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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 Ca2+ binding affinities of the high-affinity RCK1 site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔB(D2A2) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings. The dose–response relation for the effect of Ca2+ on Popen (left axis) and NPopen/NPopenmin (right axis) at negative voltage (−80 mV) is shown in C. Each point represents the average of between 6 and 16 patches at each Ca2+ concentration tested. For mutant ΔEΔB(D2A2) (open squares), log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fitted (dotted line) by Eq. 6 yielding values of KO = 4.9 μM of KC = 23.2 μM. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yields values of KOB = 5.6 μM and KCB = 26.8 μM. (D) The data were also fitted with Eq. 7, which incorporates an interaction between binding sites. For mutant ΔEΔB(D2A2) (open squares), the fit yielded values of KO = 2.8 μM, KC = 13.7 μM, and f = 0.45. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yielded KO = 1.8 μM, KC = 9.4 μM, and f = 0.27. Error bars represent SEM.
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fig6: The Ca2+ binding affinities of the high-affinity RCK1 site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔB(D2A2) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings. The dose–response relation for the effect of Ca2+ on Popen (left axis) and NPopen/NPopenmin (right axis) at negative voltage (−80 mV) is shown in C. Each point represents the average of between 6 and 16 patches at each Ca2+ concentration tested. For mutant ΔEΔB(D2A2) (open squares), log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fitted (dotted line) by Eq. 6 yielding values of KO = 4.9 μM of KC = 23.2 μM. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yields values of KOB = 5.6 μM and KCB = 26.8 μM. (D) The data were also fitted with Eq. 7, which incorporates an interaction between binding sites. For mutant ΔEΔB(D2A2) (open squares), the fit yielded values of KO = 2.8 μM, KC = 13.7 μM, and f = 0.45. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yielded KO = 1.8 μM, KC = 9.4 μM, and f = 0.27. 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 Ca2+ binding affinities of the high-affinity RCK1 site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔB(D2A2) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings. The dose–response relation for the effect of Ca2+ on Popen (left axis) and NPopen/NPopenmin (right axis) at negative voltage (−80 mV) is shown in C. Each point represents the average of between 6 and 16 patches at each Ca2+ concentration tested. For mutant ΔEΔB(D2A2) (open squares), log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fitted (dotted line) by Eq. 6 yielding values of KO = 4.9 μM of KC = 23.2 μM. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yields values of KOB = 5.6 μM and KCB = 26.8 μM. (D) The data were also fitted with Eq. 7, which incorporates an interaction between binding sites. For mutant ΔEΔB(D2A2) (open squares), the fit yielded values of KO = 2.8 μM, KC = 13.7 μM, and f = 0.45. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yielded KO = 1.8 μM, KC = 9.4 μM, and f = 0.27. Error bars represent SEM.
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fig6: The Ca2+ binding affinities of the high-affinity RCK1 site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔB(D2A2) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings. The dose–response relation for the effect of Ca2+ on Popen (left axis) and NPopen/NPopenmin (right axis) at negative voltage (−80 mV) is shown in C. Each point represents the average of between 6 and 16 patches at each Ca2+ concentration tested. For mutant ΔEΔB(D2A2) (open squares), log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fitted (dotted line) by Eq. 6 yielding values of KO = 4.9 μM of KC = 23.2 μM. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yields values of KOB = 5.6 μM and KCB = 26.8 μM. (D) The data were also fitted with Eq. 7, which incorporates an interaction between binding sites. For mutant ΔEΔB(D2A2) (open squares), the fit yielded values of KO = 2.8 μM, KC = 13.7 μM, and f = 0.45. For mutant ΔEΔB(D5N5) (closed squares), the fit (dashed line) yielded KO = 1.8 μM, KC = 9.4 μM, and f = 0.27. 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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