Cloues RK et al. (2000), Ion interactions in the high-affinity binding l...

XB-ART-10271

J Gen Physiol 2000 Oct 01;1164:569-86.

Show Gene links Show Anatomy links

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

Cloues RK , Cibulsky SM , Sather WA .

???displayArticle.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.

???displayArticle.pubmedLink??? 11004206
???displayArticle.pmcLink??? PMC2230626

???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: tnni3

???attribute.lit??? ???displayArticles.show???

	Scheme S1.
	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.
	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.
	Figure 5. Cd2+ block of Ba2+ current carried by single E→A mutants. (A) Single-channel records at 0 mV. (B) Open-time distributions, and (C) shut-time distributions. (D) On and off rates were obtained from linear regression fits (solid lines) to the mean values of the shutting (▪; kon) and opening (○; koff) rate data. At each concentration of Cd2+, n = 3–4 patches, except for EIIA at 0 Cd2+, for which n = 2. Error bars not visible are smaller than the symbols.
	Figure 4. Cd2+ block of Li+ current carried by single E→D mutants. (A) Single-channel records recorded at −100 mV. (B) Open-time distributions, and (C) shut-time distributions. (D) On and off rates were obtained from linear regression fits to the mean values of the shutting (▪; kon) and opening (○; koff) rate data (n = 3–5 patches at each concentration of Cd2+). Error bars not visible are smaller than the symbols.
	Figure 3. Cd2+ block of Ba2+ current carried by single E→D mutants. (A) Single-channel records recorded at 0 mV. (B) Open-time distributions, and (C) shut-time distributions. (D) On and off rates were obtained by fitting linear regressions (solid lines) to the mean values of the shutting (▪; kon) and opening (○; koff) rate data. At each concentration of Cd2+, n = 3–5 patches, except for EIID at 100 μM Cd2+, for which n = 2. Error bars not visible are smaller than the symbols. Time axis tic marks are 0.2 decades per tic.
	Figure 7. Ca2+ block of Li+ current in (A) WT and (B) EIIID channels. (C) On- and off-rate plots for Ca2+ block of WT and EIIID channels. Dashed lines indicate the best fits to WT data, as previously published (Cloues and Sather 2000). Membrane potential was −60 mV for WT and EIIID. Records were filtered at 2 kHz, and sampled at 10 kHz. At each concentration of Ca2+, n = 3 patches. Error bars not visible are smaller than the symbols.
	Figure 6. Cd2+ block of Li+ current carried by single E→A mutants. (A) Single-channel records recorded at −100 mV. (B) Open-time distributions, and (C) shut-time distributions. (D) On and off rates were obtained from linear regression fits to the mean values of the shutting (▪; kon) and opening (○; koff) rate data. At each concentration of Cd2+, n = 3–7 patches, except for EIIIA at 30 μM Cd2+ and EIVD at 0 Cd2+, for which n = 2 in both cases. Error bars not visible are smaller than the symbols.
	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.
	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.
	Figure 10. Relationships between unitary conductance and rates of Cd2+ block and unblock.

References [+] :

Almers, Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. 1984, Pubmed