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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+ dependence of Popen for mutant ΔE. (A) Inward potassium currents recorded 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-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings at each [Ca2+]. The dose–response relation for the effect of Ca2+ on Popen at negative voltage (−80 mV) is shown in C. For determination of Popen see Materials and methods. Each point represents the average of between 7 and 17 patches at each Ca2+ concentration tested. Error bars represent SEM.
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fig2: The Ca2+ dependence of Popen for mutant ΔE. (A) Inward potassium currents recorded 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-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings at each [Ca2+]. The dose–response relation for the effect of Ca2+ on Popen at negative voltage (−80 mV) is shown in C. For determination of Popen see Materials and methods. Each point represents the average of between 7 and 17 patches at each Ca2+ concentration tested. Error bars represent SEM.

Mentions: Fig. 2 A shows unitary ΔE currents recorded from a single membrane patch at −80 mV and four different [Ca2+]. Corresponding amplitude histograms are shown in Fig. 2 B. Although the patch contained hundreds of channels, each channel's open probability (Popen) is low in the absence of Ca2+, such that activity is observed as the infrequent and brief opening of single channels. Application of Ca2+ then caused a large increase in Popen that resulted in multi-channel openings. From data like these we derived the ΔE channel's Popen versus [Ca2+] relation (Fig. 2 C). So that all parts of the curve could be well determined, Popen was measured over five orders of magnitude with 22 Ca2+ concentrations. To do this, many patches were used and normalized by their values of NPopen at 5.3 μM, where N is the number of channels in a given patch. The data were then averaged at each [Ca2+], and the whole curve was adjusted vertically to match the BKCa channel's Popen at 5.3 μM and −80 mV, which was determined in separate experiments (see Materials and methods).


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+ dependence of Popen for mutant ΔE. (A) Inward potassium currents recorded 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-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings at each [Ca2+]. The dose–response relation for the effect of Ca2+ on Popen at negative voltage (−80 mV) is shown in C. For determination of Popen see Materials and methods. Each point represents the average of between 7 and 17 patches at each Ca2+ concentration tested. Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: The Ca2+ dependence of Popen for mutant ΔE. (A) Inward potassium currents recorded 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-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings at each [Ca2+]. The dose–response relation for the effect of Ca2+ on Popen at negative voltage (−80 mV) is shown in C. For determination of Popen see Materials and methods. Each point represents the average of between 7 and 17 patches at each Ca2+ concentration tested. Error bars represent SEM.
Mentions: Fig. 2 A shows unitary ΔE currents recorded from a single membrane patch at −80 mV and four different [Ca2+]. Corresponding amplitude histograms are shown in Fig. 2 B. Although the patch contained hundreds of channels, each channel's open probability (Popen) is low in the absence of Ca2+, such that activity is observed as the infrequent and brief opening of single channels. Application of Ca2+ then caused a large increase in Popen that resulted in multi-channel openings. From data like these we derived the ΔE channel's Popen versus [Ca2+] relation (Fig. 2 C). So that all parts of the curve could be well determined, Popen was measured over five orders of magnitude with 22 Ca2+ concentrations. To do this, many patches were used and normalized by their values of NPopen at 5.3 μM, where N is the number of channels in a given patch. The data were then averaged at each [Ca2+], and the whole curve was adjusted vertically to match the BKCa channel's Popen at 5.3 μM and −80 mV, which was determined in separate experiments (see Materials and methods).

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