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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 changes in membrane current and cytoplasmic Ca2+ concentration in a cone outer segment caused by uncaging diazo-2. The single cone was 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 double-ended arrowhead in the record, a Xenon flash uncaged diazo-2. (Top) The fluorescence excited at 380 nm and emitted in the 410–600-nm range. A rise in fluorescence intensity indicates that uncaging diazo-2 caused a rapid initial decrease in Ca2+ concentration, which then slowly drifted back towards its starting value. (○) Data in counts per 50-ms bin. The continuous line depicts the analytical function () that optimally describes the time course of Ca2+ concentration change. (Bottom) The whole-cell membrane current at −35-mV holding voltage measured simultaneously. The sudden Ca2+ sequestration caused a slow 3.75-fold increase in membrane conductance that reached to a peak in ∼1.35 s.
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Figure 6: Time course of changes in membrane current and cytoplasmic Ca2+ concentration in a cone outer segment caused by uncaging diazo-2. The single cone was 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 double-ended arrowhead in the record, a Xenon flash uncaged diazo-2. (Top) The fluorescence excited at 380 nm and emitted in the 410–600-nm range. A rise in fluorescence intensity indicates that uncaging diazo-2 caused a rapid initial decrease in Ca2+ concentration, which then slowly drifted back towards its starting value. (○) Data in counts per 50-ms bin. The continuous line depicts the analytical function () that optimally describes the time course of Ca2+ concentration change. (Bottom) The whole-cell membrane current at −35-mV holding voltage measured simultaneously. The sudden Ca2+ sequestration caused a slow 3.75-fold increase in membrane conductance that reached to a peak in ∼1.35 s.

Mentions: To test that the flash-generated current is indeed caused by CNG channel modulation, we tested the response to uncaging flashes in cones loaded with solutions modified as follows: (a) free of 8Br-cGMP (to test whether a cyclic nucleotide–independent current is activated by lowering Ca2+) and (b) diazo-2 replaced by 1 mM BAPTA (to test whether light alone, in the absence of Ca2+ changes can cause channel activation). Typical results of these tests are shown in Fig. 5. In the absence of 8Br-cGMP, the holding current at −35 mV was outward, 21.1 ± 12.3 pA (n = 35), and reflects the activity of voltage-dependent K+ channels in the inner segment. Uncaging flashes caused a decrease in Ca2+, but failed to generate changes in current. In the absence of diazo-2, 15 μM 8Br-cGMP sustained a stationary inward current and light flashes also failed to change the current. The same occurred in every cell we tested (8Br-cGMP free, n = 4; diazo-2 free, n = 5). Also, adding 10 mM BAPTA to the electrode-filling solution blocked the flash-generated current change (data not shown). Examination of the data, however, shows a large and very fast spike at the moment of flash (Fig. 5Fig. 6Fig. 7). The spikes are inevitable artifacts caused by the high-energy discharge of the capacitors in the Xe flash instrument. Thus, control experiments indicate that the slow change in current generated by uncaging diazo-2 arise specifically from activation of cyclic nucleotide–gated current caused by lowering cytoplasmic Ca2+.


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 changes in membrane current and cytoplasmic Ca2+ concentration in a cone outer segment caused by uncaging diazo-2. The single cone was 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 double-ended arrowhead in the record, a Xenon flash uncaged diazo-2. (Top) The fluorescence excited at 380 nm and emitted in the 410–600-nm range. A rise in fluorescence intensity indicates that uncaging diazo-2 caused a rapid initial decrease in Ca2+ concentration, which then slowly drifted back towards its starting value. (○) Data in counts per 50-ms bin. The continuous line depicts the analytical function () that optimally describes the time course of Ca2+ concentration change. (Bottom) The whole-cell membrane current at −35-mV holding voltage measured simultaneously. The sudden Ca2+ sequestration caused a slow 3.75-fold increase in membrane conductance that reached to a peak in ∼1.35 s.
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Figure 6: Time course of changes in membrane current and cytoplasmic Ca2+ concentration in a cone outer segment caused by uncaging diazo-2. The single cone was 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 double-ended arrowhead in the record, a Xenon flash uncaged diazo-2. (Top) The fluorescence excited at 380 nm and emitted in the 410–600-nm range. A rise in fluorescence intensity indicates that uncaging diazo-2 caused a rapid initial decrease in Ca2+ concentration, which then slowly drifted back towards its starting value. (○) Data in counts per 50-ms bin. The continuous line depicts the analytical function () that optimally describes the time course of Ca2+ concentration change. (Bottom) The whole-cell membrane current at −35-mV holding voltage measured simultaneously. The sudden Ca2+ sequestration caused a slow 3.75-fold increase in membrane conductance that reached to a peak in ∼1.35 s.
Mentions: To test that the flash-generated current is indeed caused by CNG channel modulation, we tested the response to uncaging flashes in cones loaded with solutions modified as follows: (a) free of 8Br-cGMP (to test whether a cyclic nucleotide–independent current is activated by lowering Ca2+) and (b) diazo-2 replaced by 1 mM BAPTA (to test whether light alone, in the absence of Ca2+ changes can cause channel activation). Typical results of these tests are shown in Fig. 5. In the absence of 8Br-cGMP, the holding current at −35 mV was outward, 21.1 ± 12.3 pA (n = 35), and reflects the activity of voltage-dependent K+ channels in the inner segment. Uncaging flashes caused a decrease in Ca2+, but failed to generate changes in current. In the absence of diazo-2, 15 μM 8Br-cGMP sustained a stationary inward current and light flashes also failed to change the current. The same occurred in every cell we tested (8Br-cGMP free, n = 4; diazo-2 free, n = 5). Also, adding 10 mM BAPTA to the electrode-filling solution blocked the flash-generated current change (data not shown). Examination of the data, however, shows a large and very fast spike at the moment of flash (Fig. 5Fig. 6Fig. 7). The spikes are inevitable artifacts caused by the high-energy discharge of the capacitors in the Xe flash instrument. Thus, control experiments indicate that the slow change in current generated by uncaging diazo-2 arise specifically from activation of cyclic nucleotide–gated current caused by lowering cytoplasmic Ca2+.

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