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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 affinity of mutant ΔE at −80 mV. The mean log ratio of NPopen in the presence and absence of Ca2+ determined from the data shown in Fig. 2. Log (NPopen / NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 5 yielding values of KO1 = 0.7 μM, KC1 = 3.7 μM, KO2 = 21 μM, and KC2 = 51 μM. Also shown is the fit (dashed line) assuming both types of binding sites have the same affinity for Ca2+ (KO = 1.9 μM and KC = 6.4 μM). Error bars represent SEM.
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fig3: The Ca2+ binding affinity of mutant ΔE at −80 mV. The mean log ratio of NPopen in the presence and absence of Ca2+ determined from the data shown in Fig. 2. Log (NPopen / NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 5 yielding values of KO1 = 0.7 μM, KC1 = 3.7 μM, KO2 = 21 μM, and KC2 = 51 μM. Also shown is the fit (dashed line) assuming both types of binding sites have the same affinity for Ca2+ (KO = 1.9 μM and KC = 6.4 μM). Error bars represent SEM.

Mentions: This eliminates M and leaves a curve whose properties are determined solely by the channel's Ca2+ binding constants (Horrigan and Aldrich, 2002). Thus, the curve in Fig. 2 C was normalized by its minimum value to yield a Popen (Ca2+)/Popen (0) versus [Ca2+] curve (Fig. 3) and then fitted with Eq. 5. Properties of this curve of note are: (1) Ca2+ increases Popen by a factor of 2.8 × 104; (2) Popen saturates at high [Ca2+], ∼100 μM; and (3) the curve has a shallow quality suggestive of multiple binding sites with differing affinities. Indeed the fit (solid line) yielded the following dissociation constants: SITE 1, KC1 = 3.7 ± 2.1 μM, KO1 = 0.7 ± 0.14 μM; SITE 2, KC2 = 51 ± 42 μM, KO2 = 21 ± 24 μM.


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 affinity of mutant ΔE at −80 mV. The mean log ratio of NPopen in the presence and absence of Ca2+ determined from the data shown in Fig. 2. Log (NPopen / NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 5 yielding values of KO1 = 0.7 μM, KC1 = 3.7 μM, KO2 = 21 μM, and KC2 = 51 μM. Also shown is the fit (dashed line) assuming both types of binding sites have the same affinity for Ca2+ (KO = 1.9 μM and KC = 6.4 μM). Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2571968&req=5

fig3: The Ca2+ binding affinity of mutant ΔE at −80 mV. The mean log ratio of NPopen in the presence and absence of Ca2+ determined from the data shown in Fig. 2. Log (NPopen / NPopenmin) spans the entire [Ca2+] range and is fit (solid line) by Eq. 5 yielding values of KO1 = 0.7 μM, KC1 = 3.7 μM, KO2 = 21 μM, and KC2 = 51 μM. Also shown is the fit (dashed line) assuming both types of binding sites have the same affinity for Ca2+ (KO = 1.9 μM and KC = 6.4 μM). Error bars represent SEM.
Mentions: This eliminates M and leaves a curve whose properties are determined solely by the channel's Ca2+ binding constants (Horrigan and Aldrich, 2002). Thus, the curve in Fig. 2 C was normalized by its minimum value to yield a Popen (Ca2+)/Popen (0) versus [Ca2+] curve (Fig. 3) and then fitted with Eq. 5. Properties of this curve of note are: (1) Ca2+ increases Popen by a factor of 2.8 × 104; (2) Popen saturates at high [Ca2+], ∼100 μM; and (3) the curve has a shallow quality suggestive of multiple binding sites with differing affinities. Indeed the fit (solid line) yielded the following dissociation constants: SITE 1, KC1 = 3.7 ± 2.1 μM, KO1 = 0.7 ± 0.14 μM; SITE 2, KC2 = 51 ± 42 μM, KO2 = 21 ± 24 μM.

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