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Kinetics of turn-offs of frog rod phototransduction cascade.

Astakhova LA, Firsov ML, Govardovskii VI - J. Gen. Physiol. (2008)

Bottom Line: The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs.The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding.This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

View Article: PubMed Central - PubMed

Affiliation: Sechenov Institute for Evolutionary Physiology & Biochemistry, Russian Academy of Sciences, 194223 St. Petersburg, Russia.

ABSTRACT
The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs. The two processes are among the key factors that set the speed and sensitivity of the photoresponse and whose regulation contributes to light adaptation. The aim of this study was to determine time courses of flash-induced PDE activity in frog rods that were dark adapted or subjected to nonsaturating steady background illumination. PDE activity was computed from the responses recorded from solitary rods with the suction pipette technique in Ca(2+)-clamping solution. A flash applied in the dark-adapted state elicits a wave of PDE activity whose rising and decaying phases have characteristic times near 0.5 and 2 seconds, respectively. Nonsaturating steady background shortens both phases roughly to the same extent. The acceleration may exceed fivefold at the backgrounds that suppress approximately 70% of the dark current. The time constant of the process that controls the recovery from super-saturating flashes (so-called dominant time constant) is adaptation independent and, hence, cannot be attributed to either of the processes that shape the main part of the PDE wave. We hypothesize that the dominant time constant in frog rods characterizes arrestin binding to rhodopsin partially inactivated by phosphorylation. A mathematical model of the cascade that considers two-stage rhodopsin quenching and transducin inactivation can mimic experimental PDE activity quite well. The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding. This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

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Testing the efficiency of the Ca2+-clamping procedure. (A) Assessing the stability of dark current in 0 Ca2+, 0 Na+ solution. Zero level corresponds to the dark current in normal Ringer, and outward ROS current is plotted upward. Thin line shows changes in the pipette current after jumping the cell into 0 Ca2+, 0 Na+ solution in darkness. After recovery of the dark current, the jump was repeated and the cell was stimulated with three saturating flashes (2,000 R*, marked by arrows) to determine dark current (bold trace). The dark current rose by 15% between the first and second flashes, and then decreased back. 15% change of the dark current indicates the stability of [Ca2+]in within 1.9% (see Results). Inset shows the method for drawing zero level of the response. 2-s pre-flash stretch and 7-s stretch after the response termination are fitted with a cubic parabola (smooth thin line). (B) Assessing the feedback loop gain. Curve 1 is a response to a 13 R* flash recorded in normal Ringer solution. Curve 2 is a response to the same flash in 0 Ca2+, 0 Na+ solution. Ratio of time integrals of the two curves corrected for saturation provides an estimate of gL (see Eq. 8). (A and B) Two different cells.
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fig2: Testing the efficiency of the Ca2+-clamping procedure. (A) Assessing the stability of dark current in 0 Ca2+, 0 Na+ solution. Zero level corresponds to the dark current in normal Ringer, and outward ROS current is plotted upward. Thin line shows changes in the pipette current after jumping the cell into 0 Ca2+, 0 Na+ solution in darkness. After recovery of the dark current, the jump was repeated and the cell was stimulated with three saturating flashes (2,000 R*, marked by arrows) to determine dark current (bold trace). The dark current rose by 15% between the first and second flashes, and then decreased back. 15% change of the dark current indicates the stability of [Ca2+]in within 1.9% (see Results). Inset shows the method for drawing zero level of the response. 2-s pre-flash stretch and 7-s stretch after the response termination are fitted with a cubic parabola (smooth thin line). (B) Assessing the feedback loop gain. Curve 1 is a response to a 13 R* flash recorded in normal Ringer solution. Curve 2 is a response to the same flash in 0 Ca2+, 0 Na+ solution. Ratio of time integrals of the two curves corrected for saturation provides an estimate of gL (see Eq. 8). (A and B) Two different cells.

Mentions: The thin line in Fig. 2 A shows changes of the suction pipette current in darkness after jumping the cell into the Ca2+-clamping jet. Because in this solution Na+ is substituted by choline+, the dark current is mostly carried by the outward flux of intracellular K+ and changes its sign (see Matthews, 1995, 1996, 1997; Lyubarsky et al., 1996). Shift of the junction potential and inversion of the dark current result in a fast current deviation that is followed with slow changes. The current completely recovers in <1 min after withdrawal to normal Ringer (not depicted). Then the jump is repeated, and saturating flashes are applied at various moments of time to determine the magnitude of the dark current. In this particular cell, the dark current first increased by ≈15% and then returned to its initial level. In other cells, we observed slow changes of the dark current that could be of either direction.


Kinetics of turn-offs of frog rod phototransduction cascade.

Astakhova LA, Firsov ML, Govardovskii VI - J. Gen. Physiol. (2008)

Testing the efficiency of the Ca2+-clamping procedure. (A) Assessing the stability of dark current in 0 Ca2+, 0 Na+ solution. Zero level corresponds to the dark current in normal Ringer, and outward ROS current is plotted upward. Thin line shows changes in the pipette current after jumping the cell into 0 Ca2+, 0 Na+ solution in darkness. After recovery of the dark current, the jump was repeated and the cell was stimulated with three saturating flashes (2,000 R*, marked by arrows) to determine dark current (bold trace). The dark current rose by 15% between the first and second flashes, and then decreased back. 15% change of the dark current indicates the stability of [Ca2+]in within 1.9% (see Results). Inset shows the method for drawing zero level of the response. 2-s pre-flash stretch and 7-s stretch after the response termination are fitted with a cubic parabola (smooth thin line). (B) Assessing the feedback loop gain. Curve 1 is a response to a 13 R* flash recorded in normal Ringer solution. Curve 2 is a response to the same flash in 0 Ca2+, 0 Na+ solution. Ratio of time integrals of the two curves corrected for saturation provides an estimate of gL (see Eq. 8). (A and B) Two different cells.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571975&req=5

fig2: Testing the efficiency of the Ca2+-clamping procedure. (A) Assessing the stability of dark current in 0 Ca2+, 0 Na+ solution. Zero level corresponds to the dark current in normal Ringer, and outward ROS current is plotted upward. Thin line shows changes in the pipette current after jumping the cell into 0 Ca2+, 0 Na+ solution in darkness. After recovery of the dark current, the jump was repeated and the cell was stimulated with three saturating flashes (2,000 R*, marked by arrows) to determine dark current (bold trace). The dark current rose by 15% between the first and second flashes, and then decreased back. 15% change of the dark current indicates the stability of [Ca2+]in within 1.9% (see Results). Inset shows the method for drawing zero level of the response. 2-s pre-flash stretch and 7-s stretch after the response termination are fitted with a cubic parabola (smooth thin line). (B) Assessing the feedback loop gain. Curve 1 is a response to a 13 R* flash recorded in normal Ringer solution. Curve 2 is a response to the same flash in 0 Ca2+, 0 Na+ solution. Ratio of time integrals of the two curves corrected for saturation provides an estimate of gL (see Eq. 8). (A and B) Two different cells.
Mentions: The thin line in Fig. 2 A shows changes of the suction pipette current in darkness after jumping the cell into the Ca2+-clamping jet. Because in this solution Na+ is substituted by choline+, the dark current is mostly carried by the outward flux of intracellular K+ and changes its sign (see Matthews, 1995, 1996, 1997; Lyubarsky et al., 1996). Shift of the junction potential and inversion of the dark current result in a fast current deviation that is followed with slow changes. The current completely recovers in <1 min after withdrawal to normal Ringer (not depicted). Then the jump is repeated, and saturating flashes are applied at various moments of time to determine the magnitude of the dark current. In this particular cell, the dark current first increased by ≈15% and then returned to its initial level. In other cells, we observed slow changes of the dark current that could be of either direction.

Bottom Line: The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs.The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding.This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

View Article: PubMed Central - PubMed

Affiliation: Sechenov Institute for Evolutionary Physiology & Biochemistry, Russian Academy of Sciences, 194223 St. Petersburg, Russia.

ABSTRACT
The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs. The two processes are among the key factors that set the speed and sensitivity of the photoresponse and whose regulation contributes to light adaptation. The aim of this study was to determine time courses of flash-induced PDE activity in frog rods that were dark adapted or subjected to nonsaturating steady background illumination. PDE activity was computed from the responses recorded from solitary rods with the suction pipette technique in Ca(2+)-clamping solution. A flash applied in the dark-adapted state elicits a wave of PDE activity whose rising and decaying phases have characteristic times near 0.5 and 2 seconds, respectively. Nonsaturating steady background shortens both phases roughly to the same extent. The acceleration may exceed fivefold at the backgrounds that suppress approximately 70% of the dark current. The time constant of the process that controls the recovery from super-saturating flashes (so-called dominant time constant) is adaptation independent and, hence, cannot be attributed to either of the processes that shape the main part of the PDE wave. We hypothesize that the dominant time constant in frog rods characterizes arrestin binding to rhodopsin partially inactivated by phosphorylation. A mathematical model of the cascade that considers two-stage rhodopsin quenching and transducin inactivation can mimic experimental PDE activity quite well. The effect of light adaptation on the PDE kinetics can be reproduced in the model by concomitant acceleration on both rhodopsin phosphorylation and transducin turn-off, but not by accelerated arrestin binding. This suggests that not only rhodopsin but also transducin shut-off is under adaptation control.

Show MeSH
Related in: MedlinePlus