Amplification and kinetics of the activation steps in phototransduction.

We can summarize our investigation of amplification in the activation steps of vertebrate phototransduction as follows. (1) A theoretical analysis of the activation steps of the cGMP cascade shows that after a brief flash of phi photoisomerizations the number of activated PDE molecules should rise as a delayed ramp with slope proportional to phi, and that, as a consequence, the cGMP-activated current should decay as a delayed Gaussian function of time (Eqn. 20). (i) Early in the response to a flash, the normalized response R(t) can be approximated as rising as 1/2 phi At2 (after a short delay), where A is the amplification constant characteristic of the individual photoreceptor. (ii) The delayed ramp behavior of PDE activation and the consequent decline of current in the form of the delayed Gaussian are confirmed by experiments in a variety of photoreceptors; the analysis thus yields estimates of the amplification constant from these diverse photoreceptors. (iii) Eqn. 20 further predicts that the response-intensity relation at any fixed time should saturate exponentially, as has been found experimentally. (2) The amplification constant A can be expressed as the product of amplification factors contributed by the individual activation steps of phototransduction, i.e., A = nu RG cGP beta sub n (Eqns. 9 and 21), where (i) nu RG is the rate of G* production per Rh*; (ii) cGP is the efficiency of the coupling between G* production and PDE* production; (iii) beta sub is the increment in hydrolytic rate constant produced by one PDE*, i.e., a single activated catalytic subunit of PDE; and (iv) n is the Hill coefficient of opening of the cGMP-activated channels. (3) The amplification factor beta sub includes the ratio kcat/Km, which characterizes the hydrolytic activity of the PDE in vivo where cG << Km. Two different analyses based upon photocurrents were developed which provide lower bounds for kcat/Km in vivo; these analyses establish that kcat/Km probably exceeds 10(7) M-1 s-1 (and is likely to be higher) in both amphibian and mammalian rods. Few biochemical studies (other than those using trypsin activation) have yielded such high values. A likely explanation of many of the relatively low biochemical estimates of kcat/Km is that Km may have been overestimated by a factor of about 4 in preparations in which stacks of disks are left intact, due to diffusion with hydrolysis in the stacks.(ABSTRACT TRUNCATED AT 400 WORDS)