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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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Voltage likely affects the affinity of the BKCa channel for Ca2+. Shown are a 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 fitted simultaneously with the HA model modified to include two Ca2+ binding sites (Bao et al., 2002; Horrigan and Aldrich, 2002). Using values determined from this and previous experiments in our laboratory (Bao and Cox, 2005), 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. In A, the values of allosteric factors E1 and E2 were held to a value of 1 for both Ca2+ binding sites A and B. In B, the values of E1 and E2 were allowed to vary. The best fit values of E1 and E2 were 1.43 and 1.73, respectively.
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fig8: Voltage likely affects the affinity of the BKCa channel for Ca2+. Shown are a 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 fitted simultaneously with the HA model modified to include two Ca2+ binding sites (Bao et al., 2002; Horrigan and Aldrich, 2002). Using values determined from this and previous experiments in our laboratory (Bao and Cox, 2005), 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. In A, the values of allosteric factors E1 and E2 were held to a value of 1 for both Ca2+ binding sites A and B. In B, the values of E1 and E2 were allowed to vary. The best fit values of E1 and E2 were 1.43 and 1.73, respectively.

Mentions: Another question of interest is do the binding affinities we have measured at a single voltage (−80 mV) explain the BKCa channel's sensitivity to Ca2+ over a range of voltages? Fig. 8 A shows the mSlo G-V relation at a series of [Ca2+] fit simultaneously with the BKCa-gating model of Horrigan and Aldrich (2002) (the HA model) but modified to include two sets of Ca2+ binding sites, four per set. There were no free parameters in this fit, but rather gating parameters determined from these and previous experiments (Bao and Cox, 2005) were used. The parameters were as follows: KO1 = 0.88 μM; KC1 = 3.13 μM; KO2 = 4.88 μM; KC2 = 23.2 μM; LO = 2e-06; zL= 0.41 e; Vhc = 151 mV; Vho = 27 mV; and ZJ = 0.58 e. The allosteric factors E1 and E2 were set to 1 to simulate no interaction between voltage–sensor movement and Ca2+ binding at either site. The fit is poor. The model responds to Ca2+ less than is required to move the model G-V relation along with the data. Interestingly, however, when we let E1 and E2 vary freely, that is, we allowed interactions between binding sites and voltage sensors, the fit markedly improved (Ei = 1.43; E2 = 1.73) (Fig. 8 B). This suggests that voltage-sensor movement may alter Ca2+ binding and vice versa.


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

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

Voltage likely affects the affinity of the BKCa channel for Ca2+. Shown are a 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 fitted simultaneously with the HA model modified to include two Ca2+ binding sites (Bao et al., 2002; Horrigan and Aldrich, 2002). Using values determined from this and previous experiments in our laboratory (Bao and Cox, 2005), 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. In A, the values of allosteric factors E1 and E2 were held to a value of 1 for both Ca2+ binding sites A and B. In B, the values of E1 and E2 were allowed to vary. The best fit values of E1 and E2 were 1.43 and 1.73, respectively.
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fig8: Voltage likely affects the affinity of the BKCa channel for Ca2+. Shown are a 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 fitted simultaneously with the HA model modified to include two Ca2+ binding sites (Bao et al., 2002; Horrigan and Aldrich, 2002). Using values determined from this and previous experiments in our laboratory (Bao and Cox, 2005), 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. In A, the values of allosteric factors E1 and E2 were held to a value of 1 for both Ca2+ binding sites A and B. In B, the values of E1 and E2 were allowed to vary. The best fit values of E1 and E2 were 1.43 and 1.73, respectively.
Mentions: Another question of interest is do the binding affinities we have measured at a single voltage (−80 mV) explain the BKCa channel's sensitivity to Ca2+ over a range of voltages? Fig. 8 A shows the mSlo G-V relation at a series of [Ca2+] fit simultaneously with the BKCa-gating model of Horrigan and Aldrich (2002) (the HA model) but modified to include two sets of Ca2+ binding sites, four per set. There were no free parameters in this fit, but rather gating parameters determined from these and previous experiments (Bao and Cox, 2005) were used. The parameters were as follows: KO1 = 0.88 μM; KC1 = 3.13 μM; KO2 = 4.88 μM; KC2 = 23.2 μM; LO = 2e-06; zL= 0.41 e; Vhc = 151 mV; Vho = 27 mV; and ZJ = 0.58 e. The allosteric factors E1 and E2 were set to 1 to simulate no interaction between voltage–sensor movement and Ca2+ binding at either site. The fit is poor. The model responds to Ca2+ less than is required to move the model G-V relation along with the data. Interestingly, however, when we let E1 and E2 vary freely, that is, we allowed interactions between binding sites and voltage sensors, the fit markedly improved (Ei = 1.43; E2 = 1.73) (Fig. 8 B). This suggests that voltage-sensor movement may alter Ca2+ binding and vice versa.

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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