Acceleration of key reactions as a strategy to elucidate the rate-limiting chemistry underlying phototransduction inactivation.

PURPOSE

A reconstituted system was used to establish a strategy to determine the rate-limiting chemistry responsible for recovery of the dim-flash response in rod photoreceptors.

METHODS

A general approach for identifying the rate-limiting step in a series of reactions is to evaluate the consequences of accelerating each step separately, while monitoring the rate of formation of the end product of the series. This strategy was applied to the reactions involved in quenching phototransduction in bovine rod outer segment (bROS) homogenates. The decay of photoactivated rhodopsin (R*) and inactivation of transducin by guanosine triphosphate (GTP) hydrolysis are the leading candidates for limiting the rate of phototransduction turn-off. These reactions were accelerated separately and together by adding hydroxylamine and/or the regulator of G-protein signaling-9 catalytic domain (RGS9d) while monitoring phosphodiesterase (PDE) activity triggered by a pulse of light in bROS homogenates.

RESULTS

PDE activity in bROS homogenates triggered by a flash of light returned to its dark value with a rate constant of 0.087 +/- 0.002 seconds in this system. The rate of PDE recovery increased to 0.11 +/- 0.004 seconds when R* decay was accelerated with 10 to 50 mM hydroxylamine, suggesting that R* inactivation limits the rate of phototransduction turn-off under these conditions. Adding both hydroxylamine and RGS9d, a factor that accelerates transducin inactivation, increased the rate of PDE decay even further. RGS9d had no effect on PDE recovery kinetics unless quenching of R* was also accelerated.

CONCLUSIONS

Under in vitro conditions in bROS homogenates, the quenching of R* normally limits the rate of phototransduction shut-off. If R* decay is accelerated, inactivation of transducin by GTP hydrolysis becomes rate limiting. This study offers a general approach that could be used to investigate the rate-limiting chemistry of phototransduction turn-off in vivo.