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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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Mutation of all three types of Ca2+ binding sites eliminates the Ca2+ dependence of Popen. (A) Inward K+ currents recorded for mutant ΔEΔRΔB(D5N5) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen does not increase 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. (C) Dose–response relations for the effect of Ca2+ on Popen at negative voltage (−80 mV) obtained by plotting the mean log ratio of NPopen in the presence and absence of Ca2+. For both mutant ΔEΔRΔB(D5N5) (filled circles) and mutant ΔEΔRΔB(D2A2), log (NPopen/NPopenmin) spans the entire [Ca2+] range but cannot be fitted because Popen does not vary with [Ca2+]. Each point represents the average of between 6 and 8 patches at each [Ca2+] tested. Error bars represent SEM.
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fig4: Mutation of all three types of Ca2+ binding sites eliminates the Ca2+ dependence of Popen. (A) Inward K+ currents recorded for mutant ΔEΔRΔB(D5N5) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen does not increase 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. (C) Dose–response relations for the effect of Ca2+ on Popen at negative voltage (−80 mV) obtained by plotting the mean log ratio of NPopen in the presence and absence of Ca2+. For both mutant ΔEΔRΔB(D5N5) (filled circles) and mutant ΔEΔRΔB(D2A2), log (NPopen/NPopenmin) spans the entire [Ca2+] range but cannot be fitted because Popen does not vary with [Ca2+]. Each point represents the average of between 6 and 8 patches at each [Ca2+] tested. Error bars represent SEM.

Mentions: To measure the affinities of each type of high-affinity Ca2+ binding site individually, we used mutations that selectively eliminate the effect of Ca2+ at each type of site. D367A eliminates Ca2+ sensing via RCK1 sites (Xia et al., 2002), and D897N/D898N/D899N/D900N/D901N (D5N5) or D898A/D900A (D2A2) eliminate Ca2+ sensing via the Ca2+ bowls (Schreiber and Salkoff, 1997; Bian et al., 2001; Bao et al., 2004). Before using these mutations, however, it was important to confirm that in conjunction with E399N they eliminate all Ca2+ sensing. Shown in Fig. 4 A are currents recorded at various [Ca2+] from a patch expressing the triple mutant (E399N)(D367A)(D897N/D898N/D899N/D900N/D901N), which we refer to as ΔEΔRΔB(D5N5). Corresponding amplitude histograms are superimposed in Fig. 4 B, and in Fig. 4 C the ΔEΔRΔB(D5N5) channel's Ca2+ dose-response relation is plotted at −80 mV. As is evident, the triple mutant shows virtually no response to Ca2+, which demonstrates that the three sites targeted by these mutations can together account for all of the channel's Ca2+ sensing.


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

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

Mutation of all three types of Ca2+ binding sites eliminates the Ca2+ dependence of Popen. (A) Inward K+ currents recorded for mutant ΔEΔRΔB(D5N5) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen does not increase 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. (C) Dose–response relations for the effect of Ca2+ on Popen at negative voltage (−80 mV) obtained by plotting the mean log ratio of NPopen in the presence and absence of Ca2+. For both mutant ΔEΔRΔB(D5N5) (filled circles) and mutant ΔEΔRΔB(D2A2), log (NPopen/NPopenmin) spans the entire [Ca2+] range but cannot be fitted because Popen does not vary with [Ca2+]. Each point represents the average of between 6 and 8 patches at each [Ca2+] tested. Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571968&req=5

fig4: Mutation of all three types of Ca2+ binding sites eliminates the Ca2+ dependence of Popen. (A) Inward K+ currents recorded for mutant ΔEΔRΔB(D5N5) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca2+] demonstrate that Popen does not increase 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. (C) Dose–response relations for the effect of Ca2+ on Popen at negative voltage (−80 mV) obtained by plotting the mean log ratio of NPopen in the presence and absence of Ca2+. For both mutant ΔEΔRΔB(D5N5) (filled circles) and mutant ΔEΔRΔB(D2A2), log (NPopen/NPopenmin) spans the entire [Ca2+] range but cannot be fitted because Popen does not vary with [Ca2+]. Each point represents the average of between 6 and 8 patches at each [Ca2+] tested. Error bars represent SEM.
Mentions: To measure the affinities of each type of high-affinity Ca2+ binding site individually, we used mutations that selectively eliminate the effect of Ca2+ at each type of site. D367A eliminates Ca2+ sensing via RCK1 sites (Xia et al., 2002), and D897N/D898N/D899N/D900N/D901N (D5N5) or D898A/D900A (D2A2) eliminate Ca2+ sensing via the Ca2+ bowls (Schreiber and Salkoff, 1997; Bian et al., 2001; Bao et al., 2004). Before using these mutations, however, it was important to confirm that in conjunction with E399N they eliminate all Ca2+ sensing. Shown in Fig. 4 A are currents recorded at various [Ca2+] from a patch expressing the triple mutant (E399N)(D367A)(D897N/D898N/D899N/D900N/D901N), which we refer to as ΔEΔRΔB(D5N5). Corresponding amplitude histograms are superimposed in Fig. 4 B, and in Fig. 4 C the ΔEΔRΔB(D5N5) channel's Ca2+ dose-response relation is plotted at −80 mV. As is evident, the triple mutant shows virtually no response to Ca2+, which demonstrates that the three sites targeted by these mutations can together account for all of the channel's Ca2+ sensing.

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