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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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Rate of equilibration between cone outer segment cytoplasm and lumen of tight-seal electrode. Shown are whole-cell membrane currents measured in two different cones at −35-mV holding voltage. At the arrow mark (WCR) (time = 0), whole-cell mode was attained and the contents of the cell cytoplasm began to exchange with the solution filling the tight-seal electrode. This solution contained either 15 or 30 μM 8Br-cGMP, as labeled, and 1 mM diazo-2, 1 mM free Mg2+, and 600 nM free Ca2+ (see text for details). As the cyclic nucleotide loaded the cell, CNG channels in the outer segment were activated and an inward current developed. The current reached a stationary value after ∼2 min, indicating concentration equilibrium between cell cytoplasm and the electrode lumen.
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Figure 4: Rate of equilibration between cone outer segment cytoplasm and lumen of tight-seal electrode. Shown are whole-cell membrane currents measured in two different cones at −35-mV holding voltage. At the arrow mark (WCR) (time = 0), whole-cell mode was attained and the contents of the cell cytoplasm began to exchange with the solution filling the tight-seal electrode. This solution contained either 15 or 30 μM 8Br-cGMP, as labeled, and 1 mM diazo-2, 1 mM free Mg2+, and 600 nM free Ca2+ (see text for details). As the cyclic nucleotide loaded the cell, CNG channels in the outer segment were activated and an inward current developed. The current reached a stationary value after ∼2 min, indicating concentration equilibrium between cell cytoplasm and the electrode lumen.

Mentions: To detect CNG channel modulation in the absence of ATP and GTP, we activated the channels with exogenous ligand. We added 8Br-cGMP and 0.4 mM Zaprinast to the electrode-filling solution, which also contained 1 mM diazo-2 and 0.1 mM bis-fura-2 with 1 mM free Mg2+ and 600 nM free Ca2+, a value close to that of CaKm (see above). In Fig. 4, we illustrate typical membrane currents measured in different cones at −35 mV with electrode filled with solution containing either 15 or 30 μM 8Br-cGMP. After attaining whole-cell mode, an inward current developed slowly that reached a peak and then declined to a stationary value. This time course reflects the activation of the outer segment inward current as the nucleotide loads the cell and cGMP-gated channels open, followed by channel block due to increasing cytoplasmic Ca2+ concentration. Because of this associated cytoplasmic Ca2+ load, we elected to carry out these experiments in a Ringers' solution containing 0.1 mM Ca2+, rather than the normal 1 mM. This allowed us to maintain cytoplasmic Ca2+ within reasonable bounds.


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)

Rate of equilibration between cone outer segment cytoplasm and lumen of tight-seal electrode. Shown are whole-cell membrane currents measured in two different cones at −35-mV holding voltage. At the arrow mark (WCR) (time = 0), whole-cell mode was attained and the contents of the cell cytoplasm began to exchange with the solution filling the tight-seal electrode. This solution contained either 15 or 30 μM 8Br-cGMP, as labeled, and 1 mM diazo-2, 1 mM free Mg2+, and 600 nM free Ca2+ (see text for details). As the cyclic nucleotide loaded the cell, CNG channels in the outer segment were activated and an inward current developed. The current reached a stationary value after ∼2 min, indicating concentration equilibrium between cell cytoplasm and the electrode lumen.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Rate of equilibration between cone outer segment cytoplasm and lumen of tight-seal electrode. Shown are whole-cell membrane currents measured in two different cones at −35-mV holding voltage. At the arrow mark (WCR) (time = 0), whole-cell mode was attained and the contents of the cell cytoplasm began to exchange with the solution filling the tight-seal electrode. This solution contained either 15 or 30 μM 8Br-cGMP, as labeled, and 1 mM diazo-2, 1 mM free Mg2+, and 600 nM free Ca2+ (see text for details). As the cyclic nucleotide loaded the cell, CNG channels in the outer segment were activated and an inward current developed. The current reached a stationary value after ∼2 min, indicating concentration equilibrium between cell cytoplasm and the electrode lumen.
Mentions: To detect CNG channel modulation in the absence of ATP and GTP, we activated the channels with exogenous ligand. We added 8Br-cGMP and 0.4 mM Zaprinast to the electrode-filling solution, which also contained 1 mM diazo-2 and 0.1 mM bis-fura-2 with 1 mM free Mg2+ and 600 nM free Ca2+, a value close to that of CaKm (see above). In Fig. 4, we illustrate typical membrane currents measured in different cones at −35 mV with electrode filled with solution containing either 15 or 30 μM 8Br-cGMP. After attaining whole-cell mode, an inward current developed slowly that reached a peak and then declined to a stationary value. This time course reflects the activation of the outer segment inward current as the nucleotide loads the cell and cGMP-gated channels open, followed by channel block due to increasing cytoplasmic Ca2+ concentration. Because of this associated cytoplasmic Ca2+ load, we elected to carry out these experiments in a Ringers' solution containing 0.1 mM Ca2+, rather than the normal 1 mM. This allowed us to maintain cytoplasmic Ca2+ within reasonable bounds.

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