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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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8Br-GMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on 8Br-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 = 15.1 μM and n = 1.11; at 0.2 μM Ca2+, K1/2 = 37.6 μM and n = 1.1; at 0.5 μM Ca2+, K1/2 = 49.5 μM and n = 1.67; at 2 μM Ca2+, K1/2 = 57.2 and n = 1.6; and at 20 μM Ca2+, K1/2 = 77 μM and n = 1.8. (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.
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Figure 3: 8Br-GMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on 8Br-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 = 15.1 μM and n = 1.11; at 0.2 μM Ca2+, K1/2 = 37.6 μM and n = 1.1; at 0.5 μM Ca2+, K1/2 = 49.5 μM and n = 1.67; at 2 μM Ca2+, K1/2 = 57.2 and n = 1.6; and at 20 μM Ca2+, K1/2 = 77 μM and n = 1.8. (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.

Mentions: To insure that the effects reported for cGMP are not confounded by the possible hydrolysis of cGMP by PDE in the outer segment (in spite of the presence of Zaprinast), we carried out the same class of measurements using 8Br-cGMP also in the presence of Zaprinast. 8Br-cGMP is an effective activator of cone CNG channels (Hackos and Korenbrot 1997), but a poor substrate of PDE (Zimmerman et al. 1985). We obtained the same results with 8Br-cGMP as with cGMP (Fig. 3). The normalized current amplitude increased with nucleotide concentration with a dependence well described by the Hill equation. The values of K1/2 changed with Ca2+ with a dependence well described by the modified Michaelis-Menten equation () (Fig. 3). 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 = 863 ± 51 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)

8Br-GMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on 8Br-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 = 15.1 μM and n = 1.11; at 0.2 μM Ca2+, K1/2 = 37.6 μM and n = 1.1; at 0.5 μM Ca2+, K1/2 = 49.5 μM and n = 1.67; at 2 μM Ca2+, K1/2 = 57.2 and n = 1.6; and at 20 μM Ca2+, K1/2 = 77 μM and n = 1.8. (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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230625&req=5

Figure 3: 8Br-GMP dependence of cone outer segment current as a function of cytoplasmic Ca2+. (Left) The dependence of normalized current amplitude on 8Br-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 = 15.1 μM and n = 1.11; at 0.2 μM Ca2+, K1/2 = 37.6 μM and n = 1.1; at 0.5 μM Ca2+, K1/2 = 49.5 μM and n = 1.67; at 2 μM Ca2+, K1/2 = 57.2 and n = 1.6; and at 20 μM Ca2+, K1/2 = 77 μM and n = 1.8. (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.
Mentions: To insure that the effects reported for cGMP are not confounded by the possible hydrolysis of cGMP by PDE in the outer segment (in spite of the presence of Zaprinast), we carried out the same class of measurements using 8Br-cGMP also in the presence of Zaprinast. 8Br-cGMP is an effective activator of cone CNG channels (Hackos and Korenbrot 1997), but a poor substrate of PDE (Zimmerman et al. 1985). We obtained the same results with 8Br-cGMP as with cGMP (Fig. 3). The normalized current amplitude increased with nucleotide concentration with a dependence well described by the Hill equation. The values of K1/2 changed with Ca2+ with a dependence well described by the modified Michaelis-Menten equation () (Fig. 3). 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 = 863 ± 51 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