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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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Summary of effects of individual EEEE locus mutations on the on- and off-rate constants for block by Cd2+. (▴) WT values. (A) Cd2+ off rates for E→D (○) and E→A (•) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). (B) Cd2+ on rates for E→D (□) and E→A (▪) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). Dashed lines were drawn to highlight the general effects of mutations on kon and koff. Note the differences in scaling on the ordinates between A and B: in A, koff data are displayed on a linear scale and, in B, kon data are displayed on a logarithmic scale.
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Figure 8: Summary of effects of individual EEEE locus mutations on the on- and off-rate constants for block by Cd2+. (▴) WT values. (A) Cd2+ off rates for E→D (○) and E→A (•) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). (B) Cd2+ on rates for E→D (□) and E→A (▪) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). Dashed lines were drawn to highlight the general effects of mutations on kon and koff. Note the differences in scaling on the ordinates between A and B: in A, koff data are displayed on a linear scale and, in B, kon data are displayed on a logarithmic scale.

Mentions: Fig. 4 presents data for Cd2+ block of unitary Li+ currents carried by the E→D mutant channels. The records in Fig. 4 A showed clear increases in the number of block events as [Cd2+] was raised, although the size of these increases depended upon the mutation. The most obvious effects of the mutations were profound reductions in Cd2+ on rate (Fig. 4 D; sloped lines, ▪). The EIIID mutant (1.1 × 108 M−1 s−1) was the most different from WT (7.5 ×109 M−1 s−1), exhibiting a 70-fold reduction in Cd2+ entry rate relative to WT. Decreases in Cd2+ entry rate for the other E→D mutants ranged from 10- to 20-fold, with all four mutants following the order III > IV > I > II (Table ). Off rates (horizontal lines, ○) varied somewhat relative to WT, ranging from ∼35% to ∼155% of the WT value (Table ). For Li+ currents, the general off-rate pattern across the E→D mutants was reminiscent of the off-rate pattern observed with Ba2+, but was less pronounced (illustrated in Fig. 8 A, below).


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)

Summary of effects of individual EEEE locus mutations on the on- and off-rate constants for block by Cd2+. (▴) WT values. (A) Cd2+ off rates for E→D (○) and E→A (•) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). (B) Cd2+ on rates for E→D (□) and E→A (▪) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). Dashed lines were drawn to highlight the general effects of mutations on kon and koff. Note the differences in scaling on the ordinates between A and B: in A, koff data are displayed on a linear scale and, in B, kon data are displayed on a logarithmic scale.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230626&req=5

Figure 8: Summary of effects of individual EEEE locus mutations on the on- and off-rate constants for block by Cd2+. (▴) WT values. (A) Cd2+ off rates for E→D (○) and E→A (•) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). (B) Cd2+ on rates for E→D (□) and E→A (▪) mutants are compared for Cd2+ versus Ba2+ (left) and Cd2+ versus Li+ (right). Dashed lines were drawn to highlight the general effects of mutations on kon and koff. Note the differences in scaling on the ordinates between A and B: in A, koff data are displayed on a linear scale and, in B, kon data are displayed on a logarithmic scale.
Mentions: Fig. 4 presents data for Cd2+ block of unitary Li+ currents carried by the E→D mutant channels. The records in Fig. 4 A showed clear increases in the number of block events as [Cd2+] was raised, although the size of these increases depended upon the mutation. The most obvious effects of the mutations were profound reductions in Cd2+ on rate (Fig. 4 D; sloped lines, ▪). The EIIID mutant (1.1 × 108 M−1 s−1) was the most different from WT (7.5 ×109 M−1 s−1), exhibiting a 70-fold reduction in Cd2+ entry rate relative to WT. Decreases in Cd2+ entry rate for the other E→D mutants ranged from 10- to 20-fold, with all four mutants following the order III > IV > I > II (Table ). Off rates (horizontal lines, ○) varied somewhat relative to WT, ranging from ∼35% to ∼155% of the WT value (Table ). For Li+ currents, the general off-rate pattern across the E→D mutants was reminiscent of the off-rate pattern observed with Ba2+, but was less pronounced (illustrated in Fig. 8 A, below).

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