Deactivation kinetics of the transduction cascade of vision.

Abstract

The response of the retinal rod ceil 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'-[y-thioltriphosphate was used to disdngush 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 <1 s at 22C. 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 23YC) 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.

Responses of the phototransduction cascade to dim light.

“…This is reached after a (3). As for k+, it is indeed much larger than kR and is comparable with some theoretical values reported elsewhere (28,29).…”

“…As for the difference between k+ and kR, it is huge since kR cannot be much more than a few s-1. Rokpt, for t < [3] This confirms that the rising slope is directly proportional to the illumination level R(, the proportionality constant being kp, the rate constant for the production of PDE*. Moreover, this rising phase is independent of kR, i.e.…”

1,4‐Dihydropyridines modulate GTP hydrolysis by Go in neuronal membranes

“…From studies on Gk activation of the cardiac K + currents (lktm)) [30] it has been observed that the k,~,, determined from the rate of decay oflk(M) is over 100 min -~, whereas this value is about 2 rain -I in purified preparations of Gi or Go [14,31]. A similar mechanism, where the effector (phosphodiesterase) accelerates the normally slow rate of GTP hydrolysis by a heterotrimeri¢ G-protein (transducin) has recently been proposed [32,33]. In addition, there exists a family of GAPs which stimulate the very slow endogenous GTP hydrolysis by small Q.proteins, including p21 "~ (reviewed in [14]), and may also act as effectors for this G-protein [34].…”

RGS Proteins and Signaling by Heterotrimeric G Proteins

“…Purified G␣ subunits display a measurable intrinsic rate of GTP hydrolysis, but the turnover number in vitro cannot account, in some systems, for the rate at which signaling is terminated in vivo (16,17). Other regulatory processes (such as those mediated by arrestin or phosducin) could be rate-limiting.…”