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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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The two binding sites are less than additive at 0 mV. The mean log ratio of NPopen at 0 mV in the presence and absence of Ca2+ for mutants ΔE (open circles), ΔEΔR (open triangles), and ΔEΔB(D2A2) (open squares) are plotted versus [Ca2+]. Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR (short dashed line) with Eq. 6 yielded values of KO = 0.63 μM and KC = 2.28 μM. The fit of ΔEΔB(D2A2) (long dashed curve) yielded values of KO = 1.56 μM and KC = 12.7 μM. Using the same equation, we simulated the log (NPopen/NPopenmin) versus Ca2+ relation (dark solid line) predicted by the affinities determined for each site in isolation. The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, and KC2 = 12.7 μM. Also plotted (gray curve) is a log (NPopen/NPopenmin) versus [Ca2+] fit that incorporates cooperativity between binding sites. The equation for the fit was log (NPopen/NPopenmin) = ((1+(KO1+KO2)+ KO1KO2b)4) / ((1+(KC1+ KC2)+ KC1KC2a)4). The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, KC2 = 12.7, a = 1, and b = 0.65. Error bars represent SEM.
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fig10: The two binding sites are less than additive at 0 mV. The mean log ratio of NPopen at 0 mV in the presence and absence of Ca2+ for mutants ΔE (open circles), ΔEΔR (open triangles), and ΔEΔB(D2A2) (open squares) are plotted versus [Ca2+]. Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR (short dashed line) with Eq. 6 yielded values of KO = 0.63 μM and KC = 2.28 μM. The fit of ΔEΔB(D2A2) (long dashed curve) yielded values of KO = 1.56 μM and KC = 12.7 μM. Using the same equation, we simulated the log (NPopen/NPopenmin) versus Ca2+ relation (dark solid line) predicted by the affinities determined for each site in isolation. The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, and KC2 = 12.7 μM. Also plotted (gray curve) is a log (NPopen/NPopenmin) versus [Ca2+] fit that incorporates cooperativity between binding sites. The equation for the fit was log (NPopen/NPopenmin) = ((1+(KO1+KO2)+ KO1KO2b)4) / ((1+(KC1+ KC2)+ KC1KC2a)4). The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, KC2 = 12.7, a = 1, and b = 0.65. Error bars represent SEM.

Mentions: This yielded (Fig. 9 D, solid curve) KC = 15.8 ± 3.1 μM, KO = 2.10 ± 0.4 μM (C = 7.52), and M = 1.8 × 10−5 ± 0.5 × 10−5 (see Table I). Thus, changing the voltage from −80 mV to 0 mV decreases KC at the RCK1 Ca2+ binding site by a factor of 0.7 (23.2→15.8). It decreases KO by a factor of 0.4 (4.88→2.10), and it increases C by a factor of 1.8. This increase in C makes the efficacy of the RCK1 sites an order of magnitude larger than the efficacy of the Ca2+ bowl sites at 0 mV. This is highlighted in Fig. 10, where the 0 mV Ca2+ dose-response curves for the two sites are superimposed. Also evident, at 0 mV, as we saw at −80 mV, the ΔE channel's Ca2+ dose-response curve spans a smaller range of open probabilities than is predicted (Fig. 10, dark solid curve) by the combination of the fits to each individual dose-response curve. And again, we can explain this effect by supposing negative cooperativity between the RCK1 and the Ca2+ bowl sites in each subunit. A cooperativity factor of 1 when the channel is closed (no cooperativity) and 0.65 when the channel is open (negative cooperativity) produced the best fit (Fig. 10, gray curve).


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

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

The two binding sites are less than additive at 0 mV. The mean log ratio of NPopen at 0 mV in the presence and absence of Ca2+ for mutants ΔE (open circles), ΔEΔR (open triangles), and ΔEΔB(D2A2) (open squares) are plotted versus [Ca2+]. Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR (short dashed line) with Eq. 6 yielded values of KO = 0.63 μM and KC = 2.28 μM. The fit of ΔEΔB(D2A2) (long dashed curve) yielded values of KO = 1.56 μM and KC = 12.7 μM. Using the same equation, we simulated the log (NPopen/NPopenmin) versus Ca2+ relation (dark solid line) predicted by the affinities determined for each site in isolation. The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, and KC2 = 12.7 μM. Also plotted (gray curve) is a log (NPopen/NPopenmin) versus [Ca2+] fit that incorporates cooperativity between binding sites. The equation for the fit was log (NPopen/NPopenmin) = ((1+(KO1+KO2)+ KO1KO2b)4) / ((1+(KC1+ KC2)+ KC1KC2a)4). The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, KC2 = 12.7, a = 1, and b = 0.65. Error bars represent SEM.
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fig10: The two binding sites are less than additive at 0 mV. The mean log ratio of NPopen at 0 mV in the presence and absence of Ca2+ for mutants ΔE (open circles), ΔEΔR (open triangles), and ΔEΔB(D2A2) (open squares) are plotted versus [Ca2+]. Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR (short dashed line) with Eq. 6 yielded values of KO = 0.63 μM and KC = 2.28 μM. The fit of ΔEΔB(D2A2) (long dashed curve) yielded values of KO = 1.56 μM and KC = 12.7 μM. Using the same equation, we simulated the log (NPopen/NPopenmin) versus Ca2+ relation (dark solid line) predicted by the affinities determined for each site in isolation. The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, and KC2 = 12.7 μM. Also plotted (gray curve) is a log (NPopen/NPopenmin) versus [Ca2+] fit that incorporates cooperativity between binding sites. The equation for the fit was log (NPopen/NPopenmin) = ((1+(KO1+KO2)+ KO1KO2b)4) / ((1+(KC1+ KC2)+ KC1KC2a)4). The parameters of the fit were: KO1 = 0.63 μM, KC1 = 2.28 μM, KO2 = 1.56 μM, KC2 = 12.7, a = 1, and b = 0.65. Error bars represent SEM.
Mentions: This yielded (Fig. 9 D, solid curve) KC = 15.8 ± 3.1 μM, KO = 2.10 ± 0.4 μM (C = 7.52), and M = 1.8 × 10−5 ± 0.5 × 10−5 (see Table I). Thus, changing the voltage from −80 mV to 0 mV decreases KC at the RCK1 Ca2+ binding site by a factor of 0.7 (23.2→15.8). It decreases KO by a factor of 0.4 (4.88→2.10), and it increases C by a factor of 1.8. This increase in C makes the efficacy of the RCK1 sites an order of magnitude larger than the efficacy of the Ca2+ bowl sites at 0 mV. This is highlighted in Fig. 10, where the 0 mV Ca2+ dose-response curves for the two sites are superimposed. Also evident, at 0 mV, as we saw at −80 mV, the ΔE channel's Ca2+ dose-response curve spans a smaller range of open probabilities than is predicted (Fig. 10, dark solid curve) by the combination of the fits to each individual dose-response curve. And again, we can explain this effect by supposing negative cooperativity between the RCK1 and the Ca2+ bowl sites in each subunit. A cooperativity factor of 1 when the channel is closed (no cooperativity) and 0.65 when the channel is open (negative cooperativity) produced the best fit (Fig. 10, gray curve).

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.

Show MeSH
Related in: MedlinePlus