Deactivation kinetics of the transduction cascade of vision.
The response of the retinal rod cell to a dim flash lasts less than a second. This phototransduction is mediated by a guanine nucleotide-binding (G) protein cascade in which rhodopsin is the receptor, transducin is the G-protein, and the cGMP-specific phosphodiesterase (PDE) is the effector. Photoexcited rhodopsin activates transducin which in turn activates PDE. For this underlying biochemistry to be kinetically compatible with the photoresponse, both transducin and PDE must be deactivated in subsecond times. We report here direct measurements of their deactivation kinetics. The rate of heat release when transducin and PDE hydrolyze, respectively, GTP and cGMP was measured using time-resolved microcalorimetry. With only GTP present, the heat pulse comes from the activation of transducin and its subsequent deactivation by endogenous GTP hydrolysis. The nonhydrolyzable analog guanine 5'-[gamma-thio]triphosphate was used to distinguish between these two processes: about 40% of the total heat is due to activation. From the time course of the deactivation heat, the active lifetime of transducin is less than 1 s at 22 degrees C. With both GTP and cGMP present, the highly amplified hydrolytic activity of the PDE is responsible for most of the heat produced; its rate of release is directly proportional to the amount of activated PDE. Measurements of this rate at low photoexcitation levels (e.g., 30 molecules of photoexcited rhodopsin per rod) provide much kinetic information about the cascade. Notably, deactivation of the PDE takes 0.6 s (at 23 degrees C) and absolutely requires GTP hydrolysis. This concurs with the subsecond lifetime of active transducin and means that, once GTP hydrolysis has occurred, the hitherto active PDE is quickly inhibited.
Vuong
T M
TM
Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Chabre
M
M
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry; D002417:Cattle; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D010777:Photochemistry; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013816:Thermodynamics; D015293:Transducin / Q000378:metabolism*; D014785:Vision, Ocular
D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin