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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. The mean log ratio of NPopen at −80 mV in the presence and absence of Ca2+ for mutants ΔE (solid circles), ΔEΔR (solid triangles), and ΔEΔB(D2A2) (solid squares) are plotted versus [Ca2+]. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR also displayed in Fig. 2 is shown as a short dashed curve. The fit of ΔEΔB(D2A2) also displayed in Fig. 3 is shown as a long dashed curve. We simulated the log (NPopen/NPopenmin) relation (dark solid line) predicted by the affinities determined from each of the mutants using Eq. 5. The parameters of the fit were: KO1 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, and KC2 = 23.2 μM. Also plotted (gray curve) is a fit that incorporates cooperativity between the 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 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, a = 1, and b = 0.75. Error bars represent SEM.
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fig7: The two binding sites are less than additive. The mean log ratio of NPopen at −80 mV in the presence and absence of Ca2+ for mutants ΔE (solid circles), ΔEΔR (solid triangles), and ΔEΔB(D2A2) (solid squares) are plotted versus [Ca2+]. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR also displayed in Fig. 2 is shown as a short dashed curve. The fit of ΔEΔB(D2A2) also displayed in Fig. 3 is shown as a long dashed curve. We simulated the log (NPopen/NPopenmin) relation (dark solid line) predicted by the affinities determined from each of the mutants using Eq. 5. The parameters of the fit were: KO1 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, and KC2 = 23.2 μM. Also plotted (gray curve) is a fit that incorporates cooperativity between the 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 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, a = 1, and b = 0.75. Error bars represent SEM.

Mentions: Similarly, to determine the affinities of the RCK1 site, we examined the effect of Ca2+ on the open probability of the mutant (E399N)(D898A/D900A), which we refer to as ΔEΔB(D2A2). The two D→A mutations render the Ca2+ bowl nonfunctional (Bao et al., 2004). Fig. 6 A shows unitary ΔEΔB(D2A2) currents recorded at −80 mV with various [Ca2+] from a patch that contained hundreds of channels. Corresponding amplitude histograms are shown in Fig. 6 B, and the Ca2+ dose–response relation we acquired for the ΔEΔB(D2A2) channel at −80 mV is shown in Fig. 6 C (open squares). In fact, both ΔEΔB(D2A2) and another Ca2+ bowl mutation, (D897N/D898N/D899N/D900N/D901N) (ΔEΔB(D5N5)), were analyzed (Fig. 6 C, closed squares), and both mutations behave similarly. The affinity of the RCK1 site was then estimated by fitting Eq. 6 to the two datasets in Fig. 6 C. The fits yielded similar values (KO = 4.9 ± 0.6 μM; KC = 23.2 ± 2.6 μM; C = 4.75) for ΔEΔB(D2A2) and (KO = 5.6 ± 0.8 μM; KC = 26.8 ± 3.8 μM; C = 4.75) for ΔEΔB(D5N5) (see Table I). Thus, the RCK1 site binds Ca2+ more weakly than does the Ca2+ bowl site, both when the channel is open and when it is closed (Ca2+ bowl: KO = 0.88 ± 0.06 μM; KC = 3.13 ± 0.22 μM; C = 3.55 from Fig. 5), but it has a 36% larger C value and thus a bigger effect on opening at saturating [Ca2+]. This is illustrated graphically in Fig. 7, where the ΔEΔR (closed triangles) and ΔEΔB(D2A2) (closed squares) Ca2+ dose–response curves are overlaid.


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. The mean log ratio of NPopen at −80 mV in the presence and absence of Ca2+ for mutants ΔE (solid circles), ΔEΔR (solid triangles), and ΔEΔB(D2A2) (solid squares) are plotted versus [Ca2+]. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR also displayed in Fig. 2 is shown as a short dashed curve. The fit of ΔEΔB(D2A2) also displayed in Fig. 3 is shown as a long dashed curve. We simulated the log (NPopen/NPopenmin) relation (dark solid line) predicted by the affinities determined from each of the mutants using Eq. 5. The parameters of the fit were: KO1 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, and KC2 = 23.2 μM. Also plotted (gray curve) is a fit that incorporates cooperativity between the 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 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, a = 1, and b = 0.75. Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

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fig7: The two binding sites are less than additive. The mean log ratio of NPopen at −80 mV in the presence and absence of Ca2+ for mutants ΔE (solid circles), ΔEΔR (solid triangles), and ΔEΔB(D2A2) (solid squares) are plotted versus [Ca2+]. Various fits of log (NPopen/NPopenmin) are superimposed on the data. The fit of ΔEΔR also displayed in Fig. 2 is shown as a short dashed curve. The fit of ΔEΔB(D2A2) also displayed in Fig. 3 is shown as a long dashed curve. We simulated the log (NPopen/NPopenmin) relation (dark solid line) predicted by the affinities determined from each of the mutants using Eq. 5. The parameters of the fit were: KO1 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, and KC2 = 23.2 μM. Also plotted (gray curve) is a fit that incorporates cooperativity between the 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 = 0.88 μM, KC1 = 3.13 μM, KO2 = 4.88 μM, KC2 = 23.2 μM, a = 1, and b = 0.75. Error bars represent SEM.
Mentions: Similarly, to determine the affinities of the RCK1 site, we examined the effect of Ca2+ on the open probability of the mutant (E399N)(D898A/D900A), which we refer to as ΔEΔB(D2A2). The two D→A mutations render the Ca2+ bowl nonfunctional (Bao et al., 2004). Fig. 6 A shows unitary ΔEΔB(D2A2) currents recorded at −80 mV with various [Ca2+] from a patch that contained hundreds of channels. Corresponding amplitude histograms are shown in Fig. 6 B, and the Ca2+ dose–response relation we acquired for the ΔEΔB(D2A2) channel at −80 mV is shown in Fig. 6 C (open squares). In fact, both ΔEΔB(D2A2) and another Ca2+ bowl mutation, (D897N/D898N/D899N/D900N/D901N) (ΔEΔB(D5N5)), were analyzed (Fig. 6 C, closed squares), and both mutations behave similarly. The affinity of the RCK1 site was then estimated by fitting Eq. 6 to the two datasets in Fig. 6 C. The fits yielded similar values (KO = 4.9 ± 0.6 μM; KC = 23.2 ± 2.6 μM; C = 4.75) for ΔEΔB(D2A2) and (KO = 5.6 ± 0.8 μM; KC = 26.8 ± 3.8 μM; C = 4.75) for ΔEΔB(D5N5) (see Table I). Thus, the RCK1 site binds Ca2+ more weakly than does the Ca2+ bowl site, both when the channel is open and when it is closed (Ca2+ bowl: KO = 0.88 ± 0.06 μM; KC = 3.13 ± 0.22 μM; C = 3.55 from Fig. 5), but it has a 36% larger C value and thus a bigger effect on opening at saturating [Ca2+]. This is illustrated graphically in Fig. 7, where the ΔEΔR (closed triangles) and ΔEΔB(D2A2) (closed squares) Ca2+ dose–response curves are overlaid.

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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Related in: MedlinePlus