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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 Ca2+ bowl site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔR at −80 mV and filtered at 10 kHz from a macropatch exposed to the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-point amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings as in Fig. 2. 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 11 patches at each [Ca2+] tested. Log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 6 yielding values of KO = 0.88 μM and KC = 3.13 μM. Error bars represent SEM.
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fig5: The Ca2+ binding affinities of the Ca2+ bowl site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔR at −80 mV and filtered at 10 kHz from a macropatch exposed to the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-point amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings as in Fig. 2. 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 11 patches at each [Ca2+] tested. Log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 6 yielding values of KO = 0.88 μM and KC = 3.13 μM. Error bars represent SEM.

Mentions: We then used the mutant (E399N)(D367A), which we refer to as ΔEΔR, to examine Ca2+ sensing via the Ca2+ bowl. Fig. 5 A compares BKCa currents at various [Ca2+] recorded from a single ΔEΔR patch at −80 mV. The corresponding amplitude histograms are shown in Fig. 5 B. As with ΔE, Popen is low in the absence of Ca2+, and activity is observed as the infrequent and brief opening of single channels. Application of Ca2+ then increases Popen, but the increase is not as great (∼102-fold) as it is with the ΔE channel (∼104-fold), presumably because the ΔEΔR channel has lost half of its high-affinity binding sites. A Ca2+ dose–response relation for the ΔEΔR channel at −80 mV is shown in Fig. 5 C. The affinities of the intact Ca2+ bowl site were then determined from a fit (solid line) with Eq. 6 below, which is analogous to Eq. 5, but represents the case where there is only one type of Ca2+ binding site (Horrigan and Aldrich, 2002).(6)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\frac{Popen(Ca)}{Popen(0)}=\frac{(1+[Ca]/K_{O})^{4}}{(1+[Ca]/K_{C})^{4}}\end{equation*}\end{document}


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 Ca2+ bowl site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔR at −80 mV and filtered at 10 kHz from a macropatch exposed to the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-point amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings as in Fig. 2. 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 11 patches at each [Ca2+] tested. Log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 6 yielding values of KO = 0.88 μM and KC = 3.13 μM. Error bars represent SEM.
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fig5: The Ca2+ binding affinities of the Ca2+ bowl site at −80 mV. (A) Inward K+ currents recorded from mutant ΔEΔR at −80 mV and filtered at 10 kHz from a macropatch exposed to the indicated [Ca2+] demonstrate that Popen increases in a Ca2+-dependent manner when voltage is constant. The corresponding all-point amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings as in Fig. 2. 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 11 patches at each [Ca2+] tested. Log (NPopen/NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 6 yielding values of KO = 0.88 μM and KC = 3.13 μM. Error bars represent SEM.
Mentions: We then used the mutant (E399N)(D367A), which we refer to as ΔEΔR, to examine Ca2+ sensing via the Ca2+ bowl. Fig. 5 A compares BKCa currents at various [Ca2+] recorded from a single ΔEΔR patch at −80 mV. The corresponding amplitude histograms are shown in Fig. 5 B. As with ΔE, Popen is low in the absence of Ca2+, and activity is observed as the infrequent and brief opening of single channels. Application of Ca2+ then increases Popen, but the increase is not as great (∼102-fold) as it is with the ΔE channel (∼104-fold), presumably because the ΔEΔR channel has lost half of its high-affinity binding sites. A Ca2+ dose–response relation for the ΔEΔR channel at −80 mV is shown in Fig. 5 C. The affinities of the intact Ca2+ bowl site were then determined from a fit (solid line) with Eq. 6 below, which is analogous to Eq. 5, but represents the case where there is only one type of Ca2+ binding site (Horrigan and Aldrich, 2002).(6)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\frac{Popen(Ca)}{Popen(0)}=\frac{(1+[Ca]/K_{O})^{4}}{(1+[Ca]/K_{C})^{4}}\end{equation*}\end{document}

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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