurs at ≈3 µM Ca2+i, which is similar to that found in cbv1+β1 channels. n = 5–7; each patch was excised from a different cell. *, different from control (P < 0.05); **, different from control (P < 0.01). Data are expressed as mean ± SEM.
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Figure 5. The ethanol response of β2-IR–containing BK channels is similar to the ethanol response of cbv1+β1 channels. (A) V0.5 versus [Ca2+]i plot showing that the activation to inhibition crossover for ethanol effect on cbv1+β2-IR channels occurs at ≈3 µM Ca2+i, as found with cbv1±β1 (Fig. 3 D) and wt β2 (Fig. 4 C). (B) Bar graph showing ethanol-induced change in V0.5 values from control obtained at 0.3 and 100 µM Ca2+i. n = 4–6 patches; each patch was excised from a different cell. *, different from control (P < 0.05); **, different from control (P < 0.01). Data are expressed as mean ± SEM.
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Figure 6. Ethanol responses of β3- or β4-containing BK currents mimic those of homomeric BK (cbv1) channels. (A and C) V0.5 versus [Ca2+]i plot showing that the activation to inhibition crossover for ethanol effect on cbv1+β3 (A) and cbv1+β4 channel complexes (C) occurs at ∼20 µM Ca2+i, as found for homomeric cbv1 exposed to ethanol (see Fig. 2 D). (B and D) Bar graphs representing ethanol-induced change in V0.5 values from cbv1+β3 (B) and cbv1+β4 channel complexes (D) obtained at 0.3 µM and 100 µM Ca2+i. n = 3–7 patches; each patch was excised from a different cell. *, different from control (P < 0.05); **, different from control (P < 0.01). Data are expressed as mean ± SEM.
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Figure 7. Ethanol does not alter the parameters associated with closed to open conformational change. (A) Representative unitary current recordings evoked from I/O patches in nominal zero Ca2+i, with membrane patches held at the indicated voltages. Recordings were obtained before (left) and immediately after (right) patch exposure to 50 mM ethanol. Arrows indicate the baseline (all channels in nonpermeant states). (B and C) Mean log Po-V relations over a wide range of voltages in the presence and absence of 50 mM ethanol. Below 80 mV, data points were obtained from unitary current recordings; above 80 mV, data points were obtained from macroscopic current recordings in nominal zero Ca2+i. Po-V data at far negative voltages were fitted with Eq. 2 to determine L0 and zL. Best-fit parameters (±95% confidence interval) are listed in the corresponding graphs. On average, patches contained 450 channels. n = 11; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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Figure 8. Ethanol does not alter movement of voltage sensors and other voltage-dependent parameters of cbv1 channels. (A and B) Po-V data over a wide range of voltages were fitted with Eq. 3 after constraining Vh(J), L0, and zL (Fig. 7; Horrigan and Aldrich, 2002) to determine the values of D and zJ. For ethanol data, Vh(J) was allowed to vary. Best-fit parameters (±95% confidence interval) are listed in the corresponding graphs. n = 11; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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Figure 9. Ethanol modulates parameters associated with Ca2+i binding to cbv1 channels. (A and B) The averaged (log [R0])-[Ca2+i] plots (voltage: −80 mV) in the absence (A) and presence (B) of 50 mM ethanol. (log [R0])-Ca2+i plots were fitted with the Eq. 5. Best-fit parameters (±95% confidence interval) are shown to the left of the corresponding plots. Data demonstrate that ethanol action on cbv1 is primarily caused by modulation of Kd and C. n = 4; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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Figure 10. Ethanol does not alter the allosteric interaction between Ca2+ binding and voltage sensor activation of cbv1 channels. (A and B) Averaged G/Gmax-V curves obtained over a wide range of Ca2+i in the absence (A) and presence (B) of 50 mM ethanol. G/Gmax-V plots were fitted with Eq. 1. For curves in panel A, L0, zL, Vh(J), zJ, D, Kd, and C were constrained to 2.3 × 10−6, 0.34, 155, 0.6, 19.8, 9.02, and 6.07, respectively (Figs. 7, 8, and 9), whereas E was allowed to vary. For G-V curves in panel B, L0, zL, Vh(J), zJ, D, Kd, and C were fixed to 2.1 × 10−6, 0.35, 155.1, 0.6, 19.6, 1, and 4.7, respectively, whereas E was allowed to vary. Best-fit parameters (±95% confidence interval) are shown to the left of the corresponding plots. n = 4–8; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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Figure 11. Ethanol does not alter the voltage-dependent parameters of cbv1+β1 channels. (A–C) Representaive current traces (A) and mean log Po-V relations in the absence (B) and presence (C) of 50 mM ethanol. At voltages less positive than 80 mV, data points were obtained from unitary current recordings; at voltages more positive than 80 mV, data points were obtained from macroscopic current recordings obtained at nominal zero Ca2+i. Po-V data obtained over a wide range of voltages were fitted with Eq. 2 to determine the values of Vh(J), L0, zL, D, and zJ. For control data, Vh(J) was constrained to 80 mV (according to Bao and Cox [2005]), whereas for ethanol data Vh(J) was allowed to vary. Best-fit parameters (±95% confidence interval) are listed in the corresponding graphs. n = 12; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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Figure 12. Ethanol modulates parameters associated with Ca2+i binding affinity of cbv1+β1 channels. (A and B) The averaged (log [R0])-[Ca2+i] plots (voltage: −120 mV) in the absence (A) and presence (B) of 50 mM ethanol. (log [R0])-[Ca2+i] plots were fitted with the Eq. 5. Best-fit parameters (±95% confidence interval) are shown to the left of the corresponding plots. Data demonstrate that ethanol significantly decreases the values of Kd and C. n = 4–5; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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Figure 13. Ethanol modulates allosteric interaction between Ca2+ binding and voltage sensor activation in cbv1+β1 channels. (A and B) Averaged G/Gmax-V curves obtained over a wide range of Ca2+i, in the absence (A) and presence (B) of 50 mM ethanol. These plots were fitted with Eq. 1. For curves in panel A, L0, zL, Vh(J), zJ, D, Kd, and C were fixed to 1.25 × 10−7, 0.41, 80, 0.6, 30.1, 19, and 27.2, respectively, whereas E was allowed to vary. For curves in panel B, L0, zL, Vh(J), zJ, D, and Kd were fixed to 1.3 × 10−7, 0.42, 80.83, 0.59, 29.3, and 11.3, respectively (Figs. 11 and 12), whereas E was allowed to vary. Best-fit parameters (±95% confidence interval) are shown in the corresponding plots. Data demonstrate that ethanol decreases allosteric parameter E. n = 4–8; each patch was excised from a different cell. Data are expressed as mean ± SEM.
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