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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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Time course of Ca2+-dependent CNG current modulation and control experiments. (Left) Whole-cell membrane current measured at −35-mV holding voltage in a single 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+. 1.5 s after starting the record, at the time indicated by the spike in the record, a Xenon flash uncaged diazo-2, causing a slow increase in the inward current that reached a peak in ∼1.4 s, and then returned towards its starting value. At the peak, membrane conductance was enhanced 2.26-fold. (Right) Currents measured at −35 mV in two different cones, each loaded with a modified electrode-filling solution. Two control conditions were tested: (a) omitting 8Br-cGMP (0 μM 8Br-cGMP), and (b) replacing diazo-2 with 1 mM BAPTA (0 μM Diazo-2). Free Ca2+ and Mg2+ remained at 600 nM and 1 mM, respectively. In the absence of 8Br-cGMP, the net current was outward because CNG channels are closed and current flows through voltage-gated K+ channels in the cone inner segment. The uncaging flash did not cause a current change in either of the control experiments.
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Figure 5: Time course of Ca2+-dependent CNG current modulation and control experiments. (Left) Whole-cell membrane current measured at −35-mV holding voltage in a single 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+. 1.5 s after starting the record, at the time indicated by the spike in the record, a Xenon flash uncaged diazo-2, causing a slow increase in the inward current that reached a peak in ∼1.4 s, and then returned towards its starting value. At the peak, membrane conductance was enhanced 2.26-fold. (Right) Currents measured at −35 mV in two different cones, each loaded with a modified electrode-filling solution. Two control conditions were tested: (a) omitting 8Br-cGMP (0 μM 8Br-cGMP), and (b) replacing diazo-2 with 1 mM BAPTA (0 μM Diazo-2). Free Ca2+ and Mg2+ remained at 600 nM and 1 mM, respectively. In the absence of 8Br-cGMP, the net current was outward because CNG channels are closed and current flows through voltage-gated K+ channels in the cone inner segment. The uncaging flash did not cause a current change in either of the control experiments.

Mentions: We caused a rapid (<< 50 ms, see below) decrease in cytoplasmic Ca2+ by uncaging diazo-2 in intact cone outer segment. In the presence of 15 μM cytoplasmic 8Br-cGMP, the uncaging flash caused a slow increase in current that reached a peak and then slowly returned towards its starting value (Fig. 5). Within the first 6–8 min after establishing whole-cell mode, responses of similar features could be generated repeatedly by simply waiting ∼2 min between flashes. After longer intervals, the responses became smaller and even disappeared. The change and eventual loss of the response is almost certainly due to the slow and irreversible loss of modulator, just as occurs in the ep-cones. Data presented here were collected in the interval between 3 and 6 min after achieving whole-cell mode.


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)

Time course of Ca2+-dependent CNG current modulation and control experiments. (Left) Whole-cell membrane current measured at −35-mV holding voltage in a single 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+. 1.5 s after starting the record, at the time indicated by the spike in the record, a Xenon flash uncaged diazo-2, causing a slow increase in the inward current that reached a peak in ∼1.4 s, and then returned towards its starting value. At the peak, membrane conductance was enhanced 2.26-fold. (Right) Currents measured at −35 mV in two different cones, each loaded with a modified electrode-filling solution. Two control conditions were tested: (a) omitting 8Br-cGMP (0 μM 8Br-cGMP), and (b) replacing diazo-2 with 1 mM BAPTA (0 μM Diazo-2). Free Ca2+ and Mg2+ remained at 600 nM and 1 mM, respectively. In the absence of 8Br-cGMP, the net current was outward because CNG channels are closed and current flows through voltage-gated K+ channels in the cone inner segment. The uncaging flash did not cause a current change in either of the control experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: Time course of Ca2+-dependent CNG current modulation and control experiments. (Left) Whole-cell membrane current measured at −35-mV holding voltage in a single 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+. 1.5 s after starting the record, at the time indicated by the spike in the record, a Xenon flash uncaged diazo-2, causing a slow increase in the inward current that reached a peak in ∼1.4 s, and then returned towards its starting value. At the peak, membrane conductance was enhanced 2.26-fold. (Right) Currents measured at −35 mV in two different cones, each loaded with a modified electrode-filling solution. Two control conditions were tested: (a) omitting 8Br-cGMP (0 μM 8Br-cGMP), and (b) replacing diazo-2 with 1 mM BAPTA (0 μM Diazo-2). Free Ca2+ and Mg2+ remained at 600 nM and 1 mM, respectively. In the absence of 8Br-cGMP, the net current was outward because CNG channels are closed and current flows through voltage-gated K+ channels in the cone inner segment. The uncaging flash did not cause a current change in either of the control experiments.
Mentions: We caused a rapid (<< 50 ms, see below) decrease in cytoplasmic Ca2+ by uncaging diazo-2 in intact cone outer segment. In the presence of 15 μM cytoplasmic 8Br-cGMP, the uncaging flash caused a slow increase in current that reached a peak and then slowly returned towards its starting value (Fig. 5). Within the first 6–8 min after establishing whole-cell mode, responses of similar features could be generated repeatedly by simply waiting ∼2 min between flashes. After longer intervals, the responses became smaller and even disappeared. The change and eventual loss of the response is almost certainly due to the slow and irreversible loss of modulator, just as occurs in the ep-cones. Data presented here were collected in the interval between 3 and 6 min after achieving whole-cell mode.

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