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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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Cd2+ block of unitary Ba2+ and Li+ currents carried by α1C Ca2+ channels. (A) Examples of Cd2+ block of unitary currents carried by 110 mM Ba2+, and corresponding dwell-time histograms. Test potential, 0 mV; filter corner frequency, 2 kHz; sampling rate, 10 kHz. Solid curves drawn through dwell-time histograms are the exponential fits to the binned data. Raw dwell-time constants and dwell-time constants corrected for missed events (parentheses) are indicated to the right of each histogram. (B) Examples of Cd2+ block of unitary currents carried by 100 mM Li+, and fitted dwell-time histograms. Test potential, −100 mV; corner frequency, 2 kHz; sampling rate, 10 kHz. Time axis tic marks are 0.2 decades per tic.
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Figure 1: Cd2+ block of unitary Ba2+ and Li+ currents carried by α1C Ca2+ channels. (A) Examples of Cd2+ block of unitary currents carried by 110 mM Ba2+, and corresponding dwell-time histograms. Test potential, 0 mV; filter corner frequency, 2 kHz; sampling rate, 10 kHz. Solid curves drawn through dwell-time histograms are the exponential fits to the binned data. Raw dwell-time constants and dwell-time constants corrected for missed events (parentheses) are indicated to the right of each histogram. (B) Examples of Cd2+ block of unitary currents carried by 100 mM Li+, and fitted dwell-time histograms. Test potential, −100 mV; corner frequency, 2 kHz; sampling rate, 10 kHz. Time axis tic marks are 0.2 decades per tic.

Mentions: Owing to the action of FPL 64176, open times in the absence of blocker were often tens of milliseconds in duration (see control records in Fig. 1A and Fig. B, below). Channel closures in the absence of blocker were also generally of long duration, typically >10 ms. Inclusion of blocker (Cd2+ or Ca2+) in the pipet solution produced brief interruptions (shut times <1 ms) of the FPL 64176–dependent long-duration openings. Shut times longer than 3.0 ms were excluded from the analysis because these mostly represented closures between channel openings rather than block events. Under these conditions, both the open- and the shut-time histograms were in almost all cases well-fit by single exponential functions, with the single component of the shut time largely representing the blocked time, as described 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)

Cd2+ block of unitary Ba2+ and Li+ currents carried by α1C Ca2+ channels. (A) Examples of Cd2+ block of unitary currents carried by 110 mM Ba2+, and corresponding dwell-time histograms. Test potential, 0 mV; filter corner frequency, 2 kHz; sampling rate, 10 kHz. Solid curves drawn through dwell-time histograms are the exponential fits to the binned data. Raw dwell-time constants and dwell-time constants corrected for missed events (parentheses) are indicated to the right of each histogram. (B) Examples of Cd2+ block of unitary currents carried by 100 mM Li+, and fitted dwell-time histograms. Test potential, −100 mV; corner frequency, 2 kHz; sampling rate, 10 kHz. Time axis tic marks are 0.2 decades per tic.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230626&req=5

Figure 1: Cd2+ block of unitary Ba2+ and Li+ currents carried by α1C Ca2+ channels. (A) Examples of Cd2+ block of unitary currents carried by 110 mM Ba2+, and corresponding dwell-time histograms. Test potential, 0 mV; filter corner frequency, 2 kHz; sampling rate, 10 kHz. Solid curves drawn through dwell-time histograms are the exponential fits to the binned data. Raw dwell-time constants and dwell-time constants corrected for missed events (parentheses) are indicated to the right of each histogram. (B) Examples of Cd2+ block of unitary currents carried by 100 mM Li+, and fitted dwell-time histograms. Test potential, −100 mV; corner frequency, 2 kHz; sampling rate, 10 kHz. Time axis tic marks are 0.2 decades per tic.
Mentions: Owing to the action of FPL 64176, open times in the absence of blocker were often tens of milliseconds in duration (see control records in Fig. 1A and Fig. B, below). Channel closures in the absence of blocker were also generally of long duration, typically >10 ms. Inclusion of blocker (Cd2+ or Ca2+) in the pipet solution produced brief interruptions (shut times <1 ms) of the FPL 64176–dependent long-duration openings. Shut times longer than 3.0 ms were excluded from the analysis because these mostly represented closures between channel openings rather than block events. Under these conditions, both the open- and the shut-time histograms were in almost all cases well-fit by single exponential functions, with the single component of the shut time largely representing the blocked time, as described 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