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Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit.

Bao L, Cox DH - J. Gen. Physiol. (2005)

Bottom Line: Our results may be summarized as follows.The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present.The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology Research Institute, New England Medical Center, Boston, MA 02111, USA.

ABSTRACT
Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are regulated by the tissue-specific expression of auxiliary beta subunits. Beta1 is predominantly expressed in smooth muscle, where it greatly enhances the BK(Ca) channel's Ca(2+) sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present. Furthermore, beta1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by approximately 24% (16.8-->12.8). The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca(2+) affinity brought about by beta1 at negative voltages, our studies suggest that beta1 also decreases the true Ca(2+) affinity of the closed channel, increasing its Ca(2+) dissociation constant from approximately 3.7 microM to between 4.7 and 7.1 microM, depending on how many binding sites are affected.

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β1 increases the Ca2+ sensitivity of the BKCa channel. (A and B) Macroscopic currents recorded from BKα channels (A) and BKα+β1 channels (B). Currents are from inside-out Xenopus oocyte macropatches exposed to 10 μM internal Ca2+. (C and D) G–V relations determined at the following Ca2+ concentrations: 0.003, 1, 10, and 100 μM for the BKα channel (C) and BKα+β1 channel (D). Each curve represents the average of between 4 and 22 individual curves. Error bars indicate SEM. The solid curves are Boltzmann fits with the following parameters: BKα, 3 nM Ca2+: Q = 0.93 e, V1/2 = 200.3 mV; 1 μM Ca2+: Q = 1.36 e, V1/2 = 120.6 mV; 10 μM Ca2+: Q = 1.18 e, V1/2 = 32.8 mV; 100 μM Ca2+: Q = 1.15 e, V1/2 = −2.4 mV. BKα1β1, 3 nM Ca2+: Q = 0.62 e, V1/2 = 213.1 mV; 1 μM Ca2+: Q = 0.94 e, V1/2 = 82.1 mV; 10 μM Ca2+: Q = 1.02 e, V1/2 = −59.5 mV; 100 μM Ca2+: Q = 0.96 e, V1/2 = −101 mV. (E) Plots of half-maximal activation voltage (V1/2) vs. Ca2+ concentration. The V1/2 values are from Table I. Error bars represent SEM.
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fig1: β1 increases the Ca2+ sensitivity of the BKCa channel. (A and B) Macroscopic currents recorded from BKα channels (A) and BKα+β1 channels (B). Currents are from inside-out Xenopus oocyte macropatches exposed to 10 μM internal Ca2+. (C and D) G–V relations determined at the following Ca2+ concentrations: 0.003, 1, 10, and 100 μM for the BKα channel (C) and BKα+β1 channel (D). Each curve represents the average of between 4 and 22 individual curves. Error bars indicate SEM. The solid curves are Boltzmann fits with the following parameters: BKα, 3 nM Ca2+: Q = 0.93 e, V1/2 = 200.3 mV; 1 μM Ca2+: Q = 1.36 e, V1/2 = 120.6 mV; 10 μM Ca2+: Q = 1.18 e, V1/2 = 32.8 mV; 100 μM Ca2+: Q = 1.15 e, V1/2 = −2.4 mV. BKα1β1, 3 nM Ca2+: Q = 0.62 e, V1/2 = 213.1 mV; 1 μM Ca2+: Q = 0.94 e, V1/2 = 82.1 mV; 10 μM Ca2+: Q = 1.02 e, V1/2 = −59.5 mV; 100 μM Ca2+: Q = 0.96 e, V1/2 = −101 mV. (E) Plots of half-maximal activation voltage (V1/2) vs. Ca2+ concentration. The V1/2 values are from Table I. Error bars represent SEM.

Mentions: The BKCa channel is both Ca2+ and voltage sensitive, and the effects of these stimuli together are often displayed as a series of conductance–voltage (G–V) relations determined over a series of Ca2+ concentrations (Barrett et al., 1982). Such a series, determined from currents recorded from channels expressed in Xenopus oocyte macropatches is shown in Fig. 1 C. These G–V curves were derived from channels composed of α subunits alone. When the BKCa β1 subunit is coexpressed with the α subunit, the Ca2+-induced leftward shifting evident in Fig. 1 C becomes more pronounced (Fig. 1, D and E; see also Table I) (McManus et al., 1995; Wallner et al., 1995; Meera et al., 1996; Cox and Aldrich, 2000), and thus it may be said that β1 increases the Ca2+ sensitivity of the BKCa channel in that it increases its G–V shift in response to changes in Ca2+ concentration (McManus et al., 1995).


Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit.

Bao L, Cox DH - J. Gen. Physiol. (2005)

β1 increases the Ca2+ sensitivity of the BKCa channel. (A and B) Macroscopic currents recorded from BKα channels (A) and BKα+β1 channels (B). Currents are from inside-out Xenopus oocyte macropatches exposed to 10 μM internal Ca2+. (C and D) G–V relations determined at the following Ca2+ concentrations: 0.003, 1, 10, and 100 μM for the BKα channel (C) and BKα+β1 channel (D). Each curve represents the average of between 4 and 22 individual curves. Error bars indicate SEM. The solid curves are Boltzmann fits with the following parameters: BKα, 3 nM Ca2+: Q = 0.93 e, V1/2 = 200.3 mV; 1 μM Ca2+: Q = 1.36 e, V1/2 = 120.6 mV; 10 μM Ca2+: Q = 1.18 e, V1/2 = 32.8 mV; 100 μM Ca2+: Q = 1.15 e, V1/2 = −2.4 mV. BKα1β1, 3 nM Ca2+: Q = 0.62 e, V1/2 = 213.1 mV; 1 μM Ca2+: Q = 0.94 e, V1/2 = 82.1 mV; 10 μM Ca2+: Q = 1.02 e, V1/2 = −59.5 mV; 100 μM Ca2+: Q = 0.96 e, V1/2 = −101 mV. (E) Plots of half-maximal activation voltage (V1/2) vs. Ca2+ concentration. The V1/2 values are from Table I. Error bars represent SEM.
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fig1: β1 increases the Ca2+ sensitivity of the BKCa channel. (A and B) Macroscopic currents recorded from BKα channels (A) and BKα+β1 channels (B). Currents are from inside-out Xenopus oocyte macropatches exposed to 10 μM internal Ca2+. (C and D) G–V relations determined at the following Ca2+ concentrations: 0.003, 1, 10, and 100 μM for the BKα channel (C) and BKα+β1 channel (D). Each curve represents the average of between 4 and 22 individual curves. Error bars indicate SEM. The solid curves are Boltzmann fits with the following parameters: BKα, 3 nM Ca2+: Q = 0.93 e, V1/2 = 200.3 mV; 1 μM Ca2+: Q = 1.36 e, V1/2 = 120.6 mV; 10 μM Ca2+: Q = 1.18 e, V1/2 = 32.8 mV; 100 μM Ca2+: Q = 1.15 e, V1/2 = −2.4 mV. BKα1β1, 3 nM Ca2+: Q = 0.62 e, V1/2 = 213.1 mV; 1 μM Ca2+: Q = 0.94 e, V1/2 = 82.1 mV; 10 μM Ca2+: Q = 1.02 e, V1/2 = −59.5 mV; 100 μM Ca2+: Q = 0.96 e, V1/2 = −101 mV. (E) Plots of half-maximal activation voltage (V1/2) vs. Ca2+ concentration. The V1/2 values are from Table I. Error bars represent SEM.
Mentions: The BKCa channel is both Ca2+ and voltage sensitive, and the effects of these stimuli together are often displayed as a series of conductance–voltage (G–V) relations determined over a series of Ca2+ concentrations (Barrett et al., 1982). Such a series, determined from currents recorded from channels expressed in Xenopus oocyte macropatches is shown in Fig. 1 C. These G–V curves were derived from channels composed of α subunits alone. When the BKCa β1 subunit is coexpressed with the α subunit, the Ca2+-induced leftward shifting evident in Fig. 1 C becomes more pronounced (Fig. 1, D and E; see also Table I) (McManus et al., 1995; Wallner et al., 1995; Meera et al., 1996; Cox and Aldrich, 2000), and thus it may be said that β1 increases the Ca2+ sensitivity of the BKCa channel in that it increases its G–V shift in response to changes in Ca2+ concentration (McManus et al., 1995).

Bottom Line: Our results may be summarized as follows.The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present.The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology Research Institute, New England Medical Center, Boston, MA 02111, USA.

ABSTRACT
Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are regulated by the tissue-specific expression of auxiliary beta subunits. Beta1 is predominantly expressed in smooth muscle, where it greatly enhances the BK(Ca) channel's Ca(2+) sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present. Furthermore, beta1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by approximately 24% (16.8-->12.8). The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca(2+) affinity brought about by beta1 at negative voltages, our studies suggest that beta1 also decreases the true Ca(2+) affinity of the closed channel, increasing its Ca(2+) dissociation constant from approximately 3.7 microM to between 4.7 and 7.1 microM, depending on how many binding sites are affected.

Show MeSH
Related in: MedlinePlus