Limits...
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.

Show MeSH

Related in: MedlinePlus

Modeling PDE(t) waveform with the three-stage quenching model described in the Appendix. Experimental responses from Fig. 5 B are fitted with Eqs. 3a–6a. Smooth solid lines show least-square fits obtained with the MathCad genfit procedure. Fractional activity of phosphorylated yet arrestin-unbound rhodopsin is fixed at a = 0.09. τA = 1.6 s follows from Pepperberg-type analysis on this cell. Free parameters are τR, τE, and the product of R* · νRE · βsub that determines the response amplitude. The fits yield τR = 0.96 s and τE = 1.44 s in the dark-adapted state (A). Corresponding values in light adaptation are 0.47, 0.39 s (background blocking 27% dark current, B), and 0.29, 0.18 s (42% blockade, C). Dashed lines show the fits obtained by manually varying τR and τA while keeping τE = 1.44 s at its dark-adapted level. The rising phase of the light-adapted response can only be fitted.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Modeling PDE(t) waveform with the three-stage quenching model described in the Appendix. Experimental responses from Fig. 5 B are fitted with Eqs. 3a–6a. Smooth solid lines show least-square fits obtained with the MathCad genfit procedure. Fractional activity of phosphorylated yet arrestin-unbound rhodopsin is fixed at a = 0.09. τA = 1.6 s follows from Pepperberg-type analysis on this cell. Free parameters are τR, τE, and the product of R* · νRE · βsub that determines the response amplitude. The fits yield τR = 0.96 s and τE = 1.44 s in the dark-adapted state (A). Corresponding values in light adaptation are 0.47, 0.39 s (background blocking 27% dark current, B), and 0.29, 0.18 s (42% blockade, C). Dashed lines show the fits obtained by manually varying τR and τA while keeping τE = 1.44 s at its dark-adapted level. The rising phase of the light-adapted response can only be fitted.

Mentions: One can argue that the stability of dark [Ca2+]in in the clamping solution does not guarantee its constancy during flash responses. However, free Ca2+ level in the clamping solution is in nanomolar range, so both passive Ca2+ fluxes through the light-sensitive channels and active efflux via the exchanger are greatly diminished compared with their values in normal Ringer. Therefore, possible misbalance of the fluxes due to the transient closure of the channels cannot substantially affect [Ca2+]in. The fluorescence data of Matthews and Fain (2001) support this notion. The authors show that instantaneous complete closure of the light-sensitive channels in a salamander rod bathed in Ca-clamping solution results in the [Ca2+]in decline with the time constant of ≈100 s. This is over two orders of magnitude slower than characteristic times of light-adapted responses that lie in a sub-second range (our Figs. 5, 6, and 10).


Kinetics of turn-offs of frog rod phototransduction cascade.

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

Modeling PDE(t) waveform with the three-stage quenching model described in the Appendix. Experimental responses from Fig. 5 B are fitted with Eqs. 3a–6a. Smooth solid lines show least-square fits obtained with the MathCad genfit procedure. Fractional activity of phosphorylated yet arrestin-unbound rhodopsin is fixed at a = 0.09. τA = 1.6 s follows from Pepperberg-type analysis on this cell. Free parameters are τR, τE, and the product of R* · νRE · βsub that determines the response amplitude. The fits yield τR = 0.96 s and τE = 1.44 s in the dark-adapted state (A). Corresponding values in light adaptation are 0.47, 0.39 s (background blocking 27% dark current, B), and 0.29, 0.18 s (42% blockade, C). Dashed lines show the fits obtained by manually varying τR and τA while keeping τE = 1.44 s at its dark-adapted level. The rising phase of the light-adapted response can only be fitted.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Modeling PDE(t) waveform with the three-stage quenching model described in the Appendix. Experimental responses from Fig. 5 B are fitted with Eqs. 3a–6a. Smooth solid lines show least-square fits obtained with the MathCad genfit procedure. Fractional activity of phosphorylated yet arrestin-unbound rhodopsin is fixed at a = 0.09. τA = 1.6 s follows from Pepperberg-type analysis on this cell. Free parameters are τR, τE, and the product of R* · νRE · βsub that determines the response amplitude. The fits yield τR = 0.96 s and τE = 1.44 s in the dark-adapted state (A). Corresponding values in light adaptation are 0.47, 0.39 s (background blocking 27% dark current, B), and 0.29, 0.18 s (42% blockade, C). Dashed lines show the fits obtained by manually varying τR and τA while keeping τE = 1.44 s at its dark-adapted level. The rising phase of the light-adapted response can only be fitted.
Mentions: One can argue that the stability of dark [Ca2+]in in the clamping solution does not guarantee its constancy during flash responses. However, free Ca2+ level in the clamping solution is in nanomolar range, so both passive Ca2+ fluxes through the light-sensitive channels and active efflux via the exchanger are greatly diminished compared with their values in normal Ringer. Therefore, possible misbalance of the fluxes due to the transient closure of the channels cannot substantially affect [Ca2+]in. The fluorescence data of Matthews and Fain (2001) support this notion. The authors show that instantaneous complete closure of the light-sensitive channels in a salamander rod bathed in Ca-clamping solution results in the [Ca2+]in decline with the time constant of ≈100 s. This is over two orders of magnitude slower than characteristic times of light-adapted responses that lie in a sub-second range (our Figs. 5, 6, and 10).

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