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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 the RCK1 site is affected by voltage. However, voltage does not alter the binding at the Ca2+ bowl site. (A) The mean log ratio of NPopen/NPopenmin versus [Ca2+] for mutant ΔEΔR is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 13 patches at each Ca2+ concentration tested. Shown is the fit (dashed curve) of NPopen/NPopenmin based on Eq. 6 and previously shown in Fig. 5. The values determined from the fit are: KO = 0.88 μM and KC = 3.13 μM. (B) The mean log Popen versus [Ca2+] relation for mutant ΔEΔR at both 0 and −80 mV are well fitted with the HA model using the Ca2+ binding constants determined. The values for the parameters were held as follows: KO = 0.88 μM, KC = 3.18 μM, LO = 6.3 × 10−6, zL= 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 1. (C) The NPopen/NPopenmin versus [Ca2+] relation for mutant ΔEΔB(D2A2) is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. The NPopen/NPopenmin versus Ca2+ relations are fitted with Eq. 6. The values of the fit parameters are: ΔEΔB(D2A2): −80 mV, KO = 4.9 μM and KC = 23.2 μM; ΔEΔB(D2A2): 0 mV, KO = 2.1 μM and KC = 15.6 μM. (D) The mean log Popen versus [Ca2+] relation for mutant ΔEΔB(D2A2) at both 0 and −80 mV. First, the 0 mV data were fitted by Eq. 14 to yield the values: KO = 2.1 μM and KC = 15.8 μM. The data were also fitted with the HA model. The parameters were held as follows: KO = 4.9 μM, KC = 23.2 μM, LO = 1.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 6.03.
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fig9: The Ca2+ dependence of Popen for the RCK1 site is affected by voltage. However, voltage does not alter the binding at the Ca2+ bowl site. (A) The mean log ratio of NPopen/NPopenmin versus [Ca2+] for mutant ΔEΔR is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 13 patches at each Ca2+ concentration tested. Shown is the fit (dashed curve) of NPopen/NPopenmin based on Eq. 6 and previously shown in Fig. 5. The values determined from the fit are: KO = 0.88 μM and KC = 3.13 μM. (B) The mean log Popen versus [Ca2+] relation for mutant ΔEΔR at both 0 and −80 mV are well fitted with the HA model using the Ca2+ binding constants determined. The values for the parameters were held as follows: KO = 0.88 μM, KC = 3.18 μM, LO = 6.3 × 10−6, zL= 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 1. (C) The NPopen/NPopenmin versus [Ca2+] relation for mutant ΔEΔB(D2A2) is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. The NPopen/NPopenmin versus Ca2+ relations are fitted with Eq. 6. The values of the fit parameters are: ΔEΔB(D2A2): −80 mV, KO = 4.9 μM and KC = 23.2 μM; ΔEΔB(D2A2): 0 mV, KO = 2.1 μM and KC = 15.6 μM. (D) The mean log Popen versus [Ca2+] relation for mutant ΔEΔB(D2A2) at both 0 and −80 mV. First, the 0 mV data were fitted by Eq. 14 to yield the values: KO = 2.1 μM and KC = 15.8 μM. The data were also fitted with the HA model. The parameters were held as follows: KO = 4.9 μM, KC = 23.2 μM, LO = 1.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 6.03.

Mentions: To test this hypothesis directly, we repeated the experiments so far described, but changed the voltage from −80 to 0 mV. We reasoned that at −80 mV few voltage sensors would be active (5% or less) (Stefani et al., 1997; Horrigan and Aldrich, 1999, 2002; Bao and Cox, 2005), and thus there would be very little influence of voltage-sensor movement on Ca2+ binding. But at 0 mV, where the channels' voltage sensors are active 35% of the time when the channels are open (Horrigan and Aldrich, 1999, 2002; Bao and Cox, 2005), if voltage-sensor movement affects Ca2+ binding, some influence of the change in voltage should be observed. Shown in Fig. 9 (A and C) are the Popen(Ca2+)/Popen(0) versus [Ca2+] curves derived from these experiments (open symbols) along with their counterparts determined at −80 mV (filled symbols). Examining first the ΔEΔR channel (Fig. 9 A), we see that its 0- and −80-mV Popen(Ca2+)/Popen(0) versus [Ca2+] curves superimpose. This indicates that voltage-sensor movement does not affect Ca2+ binding at the Ca2+ bowl, but rather a change in voltage simply slides the Popen versus [Ca2+] curve up the Popen axis (see Fig. 9 B). Conversely, there is a substantial effect of voltage on Ca2+ binding at the RCK1 sites (Fig. 9, C and D). The maximal effect of Ca2+ on the open probability of the ΔEΔB(D2A2) channel is ∼10-fold larger at 0 mV than it is at −80 mV (Fig. 9 C), and fitting the 0 mV curve in Fig. 9 C with Eq. 6 yields Ca2+ dissociation constants of 15.6 ± 2.5 μM and 2.1 ± 0.3 μM (C = 7.39), as compared with 23.2 ± 2.6 μM and 4.9 ± 0.6 μM (C = 4.75) at −80 mV.


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 the RCK1 site is affected by voltage. However, voltage does not alter the binding at the Ca2+ bowl site. (A) The mean log ratio of NPopen/NPopenmin versus [Ca2+] for mutant ΔEΔR is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 13 patches at each Ca2+ concentration tested. Shown is the fit (dashed curve) of NPopen/NPopenmin based on Eq. 6 and previously shown in Fig. 5. The values determined from the fit are: KO = 0.88 μM and KC = 3.13 μM. (B) The mean log Popen versus [Ca2+] relation for mutant ΔEΔR at both 0 and −80 mV are well fitted with the HA model using the Ca2+ binding constants determined. The values for the parameters were held as follows: KO = 0.88 μM, KC = 3.18 μM, LO = 6.3 × 10−6, zL= 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 1. (C) The NPopen/NPopenmin versus [Ca2+] relation for mutant ΔEΔB(D2A2) is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. The NPopen/NPopenmin versus Ca2+ relations are fitted with Eq. 6. The values of the fit parameters are: ΔEΔB(D2A2): −80 mV, KO = 4.9 μM and KC = 23.2 μM; ΔEΔB(D2A2): 0 mV, KO = 2.1 μM and KC = 15.6 μM. (D) The mean log Popen versus [Ca2+] relation for mutant ΔEΔB(D2A2) at both 0 and −80 mV. First, the 0 mV data were fitted by Eq. 14 to yield the values: KO = 2.1 μM and KC = 15.8 μM. The data were also fitted with the HA model. The parameters were held as follows: KO = 4.9 μM, KC = 23.2 μM, LO = 1.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 6.03.
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fig9: The Ca2+ dependence of Popen for the RCK1 site is affected by voltage. However, voltage does not alter the binding at the Ca2+ bowl site. (A) The mean log ratio of NPopen/NPopenmin versus [Ca2+] for mutant ΔEΔR is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 13 patches at each Ca2+ concentration tested. Shown is the fit (dashed curve) of NPopen/NPopenmin based on Eq. 6 and previously shown in Fig. 5. The values determined from the fit are: KO = 0.88 μM and KC = 3.13 μM. (B) The mean log Popen versus [Ca2+] relation for mutant ΔEΔR at both 0 and −80 mV are well fitted with the HA model using the Ca2+ binding constants determined. The values for the parameters were held as follows: KO = 0.88 μM, KC = 3.18 μM, LO = 6.3 × 10−6, zL= 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 1. (C) The NPopen/NPopenmin versus [Ca2+] relation for mutant ΔEΔB(D2A2) is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 14 patches at each [Ca2+] tested. The NPopen/NPopenmin versus Ca2+ relations are fitted with Eq. 6. The values of the fit parameters are: ΔEΔB(D2A2): −80 mV, KO = 4.9 μM and KC = 23.2 μM; ΔEΔB(D2A2): 0 mV, KO = 2.1 μM and KC = 15.6 μM. (D) The mean log Popen versus [Ca2+] relation for mutant ΔEΔB(D2A2) at both 0 and −80 mV. First, the 0 mV data were fitted by Eq. 14 to yield the values: KO = 2.1 μM and KC = 15.8 μM. The data were also fitted with the HA model. The parameters were held as follows: KO = 4.9 μM, KC = 23.2 μM, LO = 1.2 × 10−6, zL = 0.41 e, Vhc = 151 mV, Vho = 27 mV, zJ = 0.58 e, and E = 6.03.
Mentions: To test this hypothesis directly, we repeated the experiments so far described, but changed the voltage from −80 to 0 mV. We reasoned that at −80 mV few voltage sensors would be active (5% or less) (Stefani et al., 1997; Horrigan and Aldrich, 1999, 2002; Bao and Cox, 2005), and thus there would be very little influence of voltage-sensor movement on Ca2+ binding. But at 0 mV, where the channels' voltage sensors are active 35% of the time when the channels are open (Horrigan and Aldrich, 1999, 2002; Bao and Cox, 2005), if voltage-sensor movement affects Ca2+ binding, some influence of the change in voltage should be observed. Shown in Fig. 9 (A and C) are the Popen(Ca2+)/Popen(0) versus [Ca2+] curves derived from these experiments (open symbols) along with their counterparts determined at −80 mV (filled symbols). Examining first the ΔEΔR channel (Fig. 9 A), we see that its 0- and −80-mV Popen(Ca2+)/Popen(0) versus [Ca2+] curves superimpose. This indicates that voltage-sensor movement does not affect Ca2+ binding at the Ca2+ bowl, but rather a change in voltage simply slides the Popen versus [Ca2+] curve up the Popen axis (see Fig. 9 B). Conversely, there is a substantial effect of voltage on Ca2+ binding at the RCK1 sites (Fig. 9, C and D). The maximal effect of Ca2+ on the open probability of the ΔEΔB(D2A2) channel is ∼10-fold larger at 0 mV than it is at −80 mV (Fig. 9 C), and fitting the 0 mV curve in Fig. 9 C with Eq. 6 yields Ca2+ dissociation constants of 15.6 ± 2.5 μM and 2.1 ± 0.3 μM (C = 7.39), as compared with 23.2 ± 2.6 μM and 4.9 ± 0.6 μM (C = 4.75) at −80 mV.

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