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Time course and Ca(2+) dependence of sensitivity modulation in cyclic GMP-gated currents of intact cone photoreceptors.

Rebrik TI, Kotelnikova EA, Korenbrot JI - J. Gen. Physiol. (2000)

Bottom Line: Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s.Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone.Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, University of California at San Francisco, San Francisco, California 94143, USA.

ABSTRACT
We determined the Ca(2+) dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca(2+) levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K(1/2), the ligand concentration that half-saturates current, that, in turn, depend on Ca(2+). K(1/2) increases as Ca(2+) rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca(2+) with a single site without apparent cooperativity. (Ca)K(m), the Michaelis-Menten constant for Ca(2+) concentration is 857 +/- 68 nM for cGMP and 863 +/- 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca(2+) caused by sudden Ca(2+) sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 micro, Ca(2+) concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 +/- 0.95-fold with a mean time to peak of 1.25 +/- 0.23 s. We developed a model that assumes channel modulation is a pseudo-first-order process kinetically limited by free Ca(2+). Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s. Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.

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cGMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on cGMP in the presence of various Ca2+ concentrations, as labeled. Each data set was measured in a different ep-cone. The continuous line is the Hill equation that best fits the experimental data. At 0 μM Ca2+, K1/2 = 112 μM and n = 2.0; at 0.2 μM Ca2+, K1/2 = 141.5 μM and n = 2.1; at 0.5 μM Ca2+, K1/2 = 214 μM and n = 2.1; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15. (Right) The dependence of the K1/2 value of the Hill equation on Ca2+. The data points are the average (±SD) of measurements in different ep-cones. Complete statistical information is given in Table . The continuous line is a modified Michaelis-Menten function () that best fits the experimental data. The value of CaKm is given in the figure.
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Figure 2: cGMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on cGMP in the presence of various Ca2+ concentrations, as labeled. Each data set was measured in a different ep-cone. The continuous line is the Hill equation that best fits the experimental data. At 0 μM Ca2+, K1/2 = 112 μM and n = 2.0; at 0.2 μM Ca2+, K1/2 = 141.5 μM and n = 2.1; at 0.5 μM Ca2+, K1/2 = 214 μM and n = 2.1; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15. (Right) The dependence of the K1/2 value of the Hill equation on Ca2+. The data points are the average (±SD) of measurements in different ep-cones. Complete statistical information is given in Table . The continuous line is a modified Michaelis-Menten function () that best fits the experimental data. The value of CaKm is given in the figure.

Mentions: To analyze Ca2+ dependence in detail, we determined the value of K1/2 in the presence of Ca2+ at concentrations between 0 and 20 μΜ. Each Ca2+ concentration was tested in a different cone and K1/2 values were averaged (Fig. 2). Ca2+ dependence is well described by a modified Michaelis-Menten equation: 2\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K_{{1}/{2}} \left \left({\mathrm{Ca}}\right) \right = \left \left(K_{{1}/{2}}^{{\mathrm{hi}}}-K_{{1}/{2}}^{{\mathrm{low}}}\right) \right \frac{ \left \left[{\mathrm{Ca}}\right] \right }{ \left \left[{\mathrm{Ca}}\right] \right +^{{\mathrm{Ca}}}K_{{\mathrm{m}}}}+K_{{1}/{2}}^{{\mathrm{low}}}\end{equation*}\end{document} where K1/2 (Ca2+) is the value of K1/2 at a given Ca2+ concentration. K12hiand K12low are the extreme values of K1/2, at 20 and 0 μM Ca2+, respectively. CaKm is the Ca2+ concentration at the midpoint between K12hiand K12low.The values of K1/2, their errors, and details of the statistical universe sampled are presented in Table . Optimum fit of the Michaelis-Menten equation () to the mean of the data was obtained with CaKm = 857 ± 68 nM.


Time course and Ca(2+) dependence of sensitivity modulation in cyclic GMP-gated currents of intact cone photoreceptors.

Rebrik TI, Kotelnikova EA, Korenbrot JI - J. Gen. Physiol. (2000)

cGMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on cGMP in the presence of various Ca2+ concentrations, as labeled. Each data set was measured in a different ep-cone. The continuous line is the Hill equation that best fits the experimental data. At 0 μM Ca2+, K1/2 = 112 μM and n = 2.0; at 0.2 μM Ca2+, K1/2 = 141.5 μM and n = 2.1; at 0.5 μM Ca2+, K1/2 = 214 μM and n = 2.1; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15. (Right) The dependence of the K1/2 value of the Hill equation on Ca2+. The data points are the average (±SD) of measurements in different ep-cones. Complete statistical information is given in Table . The continuous line is a modified Michaelis-Menten function () that best fits the experimental data. The value of CaKm is given in the figure.
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Related In: Results  -  Collection

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Figure 2: cGMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on cGMP in the presence of various Ca2+ concentrations, as labeled. Each data set was measured in a different ep-cone. The continuous line is the Hill equation that best fits the experimental data. At 0 μM Ca2+, K1/2 = 112 μM and n = 2.0; at 0.2 μM Ca2+, K1/2 = 141.5 μM and n = 2.1; at 0.5 μM Ca2+, K1/2 = 214 μM and n = 2.1; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15. (Right) The dependence of the K1/2 value of the Hill equation on Ca2+. The data points are the average (±SD) of measurements in different ep-cones. Complete statistical information is given in Table . The continuous line is a modified Michaelis-Menten function () that best fits the experimental data. The value of CaKm is given in the figure.
Mentions: To analyze Ca2+ dependence in detail, we determined the value of K1/2 in the presence of Ca2+ at concentrations between 0 and 20 μΜ. Each Ca2+ concentration was tested in a different cone and K1/2 values were averaged (Fig. 2). Ca2+ dependence is well described by a modified Michaelis-Menten equation: 2\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}K_{{1}/{2}} \left \left({\mathrm{Ca}}\right) \right = \left \left(K_{{1}/{2}}^{{\mathrm{hi}}}-K_{{1}/{2}}^{{\mathrm{low}}}\right) \right \frac{ \left \left[{\mathrm{Ca}}\right] \right }{ \left \left[{\mathrm{Ca}}\right] \right +^{{\mathrm{Ca}}}K_{{\mathrm{m}}}}+K_{{1}/{2}}^{{\mathrm{low}}}\end{equation*}\end{document} where K1/2 (Ca2+) is the value of K1/2 at a given Ca2+ concentration. K12hiand K12low are the extreme values of K1/2, at 20 and 0 μM Ca2+, respectively. CaKm is the Ca2+ concentration at the midpoint between K12hiand K12low.The values of K1/2, their errors, and details of the statistical universe sampled are presented in Table . Optimum fit of the Michaelis-Menten equation () to the mean of the data was obtained with CaKm = 857 ± 68 nM.

Bottom Line: Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s.Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone.Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, University of California at San Francisco, San Francisco, California 94143, USA.

ABSTRACT
We determined the Ca(2+) dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca(2+) levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K(1/2), the ligand concentration that half-saturates current, that, in turn, depend on Ca(2+). K(1/2) increases as Ca(2+) rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca(2+) with a single site without apparent cooperativity. (Ca)K(m), the Michaelis-Menten constant for Ca(2+) concentration is 857 +/- 68 nM for cGMP and 863 +/- 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca(2+) caused by sudden Ca(2+) sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 micro, Ca(2+) concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 +/- 0.95-fold with a mean time to peak of 1.25 +/- 0.23 s. We developed a model that assumes channel modulation is a pseudo-first-order process kinetically limited by free Ca(2+). Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s. Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.

Show MeSH
Related in: MedlinePlus