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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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Schematic diagram illustrating a scenario that can account for the effects of permeant ion species on entry and exit rates for pore-blocking ions in WT and EEEE locus mutant channels. In the interest of clarity, numbers of oxygen atoms that might be able to interact with blocker (here indicated as Cd2+) are specifically marked. Interactions with current-carrying ions are omitted. Numbers of interactions are figurative only.
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Figure 9: Schematic diagram illustrating a scenario that can account for the effects of permeant ion species on entry and exit rates for pore-blocking ions in WT and EEEE locus mutant channels. In the interest of clarity, numbers of oxygen atoms that might be able to interact with blocker (here indicated as Cd2+) are specifically marked. Interactions with current-carrying ions are omitted. Numbers of interactions are figurative only.

Mentions: Fig. 9 presents a specific depiction of this model of selectivity filter function. The depiction is intended to capture the essence of the model only, so that some of the specifics of the structure (e.g., the precise positioning of carboxylate groups) should be taken in a figurative sense. Based on earlier Ca2+ channel work (Kuo and Hess 1993a,Kuo and Hess 1993b; Yang et al. 1993; Ellinor et al. 1995; Cloues and Sather 2000), and by analogy with other ion channels (reviewed by Khakh and Lester 1999), the selectivity filter is envisioned as a flexible structure that changes conformation as different kinds and numbers of ions pass through it. To emphasize this idea, Fig. 9 illustrates, for Ba2+ versus Li+, differing orientation angles of the glutamate side chains, differing patterns of glutamate interactions with current-carrying ions, and differing numbers of interactions made with entering Cd2+.


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)

Schematic diagram illustrating a scenario that can account for the effects of permeant ion species on entry and exit rates for pore-blocking ions in WT and EEEE locus mutant channels. In the interest of clarity, numbers of oxygen atoms that might be able to interact with blocker (here indicated as Cd2+) are specifically marked. Interactions with current-carrying ions are omitted. Numbers of interactions are figurative only.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Schematic diagram illustrating a scenario that can account for the effects of permeant ion species on entry and exit rates for pore-blocking ions in WT and EEEE locus mutant channels. In the interest of clarity, numbers of oxygen atoms that might be able to interact with blocker (here indicated as Cd2+) are specifically marked. Interactions with current-carrying ions are omitted. Numbers of interactions are figurative only.
Mentions: Fig. 9 presents a specific depiction of this model of selectivity filter function. The depiction is intended to capture the essence of the model only, so that some of the specifics of the structure (e.g., the precise positioning of carboxylate groups) should be taken in a figurative sense. Based on earlier Ca2+ channel work (Kuo and Hess 1993a,Kuo and Hess 1993b; Yang et al. 1993; Ellinor et al. 1995; Cloues and Sather 2000), and by analogy with other ion channels (reviewed by Khakh and Lester 1999), the selectivity filter is envisioned as a flexible structure that changes conformation as different kinds and numbers of ions pass through it. To emphasize this idea, Fig. 9 illustrates, for Ba2+ versus Li+, differing orientation angles of the glutamate side chains, differing patterns of glutamate interactions with current-carrying ions, and differing numbers of interactions made with entering Cd2+.

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