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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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Relationships between unitary conductance and rates of Cd2+ block and unblock.
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Figure 10: Relationships between unitary conductance and rates of Cd2+ block and unblock.

Mentions: Is conduction of current-carrying ions (Ba2+ or Li+) related in any simple way to the on and off rates of a blocking ion (Cd2+)? Fig. 10 shows that in this case it is not: for a narrow range in Cd2+ on rate, unitary Ba2+ flux did not vary in any systematic way over an eightfold range in Cd2+ off rate, nor did unitary Li+ flux vary systematically over a 4.5-fold range in Cd2+ off rate and 3,600-fold range in Cd2+ on rate. The absence of a simple pattern in Fig. 10 indicates that the interactions between selectivity filter O atoms and current-carrying ions that occur during ion conduction must be different from those that occur between selectivity filter O atoms, current-carrying ions, and a blocking ion. In contrast to this conclusion for EEEE locus substitution mutants, we have previously found for subconductance states of the WT channel that the amplitudes of unitary Ca2+ currents carried by the various substates could be predicted from substate-specific on and off rates for Ca2+ block of Li+ current (Cloues and Sather 2000). These divergent conclusions might be reconciled by noting that the structure of the EEEE locus was significantly altered in the D and A substitution mutants, whereas for the WT subconductance states, EEEE locus glutamates were preserved.


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)

Relationships between unitary conductance and rates of Cd2+ block and unblock.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Relationships between unitary conductance and rates of Cd2+ block and unblock.
Mentions: Is conduction of current-carrying ions (Ba2+ or Li+) related in any simple way to the on and off rates of a blocking ion (Cd2+)? Fig. 10 shows that in this case it is not: for a narrow range in Cd2+ on rate, unitary Ba2+ flux did not vary in any systematic way over an eightfold range in Cd2+ off rate, nor did unitary Li+ flux vary systematically over a 4.5-fold range in Cd2+ off rate and 3,600-fold range in Cd2+ on rate. The absence of a simple pattern in Fig. 10 indicates that the interactions between selectivity filter O atoms and current-carrying ions that occur during ion conduction must be different from those that occur between selectivity filter O atoms, current-carrying ions, and a blocking ion. In contrast to this conclusion for EEEE locus substitution mutants, we have previously found for subconductance states of the WT channel that the amplitudes of unitary Ca2+ currents carried by the various substates could be predicted from substate-specific on and off rates for Ca2+ block of Li+ current (Cloues and Sather 2000). These divergent conclusions might be reconciled by noting that the structure of the EEEE locus was significantly altered in the D and A substitution mutants, whereas for the WT subconductance states, EEEE locus glutamates were preserved.

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