Limits...
The EEEE locus is the sole high-affinity Ca(2+) binding structure in the pore of a voltage-gated Ca(2+) channel: block by ca(2+) entering from the intracellular pore entrance.

Cibulsky SM, Sather WA - J. Gen. Physiol. (2000)

Bottom Line: First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed.Using inside-out patches, we found that, whereas micromolar Ca(2+) produced substantial block of outward Li(+) current in wild-type channels, internal Ca(2+) concentrations up to 1 mM did not produce detectable block of outward Li(+) current in the AAAA or QQQQ mutants.These results indicate that the EEEE locus is indeed the sole high-affinity Ca(2+) binding locus in the pore of voltage-gated Ca(2+) channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

ABSTRACT
Selective permeability in voltage-gated Ca(2+) channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca(2+) binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca(2+) binding sites. The current view is based on site-directed mutagenesis work in which Ca(2+) binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca(2+) channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca(2+) produced substantial block of outward Li(+) current in wild-type channels, internal Ca(2+) concentrations up to 1 mM did not produce detectable block of outward Li(+) current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca(2+) binding locus in the pore of voltage-gated Ca(2+) channels.

Show MeSH

Related in: MedlinePlus

Concentration and voltage dependence of Ca2+ block of outward Li+ current carried through WT α1C channels in inside-out patches excised from HEK293 cells. (A) Internal Ca2+ blocked outward Li+ currents through single WT channels with high affinity. The bath solution contained 300 mM Li+ plus various [Ca2+], and the patch pipet contained 55 mM Li+. Control Li+ solution contained 3 nM Ca2+. Single-channel currents were recorded during a test depolarization to +20 mV from a holding potential of −100 mV. In some cases, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates are plotted as mean ± SEM versus [Ca2+] for n = 3–5. The linear regression fit through the on-rate data points has a slope of 2.3 × 108 M−1 s−1. The horizontal line fit to the off-rate data indicates the average off rate was 2,965 s−1. (B) Voltage dependence of internal Ca2+ block of outward Li+ currents through single WT channels. The bath solution contained 300 mM Li+ and 3 μM Ca2+, while the pipet solution contained 55 mM Li+. Single-channel currents were recorded during a step depolarization from the holding potential of −100 mV. In some experiments, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates plotted as mean ± SEM versus test potential (n = 4–5). The horizontal line fit to the on-rate data indicates the average on rate was 846 s−1. The exponential curve fit to the off-rate data indicates that off rate increased e-fold per 19 mV.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233694&req=5

Figure 5: Concentration and voltage dependence of Ca2+ block of outward Li+ current carried through WT α1C channels in inside-out patches excised from HEK293 cells. (A) Internal Ca2+ blocked outward Li+ currents through single WT channels with high affinity. The bath solution contained 300 mM Li+ plus various [Ca2+], and the patch pipet contained 55 mM Li+. Control Li+ solution contained 3 nM Ca2+. Single-channel currents were recorded during a test depolarization to +20 mV from a holding potential of −100 mV. In some cases, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates are plotted as mean ± SEM versus [Ca2+] for n = 3–5. The linear regression fit through the on-rate data points has a slope of 2.3 × 108 M−1 s−1. The horizontal line fit to the off-rate data indicates the average off rate was 2,965 s−1. (B) Voltage dependence of internal Ca2+ block of outward Li+ currents through single WT channels. The bath solution contained 300 mM Li+ and 3 μM Ca2+, while the pipet solution contained 55 mM Li+. Single-channel currents were recorded during a step depolarization from the holding potential of −100 mV. In some experiments, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates plotted as mean ± SEM versus test potential (n = 4–5). The horizontal line fit to the on-rate data indicates the average on rate was 846 s−1. The exponential curve fit to the off-rate data indicates that off rate increased e-fold per 19 mV.

Mentions: Recordings of outward Li+ currents through single α1C channels in inside-out patches from HEK293 cells are shown in Fig. 5 A. These channels exhibited multiple conductance states, a well-documented behavior of voltage-gated Ca2+ channels (e.g., Cloues and Sather 2000). The amplitude to which they opened most frequently was the largest one, and this amplitude was analyzed for kinetics of pore block. WT α1C channels were blocked when at least micromolar Ca2+ was present in the solution bathing the cytosolic surface of the patch. Representative WT single-channel records illustrate the flicker block that was introduced by internal Ca2+, with the number of flicker block events increasing with [Ca2+] (Fig. 5 A). Quantitation of open and shut times yielded Ca2+ on rates (1/τopen) that increased with [Ca2+] and off rates (1/τshut) that were concentration independent, findings that are indicative of a bimolecular reaction. At a membrane potential of +20 mV, the average off rate was 2,965 s−1 and the on-rate coefficient, determined as the slope of a linear regression fit, was 2.3 × 108 M−1 s−1. The WT channel exhibited high-affinity binding of Ca2+ in this configuration, as the apparent Kd is calculated to be ∼13 μM (koff/kon). The concentration-dependent on rate is similar to that of Kuo and Hess 1993a, who studied Ca2+ block of Li+ current through endogenous Ca2+ channels in PC12 cells. Likewise, the magnitude and concentration independence of the off rate agree with that measured by Kuo and Hess 1993a. The voltage dependence of block by Ca2+ was determined over the voltage range 0 to +20 mV. The on rate was voltage independent, whereas the off rate increased with depolarization, e-fold/19 mV (Fig. 5 B). These characteristics match those of on and off rates obtained by Kuo and Hess 1993a, as expected if the channels we have studied are WT α1C channels.


The EEEE locus is the sole high-affinity Ca(2+) binding structure in the pore of a voltage-gated Ca(2+) channel: block by ca(2+) entering from the intracellular pore entrance.

Cibulsky SM, Sather WA - J. Gen. Physiol. (2000)

Concentration and voltage dependence of Ca2+ block of outward Li+ current carried through WT α1C channels in inside-out patches excised from HEK293 cells. (A) Internal Ca2+ blocked outward Li+ currents through single WT channels with high affinity. The bath solution contained 300 mM Li+ plus various [Ca2+], and the patch pipet contained 55 mM Li+. Control Li+ solution contained 3 nM Ca2+. Single-channel currents were recorded during a test depolarization to +20 mV from a holding potential of −100 mV. In some cases, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates are plotted as mean ± SEM versus [Ca2+] for n = 3–5. The linear regression fit through the on-rate data points has a slope of 2.3 × 108 M−1 s−1. The horizontal line fit to the off-rate data indicates the average off rate was 2,965 s−1. (B) Voltage dependence of internal Ca2+ block of outward Li+ currents through single WT channels. The bath solution contained 300 mM Li+ and 3 μM Ca2+, while the pipet solution contained 55 mM Li+. Single-channel currents were recorded during a step depolarization from the holding potential of −100 mV. In some experiments, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates plotted as mean ± SEM versus test potential (n = 4–5). The horizontal line fit to the on-rate data indicates the average on rate was 846 s−1. The exponential curve fit to the off-rate data indicates that off rate increased e-fold per 19 mV.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233694&req=5

Figure 5: Concentration and voltage dependence of Ca2+ block of outward Li+ current carried through WT α1C channels in inside-out patches excised from HEK293 cells. (A) Internal Ca2+ blocked outward Li+ currents through single WT channels with high affinity. The bath solution contained 300 mM Li+ plus various [Ca2+], and the patch pipet contained 55 mM Li+. Control Li+ solution contained 3 nM Ca2+. Single-channel currents were recorded during a test depolarization to +20 mV from a holding potential of −100 mV. In some cases, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates are plotted as mean ± SEM versus [Ca2+] for n = 3–5. The linear regression fit through the on-rate data points has a slope of 2.3 × 108 M−1 s−1. The horizontal line fit to the off-rate data indicates the average off rate was 2,965 s−1. (B) Voltage dependence of internal Ca2+ block of outward Li+ currents through single WT channels. The bath solution contained 300 mM Li+ and 3 μM Ca2+, while the pipet solution contained 55 mM Li+. Single-channel currents were recorded during a step depolarization from the holding potential of −100 mV. In some experiments, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates plotted as mean ± SEM versus test potential (n = 4–5). The horizontal line fit to the on-rate data indicates the average on rate was 846 s−1. The exponential curve fit to the off-rate data indicates that off rate increased e-fold per 19 mV.
Mentions: Recordings of outward Li+ currents through single α1C channels in inside-out patches from HEK293 cells are shown in Fig. 5 A. These channels exhibited multiple conductance states, a well-documented behavior of voltage-gated Ca2+ channels (e.g., Cloues and Sather 2000). The amplitude to which they opened most frequently was the largest one, and this amplitude was analyzed for kinetics of pore block. WT α1C channels were blocked when at least micromolar Ca2+ was present in the solution bathing the cytosolic surface of the patch. Representative WT single-channel records illustrate the flicker block that was introduced by internal Ca2+, with the number of flicker block events increasing with [Ca2+] (Fig. 5 A). Quantitation of open and shut times yielded Ca2+ on rates (1/τopen) that increased with [Ca2+] and off rates (1/τshut) that were concentration independent, findings that are indicative of a bimolecular reaction. At a membrane potential of +20 mV, the average off rate was 2,965 s−1 and the on-rate coefficient, determined as the slope of a linear regression fit, was 2.3 × 108 M−1 s−1. The WT channel exhibited high-affinity binding of Ca2+ in this configuration, as the apparent Kd is calculated to be ∼13 μM (koff/kon). The concentration-dependent on rate is similar to that of Kuo and Hess 1993a, who studied Ca2+ block of Li+ current through endogenous Ca2+ channels in PC12 cells. Likewise, the magnitude and concentration independence of the off rate agree with that measured by Kuo and Hess 1993a. The voltage dependence of block by Ca2+ was determined over the voltage range 0 to +20 mV. The on rate was voltage independent, whereas the off rate increased with depolarization, e-fold/19 mV (Fig. 5 B). These characteristics match those of on and off rates obtained by Kuo and Hess 1993a, as expected if the channels we have studied are WT α1C channels.

Bottom Line: First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed.Using inside-out patches, we found that, whereas micromolar Ca(2+) produced substantial block of outward Li(+) current in wild-type channels, internal Ca(2+) concentrations up to 1 mM did not produce detectable block of outward Li(+) current in the AAAA or QQQQ mutants.These results indicate that the EEEE locus is indeed the sole high-affinity Ca(2+) binding locus in the pore of voltage-gated Ca(2+) channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

ABSTRACT
Selective permeability in voltage-gated Ca(2+) channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca(2+) binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca(2+) binding sites. The current view is based on site-directed mutagenesis work in which Ca(2+) binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca(2+) channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca(2+) produced substantial block of outward Li(+) current in wild-type channels, internal Ca(2+) concentrations up to 1 mM did not produce detectable block of outward Li(+) current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca(2+) binding locus in the pore of voltage-gated Ca(2+) channels.

Show MeSH
Related in: MedlinePlus