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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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Determining dark cGMP turnover rate by IBMX application. (A) Saturating flash (downward arrow) is applied to determine zero level of the rod current. After recovery, the cell was instantaneously transferred to the jet of Ringer containing 0.5 mM IBMX (upward arrow). (B, inset) Current changes after IBMX application shown on an expanded time scale. Ordinate, cubic root of the current normalized to the dark level before the jump. Straight line is fit to the central part of the transition curve. Its slope is supposed to yield βdark.
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fig1: Determining dark cGMP turnover rate by IBMX application. (A) Saturating flash (downward arrow) is applied to determine zero level of the rod current. After recovery, the cell was instantaneously transferred to the jet of Ringer containing 0.5 mM IBMX (upward arrow). (B, inset) Current changes after IBMX application shown on an expanded time scale. Ordinate, cubic root of the current normalized to the dark level before the jump. Straight line is fit to the central part of the transition curve. Its slope is supposed to yield βdark.

Mentions: The PDE rate was measured by instant transfer of the cell from normal Ringer solution into the jet of Ringer containing 0.5 mM IBMX. The cell was first stimulated with a saturating flash to determine the zero level of the ROS current in normal Ringer. After recovery of the dark current, the cell was exposed to the IBMX-containing jet that resulted in a fast approximately twofold increase of the current (Fig. 1 A). Plotting\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}(j(t)/j_{d})^{1/n_{cG}}\end{equation*}\end{document}versus time yields the curve whose initial linear part has a slope of 3.4 s−1 (Fig. 1 B). (Here, we assume ncG = 3, as follows from further analysis). Four trials made with this cell gave averaged value for βd = 3.7 ± 0.4 s−1 (mean ± SD). Mean value for six cells yields βd = 3.4 ± 0.8 s−1.


Kinetics of turn-offs of frog rod phototransduction cascade.

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

Determining dark cGMP turnover rate by IBMX application. (A) Saturating flash (downward arrow) is applied to determine zero level of the rod current. After recovery, the cell was instantaneously transferred to the jet of Ringer containing 0.5 mM IBMX (upward arrow). (B, inset) Current changes after IBMX application shown on an expanded time scale. Ordinate, cubic root of the current normalized to the dark level before the jump. Straight line is fit to the central part of the transition curve. Its slope is supposed to yield βdark.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Determining dark cGMP turnover rate by IBMX application. (A) Saturating flash (downward arrow) is applied to determine zero level of the rod current. After recovery, the cell was instantaneously transferred to the jet of Ringer containing 0.5 mM IBMX (upward arrow). (B, inset) Current changes after IBMX application shown on an expanded time scale. Ordinate, cubic root of the current normalized to the dark level before the jump. Straight line is fit to the central part of the transition curve. Its slope is supposed to yield βdark.
Mentions: The PDE rate was measured by instant transfer of the cell from normal Ringer solution into the jet of Ringer containing 0.5 mM IBMX. The cell was first stimulated with a saturating flash to determine the zero level of the ROS current in normal Ringer. After recovery of the dark current, the cell was exposed to the IBMX-containing jet that resulted in a fast approximately twofold increase of the current (Fig. 1 A). Plotting\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}(j(t)/j_{d})^{1/n_{cG}}\end{equation*}\end{document}versus time yields the curve whose initial linear part has a slope of 3.4 s−1 (Fig. 1 B). (Here, we assume ncG = 3, as follows from further analysis). Four trials made with this cell gave averaged value for βd = 3.7 ± 0.4 s−1 (mean ± SD). Mean value for six cells yields βd = 3.4 ± 0.8 s−1.

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