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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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Activation of cGMP-dependent currents in the intact outer segment of electropermeabilized cones. In each panel are shown inward outer-segment currents activated by step increments in cytoplasmic cGMP concentrations. Data in each panel were measured in different cells and measurements were completed within 180 s of the moment of electropermeabilization. Each panel illustrates data measured in the presence of different Ca2+ concentrations between 0 and 20 μM, as labeled. Solutions also included constant concentrations of Mg2+ (1 mM) and Zaprinast (0.4 mM), a phosphodiesterase inhibitor. cGMP concentrations tested at 0 Ca2+ were 0, 80, 160, 320, and 1,000 μM. At all other Ca2+ concentrations, cGMP tested were 0, 62.5, 125, 250, 500, and 1,000 μM. The data in each panel are analyzed to illustrate the dependence of peak current amplitude on cGMP. The continuous lines are Hill functions () optimally fit to the experimental data. For the data shown: 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 = 171 μM and n = 1.8; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; at 5 μM Ca2+, K1/2 = 295 μM and n = 2.5; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15.
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Figure 1: Activation of cGMP-dependent currents in the intact outer segment of electropermeabilized cones. In each panel are shown inward outer-segment currents activated by step increments in cytoplasmic cGMP concentrations. Data in each panel were measured in different cells and measurements were completed within 180 s of the moment of electropermeabilization. Each panel illustrates data measured in the presence of different Ca2+ concentrations between 0 and 20 μM, as labeled. Solutions also included constant concentrations of Mg2+ (1 mM) and Zaprinast (0.4 mM), a phosphodiesterase inhibitor. cGMP concentrations tested at 0 Ca2+ were 0, 80, 160, 320, and 1,000 μM. At all other Ca2+ concentrations, cGMP tested were 0, 62.5, 125, 250, 500, and 1,000 μM. The data in each panel are analyzed to illustrate the dependence of peak current amplitude on cGMP. The continuous lines are Hill functions () optimally fit to the experimental data. For the data shown: 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 = 171 μM and n = 1.8; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; at 5 μM Ca2+, K1/2 = 295 μM and n = 2.5; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15.

Mentions: We measured the Ca2+ dependence of ligand sensitivity in cGMP-gated currents of electropermeabilized, single cones (ep-cones). In this preparation, outer segment membrane currents are measured with suction electrodes at 0-mV holding potential. To generate an electromotive force that drives the cGMP-gated currents, measurements are conducted under an ion concentration gradient with Na+, a permeable cation, in the extracellular medium and choline+, an impermeant cation, in the intracellular medium. Also, there is only 1 μM Ca2+ in the extracellular medium to reduce the ion's influx through open CNG, and thus to minimize intracellular Ca2+ loading. Under these conditions, activation of cyclic nucleotide-gated channels generates a sustained, inward current (Fig. 1).


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)

Activation of cGMP-dependent currents in the intact outer segment of electropermeabilized cones. In each panel are shown inward outer-segment currents activated by step increments in cytoplasmic cGMP concentrations. Data in each panel were measured in different cells and measurements were completed within 180 s of the moment of electropermeabilization. Each panel illustrates data measured in the presence of different Ca2+ concentrations between 0 and 20 μM, as labeled. Solutions also included constant concentrations of Mg2+ (1 mM) and Zaprinast (0.4 mM), a phosphodiesterase inhibitor. cGMP concentrations tested at 0 Ca2+ were 0, 80, 160, 320, and 1,000 μM. At all other Ca2+ concentrations, cGMP tested were 0, 62.5, 125, 250, 500, and 1,000 μM. The data in each panel are analyzed to illustrate the dependence of peak current amplitude on cGMP. The continuous lines are Hill functions () optimally fit to the experimental data. For the data shown: 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 = 171 μM and n = 1.8; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; at 5 μM Ca2+, K1/2 = 295 μM and n = 2.5; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15.
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Related In: Results  -  Collection

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Figure 1: Activation of cGMP-dependent currents in the intact outer segment of electropermeabilized cones. In each panel are shown inward outer-segment currents activated by step increments in cytoplasmic cGMP concentrations. Data in each panel were measured in different cells and measurements were completed within 180 s of the moment of electropermeabilization. Each panel illustrates data measured in the presence of different Ca2+ concentrations between 0 and 20 μM, as labeled. Solutions also included constant concentrations of Mg2+ (1 mM) and Zaprinast (0.4 mM), a phosphodiesterase inhibitor. cGMP concentrations tested at 0 Ca2+ were 0, 80, 160, 320, and 1,000 μM. At all other Ca2+ concentrations, cGMP tested were 0, 62.5, 125, 250, 500, and 1,000 μM. The data in each panel are analyzed to illustrate the dependence of peak current amplitude on cGMP. The continuous lines are Hill functions () optimally fit to the experimental data. For the data shown: 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 = 171 μM and n = 1.8; at 2 μM Ca2+, K1/2 = 259.8 and n = 1.98; at 5 μM Ca2+, K1/2 = 295 μM and n = 2.5; and at 20 μM Ca2+, K1/2 = 331 μM and n = 2.15.
Mentions: We measured the Ca2+ dependence of ligand sensitivity in cGMP-gated currents of electropermeabilized, single cones (ep-cones). In this preparation, outer segment membrane currents are measured with suction electrodes at 0-mV holding potential. To generate an electromotive force that drives the cGMP-gated currents, measurements are conducted under an ion concentration gradient with Na+, a permeable cation, in the extracellular medium and choline+, an impermeant cation, in the intracellular medium. Also, there is only 1 μM Ca2+ in the extracellular medium to reduce the ion's influx through open CNG, and thus to minimize intracellular Ca2+ loading. Under these conditions, activation of cyclic nucleotide-gated channels generates a sustained, inward current (Fig. 1).

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