Orio P, Torres Y, Rojas P, Carvacho I, Garcia ML, Toro L, Valverde MA, Latorre R.

HL54970 NHLBI NIH HHS , GM56312 NIGMS NIH HHS , R01 HL054970 NHLBI NIH HHS

	Figure 1. Effects of β1 and β2IR subunits on BK channel steady-state activation parameters. (A) Macroscopic currents recorded in the inside-out configuration at 5 nM (left) and 3 μM (right) intracellular calcium. The voltage protocol for 5 nM was: holding potential (HP), 0 mV; prepulse (PP), −80 mV; test pulse (TP), −10 to 250 mV in 10-mV steps; tail, −60 mV. The voltage protocol for 3 μM was: HP, 0 mV; PP, −150 mV; TP, −150 to 140 in 10-mV steps; tail, −60 mV. (B) Normalized G/V curves at 3 μM (closed symbols) and 5 nM Ca2+ (open symbols) for the α, α + β1 and α + β2IR current traces shown in A. Lines are the best fit of a Boltzmann distribution (Eq. 1). Fit parameters are as follows. α, V0.5 = 215 mV, z = 0.95 (5 nM); V0.5 = 91 mV, z = 1.19 (3 μM). α + β1, V0.5 = 243 mV, z = 0.61 (5 nM); V0.5 = −7 mV, z = 1.11 (3 μM). α + β2IR, V0.5 = 189 mV, z = 0.91 (5 nM); V0.5 = −37 mV, z = 1.58 (3 μM). (C) Average of the obtained V0.5 values plotted against calcium concentration. A best fit sigmoid concentration–effect curve was added as reference for visual comparison in the following figures. (D) Average of the obtained z values plotted against calcium concentration. A best fit dual effect sigmoid curve was added as reference for visual comparison in the following figures. Error bars are SD, n = 7–8. When SD bars are not visible, they are smaller than symbol size.
	Figure 4. Effect of the β1Lβ2 and β2Lβ1 subunits on BK channel macroscopic kinetics in the absence of calcium. (A) Current traces evoked by a 200 mV pulse after a −80 mV prepulse. Over the traces, the best fit to an exponential raise is shown. Current magnitude of each trace was normalized by the steady-state current predicted by the fit. For α, the fit was extrapolated to show that the traces are effectively normalized to the same maximum. (B) Tail current traces evoked by a −60 mV pulse after opening the channels with a +200 mV activation pulse. Current magnitude of each trace was normalized by the peak of the tail current. Over the traces, the best fit to an exponential decay is shown. (C) Activation time constant (τact) plotted against activation voltage. Symbols represent mean ± SD. n = 7–10. (D) Deactivation time constant (τdeact) plotted against voltage. Symbols represent mean ± SD. n = 3–5. In the case of α, α + β1, and α + β2IR, only the mean is shown. Lines represent the best fit of a single exponential function to the data between −50 and +60 mV extrapolated from −80 to +100 mV.
	Figure 2. Effects of β1Lβ2 and β2Lβ1 subunits on BK channel steady-state activation parameters. (A) Schematic description of the β1Lβ2 and β2Lβ1 chimeras. (B) Macroscopic currents recorded in the inside-out configuration at 5 nM (left) and 3.3 μM (right) intracellular calcium, for α + β1Lβ2 (top) and α + β2Lβ1 (bottom) channels. The voltage protocols are as described in legend of Fig. 1. (C) Normalized G/V curves at 3.3 μM (closed symbols) and 5 nM Ca2+ (open symbols) for the current traces shown in B. Lines are the best fit of a Boltzmann distribution (Eq. 1). Fit parameters are as follows. α + β1Lβ2, V0.5 = 267 mV, z = 0.7 (5 nM); V0.5 = 2 mV, z = 0.92 (3.3 μM). α + β2Lβ1, V0.5 = 226 mV, z = 0.65 (5 nM); V0.5 = −42 mV, z = 1.7 (3.3 μM).
	Figure 3. Steady-state activation parameters for α + β1Lβ2 and α + β2Lβ1 channels. (A) Average of the obtained V0.5 values plotted against calcium concentration. For α, α + β1, and α + β2IR only the best fit sigmoid concentration–effect curve (see Fig. 1 C) is depicted. (B) Average of the obtained z values plotted against calcium concentration. For α, α + β1, and α + β2IR only the best fit curve (see Fig. 1 D) is depicted. Error bars are SD, n = 5–6. When SD bars are not visible, they are smaller than symbol size.
	Figure 5. Effects of β1NCβ2 and β2NCβ1 subunits on BK channel steady-state activation parameters. (A) Schematic description of the β1NCβ2 and β2NCβ1 chimeras. (B) Macroscopic currents recorded in the inside-out configuration at 5 nM (left) and 3.3 μM (right) intracellular calcium, for α + β1NCβ2 (top) and α + β2NCβ1 (bottom) channels. The voltage protocols are as described in legend of Fig. 1. (C) Normalized G/V curves at 3.3 μM (closed symbols) and 5 nM Ca2+ (open symbols) for the current traces shown in B. Lines are the best fit of a Boltzmann distribution (Eq. 1). Fit parameters are as follows. α + β1NCβ2, V0.5 = 204 mV, z = 0.7 (5 nM); V0.5 = −95 mV, z = 2.0 (3.3 μM). α + β2NCβ1, V0.5 = 208 mV, z = 0.9 (5 nM); V0.5 = −10 mV, z = 1.5 (3.3 μM).
	Figure 6. Activation parameters for α + β1NCβ2 and α + β2NCβ1 channels. (A) Average of the obtained V0.5 values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit sigmoid concentration–effect curve (see Fig. 1 C) is depicted. (B) Average of the obtained z values plotted against calcium concentration. For α, α + β1, and α + β2IR only the best fit curve (see Fig. 1 D) is depicted. Error bars are SD, n = 4–7. When SD bars are not visible, they are smaller than symbol size. (C) Activation time constant (τact) at 5 nM intracellular [Ca2+] plotted against activation voltage. Symbols represent mean ± SD. n = 3–5. (D) Deactivation time constant (τdeact) at 5 nM intracellular [Ca2+] plotted against voltage. Symbols represent mean ± SD. n = 3–7. In the case of α, α + β1, and α + β2IR, only the mean is shown. Lines are defined as in Fig. 4.
	Figure 7. Effects of β1TMsβ2 and β2TMsβ1 subunits on BK channel steady-state activation parameters. (A) Schematic description of the β1TMsβ2 and β2TMsβ1 chimeras. (B) Macroscopic currents recorded in the inside-out configuration at 5 nM (left) and 3.3 μM (right) intracellular calcium, for α + β1TMsβ2 (top) and α + β2TMsβ1 (bottom) channels. The voltage protocols are as described in legend of Fig. 1. (C) Normalized G/V curves at 3.3 μM (closed symbols) and 5 nM Ca2+ (open symbols) for the current traces shown in B. Lines are the best fit of a Boltzmann distribution (Eq. 1). Fit parameters are as follows. α + β1TMsβ2, V0.5 = 251 mV, z = 0.7 (5 nM); V0.5 = −9 mV, z = 1.2 (3.3 μM). α + β2TMsβ1, V0.5 = 191 mV, z = 0.7 (5 nM); V0.5 = −90 mV, z = 1.7 (3.3 μM).
	Figure 8. Activation parameters for α + β1TMsβ2 and α + β2TMsβ1 channels. (A) Average of the obtained V0.5 values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit sigmoid concentration–effect curve (see Fig. 1 C) is depicted. (B) Average of the obtained z values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit curve (see Fig. 1 D) is depicted. Error bars are SD, n = 5–6. Missing error bars are smaller than symbol size. (C) Activation time constant (τact) at 5 nM intracellular [Ca2+] plotted against activation voltage. Symbols represent mean ± SD. n = 6–8. (D) Deactivation time constant (τdeact) at 5 nM intracellular [Ca2+] plotted against voltage. Symbols represent mean ± SD. n = 4–5. In the case of α, α + β1, and α + β2IR, only the mean is shown. Lines are defined as in Fig. 4. The exponent factors expressed as electronic charges (z = slope × RT/F) are α + β1TMsβ2, 0.39 ± 0.01; α + β2TMsβ1, 0.39 ± 0.02.
	Figure 9. Activation parameters for α + β1Nβ2 and α + β2Nβ1 channels. (A) Schematic description of the β1Nβ2 and β2Nβ1 chimeras. (B) Average of the obtained V0.5 values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit sigmoid concentration–effect curve (see Fig. 1 C) is depicted. (C) Average of the obtained z values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit curve (see Fig. 1 D) is depicted. Error bars are SD, n = 4–6. Missing error bars are smaller than symbol size. (D) Activation time constant (τact) at 5 nM intracellular [Ca2+] plotted against activation voltage. Symbols represent mean ± SD. n = 4–11. (E) Deactivation time constant (τdeact) at 5 nM intracellular [Ca2+] plotted against voltage. Symbols represent mean ± SD. n = 4–6. In the case of α, α + β1, and α + β2IR, only the mean is shown. Lines represent the best fit of a simple exponential function to the data between −50 and +60 mV, extrapolated from −80 to +100 mV. The exponential factors expressed as electronic charges (z = slope×RT/F) are α + β1Nβ2, 0.28 ± 0.01; α + β2Nβ1, 0.34 ± 0.01.
	Figure 10. Activation parameters for α + β1Cβ2 and α + β2Cβ1 channels. (A) Schematic description of the β1Cβ2 and β2Cβ1 chimeras. (B) Average of the obtained V0.5 values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit sigmoid concentration–effect curve (see Fig. 1 C) is depicted. (C) Average of the obtained z values plotted against calcium concentration. For α, α + β1, and α + β2IR, only the best fit curve (see Fig. 1 D) is depicted. Error bars are SD, n = 9–11. Missing error bars are smaller than symbol size. (D) Activation time constant (τact) at 5 nM intracellular [Ca2+] plotted against activation voltage. Symbols represent mean ± SD. n = 8–12. (E) Deactivation time constant (τdeact) at 5 nM intracellular [Ca2+] plotted against voltage. Symbols represent mean ± SD. n = 4–8. In the case of α, α + β1, and α + β2IR, only the mean is shown. Lines represent the best fit of a simple exponential function to the data between −50 and +60 mV, extrapolated from −80 to +100 mV. The exponential factors expressed as electronic charges (z = slope×RT/F) are α + β1Cβ2, 0.29 ± 0.01; α + β2Cβ1, 0.43 ± 0.01.

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