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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 also affects Ca2+ binding. (A) BKα G–V relations at a series of Ca2+ concentrations fitted simultaneously (solid curves) with Eq. 14. Only KC and KO were allowed to vary. The fit yielded KC = 3.71 μM and KO = 0.88 μM. The other parameters of the fit were determined from experiments performed with nominally 0 Ca2+ (3 nM) (Vhc = 151 mV, L = 2.2 × 10−6, Vho = 27 mV, zJ = 0.58, zL = 0.41). (B) The fit from A is superimposed on a series of BKα+β1 G–V curves. (C) The voltage-sensing parameters of the model were altered to reflect the changes that occur as β1 binds, Vho = (27→−34 mV), Vhc = (151→80 mV), L = (2.2 × 10−6→2.5 × 10−6). (D) With BKα+β1 voltage-sensing parameters KC and KO were allowed to vary freely, yielding the fit shown and KC = 4.72 μM, KO = 0.82 μM. (E) Here, the BKα+β1 voltage-sensing parameters were used for the fit, and β1 was allowed to influence only half of the channels' eight Ca2+-binding sites. The data are fit with Eq. 13. KC1 and KO1 were held at 3.71 μM and 0.88 μM, respectively. KC2 and KO2 were allowed to vary freely, yielding KC2 = 5.78 μM, KO2 = 0.73 μM. (F) The data are again fit with Eq. 13 but now KO2 was held at 0.88 μM and only KC2 was allowed to vary. This yielded KC2 = 7.14 μM.
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fig9: β1 also affects Ca2+ binding. (A) BKα G–V relations at a series of Ca2+ concentrations fitted simultaneously (solid curves) with Eq. 14. Only KC and KO were allowed to vary. The fit yielded KC = 3.71 μM and KO = 0.88 μM. The other parameters of the fit were determined from experiments performed with nominally 0 Ca2+ (3 nM) (Vhc = 151 mV, L = 2.2 × 10−6, Vho = 27 mV, zJ = 0.58, zL = 0.41). (B) The fit from A is superimposed on a series of BKα+β1 G–V curves. (C) The voltage-sensing parameters of the model were altered to reflect the changes that occur as β1 binds, Vho = (27→−34 mV), Vhc = (151→80 mV), L = (2.2 × 10−6→2.5 × 10−6). (D) With BKα+β1 voltage-sensing parameters KC and KO were allowed to vary freely, yielding the fit shown and KC = 4.72 μM, KO = 0.82 μM. (E) Here, the BKα+β1 voltage-sensing parameters were used for the fit, and β1 was allowed to influence only half of the channels' eight Ca2+-binding sites. The data are fit with Eq. 13. KC1 and KO1 were held at 3.71 μM and 0.88 μM, respectively. KC2 and KO2 were allowed to vary freely, yielding KC2 = 5.78 μM, KO2 = 0.73 μM. (F) The data are again fit with Eq. 13 but now KO2 was held at 0.88 μM and only KC2 was allowed to vary. This yielded KC2 = 7.14 μM.

Mentions: To see, then, if the changes in Vhc and Vho that we have observed upon β1 coexpression are sufficient to account for the BKα channel's enhanced Ca2+ response, for each channel type, we fitted a series of G–V relations with Eq. 14 (Fig. 9). For the BKα fit (Fig. 9 A), Vhc, Vho, zJ, L, and zL were held at the values we determined from our experiments in the absence of Ca2+ (Table II), and only KC and KO were allowed to vary. Still a reasonably good fit was obtained that captured well the shifting nature of the BKα channel's G–V relation as a function of Ca2+ concentration. This fit yielded KC = 3.71 μM and KO = 0.88 μM, similar to our previous estimates (Bao et al., 2002).


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 also affects Ca2+ binding. (A) BKα G–V relations at a series of Ca2+ concentrations fitted simultaneously (solid curves) with Eq. 14. Only KC and KO were allowed to vary. The fit yielded KC = 3.71 μM and KO = 0.88 μM. The other parameters of the fit were determined from experiments performed with nominally 0 Ca2+ (3 nM) (Vhc = 151 mV, L = 2.2 × 10−6, Vho = 27 mV, zJ = 0.58, zL = 0.41). (B) The fit from A is superimposed on a series of BKα+β1 G–V curves. (C) The voltage-sensing parameters of the model were altered to reflect the changes that occur as β1 binds, Vho = (27→−34 mV), Vhc = (151→80 mV), L = (2.2 × 10−6→2.5 × 10−6). (D) With BKα+β1 voltage-sensing parameters KC and KO were allowed to vary freely, yielding the fit shown and KC = 4.72 μM, KO = 0.82 μM. (E) Here, the BKα+β1 voltage-sensing parameters were used for the fit, and β1 was allowed to influence only half of the channels' eight Ca2+-binding sites. The data are fit with Eq. 13. KC1 and KO1 were held at 3.71 μM and 0.88 μM, respectively. KC2 and KO2 were allowed to vary freely, yielding KC2 = 5.78 μM, KO2 = 0.73 μM. (F) The data are again fit with Eq. 13 but now KO2 was held at 0.88 μM and only KC2 was allowed to vary. This yielded KC2 = 7.14 μM.
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fig9: β1 also affects Ca2+ binding. (A) BKα G–V relations at a series of Ca2+ concentrations fitted simultaneously (solid curves) with Eq. 14. Only KC and KO were allowed to vary. The fit yielded KC = 3.71 μM and KO = 0.88 μM. The other parameters of the fit were determined from experiments performed with nominally 0 Ca2+ (3 nM) (Vhc = 151 mV, L = 2.2 × 10−6, Vho = 27 mV, zJ = 0.58, zL = 0.41). (B) The fit from A is superimposed on a series of BKα+β1 G–V curves. (C) The voltage-sensing parameters of the model were altered to reflect the changes that occur as β1 binds, Vho = (27→−34 mV), Vhc = (151→80 mV), L = (2.2 × 10−6→2.5 × 10−6). (D) With BKα+β1 voltage-sensing parameters KC and KO were allowed to vary freely, yielding the fit shown and KC = 4.72 μM, KO = 0.82 μM. (E) Here, the BKα+β1 voltage-sensing parameters were used for the fit, and β1 was allowed to influence only half of the channels' eight Ca2+-binding sites. The data are fit with Eq. 13. KC1 and KO1 were held at 3.71 μM and 0.88 μM, respectively. KC2 and KO2 were allowed to vary freely, yielding KC2 = 5.78 μM, KO2 = 0.73 μM. (F) The data are again fit with Eq. 13 but now KO2 was held at 0.88 μM and only KC2 was allowed to vary. This yielded KC2 = 7.14 μM.
Mentions: To see, then, if the changes in Vhc and Vho that we have observed upon β1 coexpression are sufficient to account for the BKα channel's enhanced Ca2+ response, for each channel type, we fitted a series of G–V relations with Eq. 14 (Fig. 9). For the BKα fit (Fig. 9 A), Vhc, Vho, zJ, L, and zL were held at the values we determined from our experiments in the absence of Ca2+ (Table II), and only KC and KO were allowed to vary. Still a reasonably good fit was obtained that captured well the shifting nature of the BKα channel's G–V relation as a function of Ca2+ concentration. This fit yielded KC = 3.71 μM and KO = 0.88 μM, similar to our previous estimates (Bao et al., 2002).

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