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Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage.

Sweet TB, Cox DH - J. Gen. Physiol. (2008)

Bottom Line: Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation.To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude.Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA.

ABSTRACT
It has been established that the large conductance Ca(2+)-activated K(+) channel contains two types of high-affinity Ca(2+) binding sites, termed the Ca(2+) bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance-voltage relations determined over a series of Ca(2+) concentrations with models of channel gating that include both voltage sensing and Ca(2+) binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation. To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

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A modified HA model can explain the effect of Ca2+ on the steady-state gating properties of the BKCa channel. Shown are series of mSlo1α G-V relations determined at the following [Ca2+]: 0.003, 0.070, 0.130, 0.360, 0.8, 10, and 100 μM and fit simultaneously with the modified model that includes two types of Ca2+ binding sites, one type interacts with the voltage sensor, and the other type is independent. The data are the same as shown in Fig. 8. The parameters were held as follows: KO1 = 0.88 μM, KC1 = 3.18 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, LO = 2.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, and zJ = 0.58 e. The value of E coupling voltage sensor activation and Ca2+ binding at one type of site was allowed to vary. Shown is the best fit obtained. The value of E was calculated to be 2.84 ± 0.13.
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fig11: A modified HA model can explain the effect of Ca2+ on the steady-state gating properties of the BKCa channel. Shown are series of mSlo1α G-V relations determined at the following [Ca2+]: 0.003, 0.070, 0.130, 0.360, 0.8, 10, and 100 μM and fit simultaneously with the modified model that includes two types of Ca2+ binding sites, one type interacts with the voltage sensor, and the other type is independent. The data are the same as shown in Fig. 8. The parameters were held as follows: KO1 = 0.88 μM, KC1 = 3.18 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, LO = 2.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, and zJ = 0.58 e. The value of E coupling voltage sensor activation and Ca2+ binding at one type of site was allowed to vary. Shown is the best fit obtained. The value of E was calculated to be 2.84 ± 0.13.

Mentions: We held all parameters but E to values that have been independently determined either here or previously (Bao and Cox, 2005) and allowed only E to vary. The resulting best fit from this approach is shown in Fig. 11. It shows that even with these severe constraints, the two-site HA model with our newly determined Ca2+ binding constants can approximate the shifting of the mSlo channel's G-V relation as a function of [Ca2+], and remarkably the fit yields E = 2.8, a value that is very similar to the value of E (2.4) estimated independently by Horrigan and Aldrich (2002) from measurements of the channel's gating charge movement as a function of voltage at 0 and 70 μM [Ca2+]. Thus, we currently favor this estimate.


Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage.

Sweet TB, Cox DH - J. Gen. Physiol. (2008)

A modified HA model can explain the effect of Ca2+ on the steady-state gating properties of the BKCa channel. Shown are series of mSlo1α G-V relations determined at the following [Ca2+]: 0.003, 0.070, 0.130, 0.360, 0.8, 10, and 100 μM and fit simultaneously with the modified model that includes two types of Ca2+ binding sites, one type interacts with the voltage sensor, and the other type is independent. The data are the same as shown in Fig. 8. The parameters were held as follows: KO1 = 0.88 μM, KC1 = 3.18 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, LO = 2.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, and zJ = 0.58 e. The value of E coupling voltage sensor activation and Ca2+ binding at one type of site was allowed to vary. Shown is the best fit obtained. The value of E was calculated to be 2.84 ± 0.13.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2571968&req=5

fig11: A modified HA model can explain the effect of Ca2+ on the steady-state gating properties of the BKCa channel. Shown are series of mSlo1α G-V relations determined at the following [Ca2+]: 0.003, 0.070, 0.130, 0.360, 0.8, 10, and 100 μM and fit simultaneously with the modified model that includes two types of Ca2+ binding sites, one type interacts with the voltage sensor, and the other type is independent. The data are the same as shown in Fig. 8. The parameters were held as follows: KO1 = 0.88 μM, KC1 = 3.18 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, LO = 2.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, and zJ = 0.58 e. The value of E coupling voltage sensor activation and Ca2+ binding at one type of site was allowed to vary. Shown is the best fit obtained. The value of E was calculated to be 2.84 ± 0.13.
Mentions: We held all parameters but E to values that have been independently determined either here or previously (Bao and Cox, 2005) and allowed only E to vary. The resulting best fit from this approach is shown in Fig. 11. It shows that even with these severe constraints, the two-site HA model with our newly determined Ca2+ binding constants can approximate the shifting of the mSlo channel's G-V relation as a function of [Ca2+], and remarkably the fit yields E = 2.8, a value that is very similar to the value of E (2.4) estimated independently by Horrigan and Aldrich (2002) from measurements of the channel's gating charge movement as a function of voltage at 0 and 70 μM [Ca2+]. Thus, we currently favor this estimate.

Bottom Line: Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation.To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude.Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology Research Institute, Tufts Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA.

ABSTRACT
It has been established that the large conductance Ca(2+)-activated K(+) channel contains two types of high-affinity Ca(2+) binding sites, termed the Ca(2+) bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance-voltage relations determined over a series of Ca(2+) concentrations with models of channel gating that include both voltage sensing and Ca(2+) binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca(2+) dose-response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca(2+) binding site) and for channels that have had one or the other Ca(2+) binding site disabled via mutation. To accurately determine these dose-response curves we have used a series of 22 Ca(2+) concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at -80 mV the Ca(2+) bowl has higher affinity for Ca(2+) than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca(2+) to the RCK1 site is voltage dependent, whereas at the Ca(2+) bowl it is not.

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