The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse

We investigated the kinetics and sensitivity of photocurrent responses of s alamander rods, both in darkness and during adaptation to steady background s producing 20-3,000 photoisomerizations per second, using suction pipet re cordings. The most intense backgrounds suppressed 80% of the circulating da rk current and decreased the flash sensitivity similar to 30-fold. To inves tigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the b ackground. The fractional responses to flashes of any fixed intensity began rising along a common trajectory regardless of background intensity. We in terpret these invariant initial trajectories to indicate that, at these bac kground intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different in tensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier t he time of peeling off. This behavior is consistent with a background-induc ed reduction in the effective lifetime of at least one of the three major i ntegrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) th e inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha -subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on a verage to similar to 20 times its dark-adapted value; and our theoretical a nalysis indicates that this increase in beta is the primary mechanism under lying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.