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Ion interactions in the high-affinity binding locus of a voltage-gated Ca(2+) channel.

Cloues RK, Cibulsky SM, Sather WA - J. Gen. Physiol. (2000)

Bottom Line: For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate.Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs.Li(+)).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

ABSTRACT
The selectivity filter of voltage-gated Ca(2+) channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca(2+) channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca(2+) channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd(2+), which binds very tightly within the EEEE locus, and Ba(2+) or Li(+), which bind less tightly and hence exhibit high flux rates: Cd(2+) binds to the EEEE locus approximately 10(4)x more tightly than does Ba(2+), and approximately 10(8)x more tightly than does Li(+). For wild-type channels, Cd(2+) entry into the EEEE locus was 400x faster when Li(+) rather than Ba(2+) was the current carrier, reflecting the large difference between Ba(2+) and Li(+) in affinity for the EEEE locus. For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba(2+) vs. Li(+)). The dependence of Cd(2+) block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.

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For WT channels, comparison of kinetics of Cd2+ block of Ba2+ and Li+ currents. (Left) Plot of reciprocals of corrected open-time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Linear regression fits (solid lines) to these data yield rate constants for Cd2+ block of Ba2+ (slope = 1.8 × 107 M−1 s−1) and of Li+ (slope = 8.2 × 109 M−1 s−1) currents. (Inset) Cd2+ block of Li+ data on an expanded scale. (Right) Plot of reciprocals of shut time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Horizontal lines are drawn through the mean of all the data points and yield rate constants for Cd2+ block of Ba2+ (1,309 s−1) and of Li+ (3,538 s−1) currents. For Ba2+ currents, n = 3–5 patches at each concentration of Cd2+. For Li+ currents, n = 3 patches at each Cd2+ concentration, except for 0.1 μM Cd2+, for which n = 2 patches.
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Figure 2: For WT channels, comparison of kinetics of Cd2+ block of Ba2+ and Li+ currents. (Left) Plot of reciprocals of corrected open-time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Linear regression fits (solid lines) to these data yield rate constants for Cd2+ block of Ba2+ (slope = 1.8 × 107 M−1 s−1) and of Li+ (slope = 8.2 × 109 M−1 s−1) currents. (Inset) Cd2+ block of Li+ data on an expanded scale. (Right) Plot of reciprocals of shut time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Horizontal lines are drawn through the mean of all the data points and yield rate constants for Cd2+ block of Ba2+ (1,309 s−1) and of Li+ (3,538 s−1) currents. For Ba2+ currents, n = 3–5 patches at each concentration of Cd2+. For Li+ currents, n = 3 patches at each Cd2+ concentration, except for 0.1 μM Cd2+, for which n = 2 patches.

Mentions: The block rate, kon, was obtained as the slope of a linear regression fit to plots of τO−1 versus [Cd2+], and the [Cd2+]-independent closing rate (kC; given by the fit intercept on the ordinate) consequently did not significantly distort estimation of kon. Furthermore, in the absence of a blocking ion, pure closing rates (kC) were obtained and compared with τO−1 values when blocker was included in the pipet. Closing rates for wild-type and mutant channels ranged from 26 to 323 s−1 for Ba2+ currents, and from 122 to 357 s−1 for Li+ currents. When a blocking ion, such as Cd2+, was included in the pipet solution, a combination of channel closing and block transitions were recorded, and the frequency of these combined events is referred to here as the shutting rate. The closing rates listed in Table can be compared with shutting rates that ranged from hundreds to thousands per second, depending upon the concentration of blocker used (see Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7). Estimates of kC obtained from the intercept of the fit with the ordinate ([X2+] = 0) were comparable with values of kC obtained in the absence of blocker. Overall, estimated rates of block were little affected by contamination with channel closing events.


Ion interactions in the high-affinity binding locus of a voltage-gated Ca(2+) channel.

Cloues RK, Cibulsky SM, Sather WA - J. Gen. Physiol. (2000)

For WT channels, comparison of kinetics of Cd2+ block of Ba2+ and Li+ currents. (Left) Plot of reciprocals of corrected open-time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Linear regression fits (solid lines) to these data yield rate constants for Cd2+ block of Ba2+ (slope = 1.8 × 107 M−1 s−1) and of Li+ (slope = 8.2 × 109 M−1 s−1) currents. (Inset) Cd2+ block of Li+ data on an expanded scale. (Right) Plot of reciprocals of shut time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Horizontal lines are drawn through the mean of all the data points and yield rate constants for Cd2+ block of Ba2+ (1,309 s−1) and of Li+ (3,538 s−1) currents. For Ba2+ currents, n = 3–5 patches at each concentration of Cd2+. For Li+ currents, n = 3 patches at each Cd2+ concentration, except for 0.1 μM Cd2+, for which n = 2 patches.
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Figure 2: For WT channels, comparison of kinetics of Cd2+ block of Ba2+ and Li+ currents. (Left) Plot of reciprocals of corrected open-time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Linear regression fits (solid lines) to these data yield rate constants for Cd2+ block of Ba2+ (slope = 1.8 × 107 M−1 s−1) and of Li+ (slope = 8.2 × 109 M−1 s−1) currents. (Inset) Cd2+ block of Li+ data on an expanded scale. (Right) Plot of reciprocals of shut time constants versus [Cd2+] for Ba2+ (▪; VM = 0 mV) and for Li+ (○; VM = −100 mV). Horizontal lines are drawn through the mean of all the data points and yield rate constants for Cd2+ block of Ba2+ (1,309 s−1) and of Li+ (3,538 s−1) currents. For Ba2+ currents, n = 3–5 patches at each concentration of Cd2+. For Li+ currents, n = 3 patches at each Cd2+ concentration, except for 0.1 μM Cd2+, for which n = 2 patches.
Mentions: The block rate, kon, was obtained as the slope of a linear regression fit to plots of τO−1 versus [Cd2+], and the [Cd2+]-independent closing rate (kC; given by the fit intercept on the ordinate) consequently did not significantly distort estimation of kon. Furthermore, in the absence of a blocking ion, pure closing rates (kC) were obtained and compared with τO−1 values when blocker was included in the pipet. Closing rates for wild-type and mutant channels ranged from 26 to 323 s−1 for Ba2+ currents, and from 122 to 357 s−1 for Li+ currents. When a blocking ion, such as Cd2+, was included in the pipet solution, a combination of channel closing and block transitions were recorded, and the frequency of these combined events is referred to here as the shutting rate. The closing rates listed in Table can be compared with shutting rates that ranged from hundreds to thousands per second, depending upon the concentration of blocker used (see Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7). Estimates of kC obtained from the intercept of the fit with the ordinate ([X2+] = 0) were comparable with values of kC obtained in the absence of blocker. Overall, estimated rates of block were little affected by contamination with channel closing events.

Bottom Line: For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate.Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs.Li(+)).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

ABSTRACT
The selectivity filter of voltage-gated Ca(2+) channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca(2+) channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca(2+) channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd(2+), which binds very tightly within the EEEE locus, and Ba(2+) or Li(+), which bind less tightly and hence exhibit high flux rates: Cd(2+) binds to the EEEE locus approximately 10(4)x more tightly than does Ba(2+), and approximately 10(8)x more tightly than does Li(+). For wild-type channels, Cd(2+) entry into the EEEE locus was 400x faster when Li(+) rather than Ba(2+) was the current carrier, reflecting the large difference between Ba(2+) and Li(+) in affinity for the EEEE locus. For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba(2+) vs. Li(+)). The dependence of Cd(2+) block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.

Show MeSH
Related in: MedlinePlus