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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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BKCa gating currents. (Top traces) Gating current families recorded from BKα (A) and BKα+β1 (B) channels with 0.5 nM internal Ca2+. The second and third traces in A and B demonstrate that gating currents are not observed in patches from oocytes that were not injected with BKCa cRNA (second) or with hyperpolarizing voltage steps (third). The lowest traces in A and B are gating currents recorded with pulses to +160 mV. Repolarizations are to −80 mV. Exponential fits to the on and off currents are indicated with dashed line. (C and D) Comparisons of on-gating current (Ig) and potassium current (IK) from BKα (C) and BKα+β1 (D) channels. Pulses were to +160 mV. Ca2+ = 0.5 nM. The gating and ionic currents compared in C and D are from different patches.
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fig3: BKCa gating currents. (Top traces) Gating current families recorded from BKα (A) and BKα+β1 (B) channels with 0.5 nM internal Ca2+. The second and third traces in A and B demonstrate that gating currents are not observed in patches from oocytes that were not injected with BKCa cRNA (second) or with hyperpolarizing voltage steps (third). The lowest traces in A and B are gating currents recorded with pulses to +160 mV. Repolarizations are to −80 mV. Exponential fits to the on and off currents are indicated with dashed line. (C and D) Comparisons of on-gating current (Ig) and potassium current (IK) from BKα (C) and BKα+β1 (D) channels. Pulses were to +160 mV. Ca2+ = 0.5 nM. The gating and ionic currents compared in C and D are from different patches.

Mentions: Here, to directly test this prediction we have examined BKCa gating currents in the absence and presence of β1. A family of gating currents for the BKα channel is shown in Fig. 3 A. These currents were recorded in the essential absence of Ca2+ (0.5 nM) with 1-ms voltage steps. Most notable, they are small and fast, 500–1,000 times smaller than the ionic currents we typically observe under the same conditions of channel expression (Fig. 1 A), and at +160 mV (Fig. 3 A, fourth trace down) the ON gating current decays with a time constant of 57.2 ± 4.0 μs (n = 16), and the OFF gating current at −80 mV is similarly fast (τ(off) = 31.2 ± 3.3 μs, n = 20). Thus, care had to be taken to ensure that what we were observing was in fact gating current and not the result of capacity current subtraction errors. We are confident, however, that these currents are indeed gating currents, as they are not seen in uninjected oocytes (second trace down). They are not seen in response to voltage pulses of equal magnitude but opposite polarity (third trace down), and they have characteristics very similar to those reported previously for the BKα channel (Horrigan and Aldrich, 1999, 2002) (Table II).


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)

BKCa gating currents. (Top traces) Gating current families recorded from BKα (A) and BKα+β1 (B) channels with 0.5 nM internal Ca2+. The second and third traces in A and B demonstrate that gating currents are not observed in patches from oocytes that were not injected with BKCa cRNA (second) or with hyperpolarizing voltage steps (third). The lowest traces in A and B are gating currents recorded with pulses to +160 mV. Repolarizations are to −80 mV. Exponential fits to the on and off currents are indicated with dashed line. (C and D) Comparisons of on-gating current (Ig) and potassium current (IK) from BKα (C) and BKα+β1 (D) channels. Pulses were to +160 mV. Ca2+ = 0.5 nM. The gating and ionic currents compared in C and D are from different patches.
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getmorefigures.php?uid=PMC2266624&req=5

fig3: BKCa gating currents. (Top traces) Gating current families recorded from BKα (A) and BKα+β1 (B) channels with 0.5 nM internal Ca2+. The second and third traces in A and B demonstrate that gating currents are not observed in patches from oocytes that were not injected with BKCa cRNA (second) or with hyperpolarizing voltage steps (third). The lowest traces in A and B are gating currents recorded with pulses to +160 mV. Repolarizations are to −80 mV. Exponential fits to the on and off currents are indicated with dashed line. (C and D) Comparisons of on-gating current (Ig) and potassium current (IK) from BKα (C) and BKα+β1 (D) channels. Pulses were to +160 mV. Ca2+ = 0.5 nM. The gating and ionic currents compared in C and D are from different patches.
Mentions: Here, to directly test this prediction we have examined BKCa gating currents in the absence and presence of β1. A family of gating currents for the BKα channel is shown in Fig. 3 A. These currents were recorded in the essential absence of Ca2+ (0.5 nM) with 1-ms voltage steps. Most notable, they are small and fast, 500–1,000 times smaller than the ionic currents we typically observe under the same conditions of channel expression (Fig. 1 A), and at +160 mV (Fig. 3 A, fourth trace down) the ON gating current decays with a time constant of 57.2 ± 4.0 μs (n = 16), and the OFF gating current at −80 mV is similarly fast (τ(off) = 31.2 ± 3.3 μs, n = 20). Thus, care had to be taken to ensure that what we were observing was in fact gating current and not the result of capacity current subtraction errors. We are confident, however, that these currents are indeed gating currents, as they are not seen in uninjected oocytes (second trace down). They are not seen in response to voltage pulses of equal magnitude but opposite polarity (third trace down), and they have characteristics very similar to those reported previously for the BKα channel (Horrigan and Aldrich, 1999, 2002) (Table II).

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