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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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Experimental and simulated changes in membrane current caused by sudden Ca2+ loss in a cone outer segment in the presence of unchanging 8Br-cGMP. Experimental data (noisy traces) are currents measured at −35 mV in a cone loaded with standard electrode-filling solution containing 15 μM 8Br-cGMP, 0.4 mM Zaprinast, 1 mM diazo-2, 0.1 mM bis-fura-2 with 600 nM free Ca2+, and 1 mM free Mg2+. At the arrow, Ca2+ was sequestered by uncaging diazo-2. The Ca2+ concentration change in the cell was measured (not shown) and described by  with Capeak2+Cadark2+= 0.72, Cass2+Cadark2+= 0.91, and τCa = 5.55 s. Simulated data (smooth traces) are the results of simulations that assume channel modulation is a pseudo–first-order process rate limited by the kinetics of Ca2+ concentration change, with time constant τg = 0.32 s. The same data are shown at two different time resolutions.
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Figure 8: Experimental and simulated changes in membrane current caused by sudden Ca2+ loss in a cone outer segment in the presence of unchanging 8Br-cGMP. Experimental data (noisy traces) are currents measured at −35 mV in a cone loaded with standard electrode-filling solution containing 15 μM 8Br-cGMP, 0.4 mM Zaprinast, 1 mM diazo-2, 0.1 mM bis-fura-2 with 600 nM free Ca2+, and 1 mM free Mg2+. At the arrow, Ca2+ was sequestered by uncaging diazo-2. The Ca2+ concentration change in the cell was measured (not shown) and described by with Capeak2+Cadark2+= 0.72, Cass2+Cadark2+= 0.91, and τCa = 5.55 s. Simulated data (smooth traces) are the results of simulations that assume channel modulation is a pseudo–first-order process rate limited by the kinetics of Ca2+ concentration change, with time constant τg = 0.32 s. The same data are shown at two different time resolutions.

Mentions: To test the adequacy of the model, we fit data simulated by the model to experimental results. To this end, and for each set of experimental data: (a) we fit to the measured fluorescence change and used the resulting analytical expression to describe Ca(t) for that data set. (b) We simulated changes in membrane conductance using the model above; the simulation predicts the change in conductance caused by a defined change Ca2+ provided as an input. (c) We systematically varied the values of τg to best match simulated and experimental data. The kinetic model could be made to simulate experimental data well (Fig. 8). For seven cells, with fits of similar quality to that shown in Fig. 8, mean value was τg = 0.40 ± 0.14 s.


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)

Experimental and simulated changes in membrane current caused by sudden Ca2+ loss in a cone outer segment in the presence of unchanging 8Br-cGMP. Experimental data (noisy traces) are currents measured at −35 mV in a cone loaded with standard electrode-filling solution containing 15 μM 8Br-cGMP, 0.4 mM Zaprinast, 1 mM diazo-2, 0.1 mM bis-fura-2 with 600 nM free Ca2+, and 1 mM free Mg2+. At the arrow, Ca2+ was sequestered by uncaging diazo-2. The Ca2+ concentration change in the cell was measured (not shown) and described by  with Capeak2+Cadark2+= 0.72, Cass2+Cadark2+= 0.91, and τCa = 5.55 s. Simulated data (smooth traces) are the results of simulations that assume channel modulation is a pseudo–first-order process rate limited by the kinetics of Ca2+ concentration change, with time constant τg = 0.32 s. The same data are shown at two different time resolutions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Experimental and simulated changes in membrane current caused by sudden Ca2+ loss in a cone outer segment in the presence of unchanging 8Br-cGMP. Experimental data (noisy traces) are currents measured at −35 mV in a cone loaded with standard electrode-filling solution containing 15 μM 8Br-cGMP, 0.4 mM Zaprinast, 1 mM diazo-2, 0.1 mM bis-fura-2 with 600 nM free Ca2+, and 1 mM free Mg2+. At the arrow, Ca2+ was sequestered by uncaging diazo-2. The Ca2+ concentration change in the cell was measured (not shown) and described by with Capeak2+Cadark2+= 0.72, Cass2+Cadark2+= 0.91, and τCa = 5.55 s. Simulated data (smooth traces) are the results of simulations that assume channel modulation is a pseudo–first-order process rate limited by the kinetics of Ca2+ concentration change, with time constant τg = 0.32 s. The same data are shown at two different time resolutions.
Mentions: To test the adequacy of the model, we fit data simulated by the model to experimental results. To this end, and for each set of experimental data: (a) we fit to the measured fluorescence change and used the resulting analytical expression to describe Ca(t) for that data set. (b) We simulated changes in membrane conductance using the model above; the simulation predicts the change in conductance caused by a defined change Ca2+ provided as an input. (c) We systematically varied the values of τg to best match simulated and experimental data. The kinetic model could be made to simulate experimental data well (Fig. 8). For seven cells, with fits of similar quality to that shown in Fig. 8, mean value was τg = 0.40 ± 0.14 s.

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