Trevor D. Lamb

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)

A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors.

1. We have undertaken a theoretical analysis of the steps contributing to the phototransduction cascade in vertebrate photoreceptors. We have explicitly considered only the activation steps, i.e. we have not dealt with the inactivation reactions. 2. From the theoretical analysis we conclude that a single photoisomerization leads to activation of the phosphodiesterase (PDE) with a time course which approximates a delayed ramp; the delay is contributed by several short first‐order delay stages. 3. We derive a method for extracting the time course of PDE activation from the measured electrical response, and we apply this method to recordings of the photoresponse from salamander rods. The results confirm the prediction that the time course of PDE activation is a delayed ramp, with slope proportional to light intensity; the initial delay is about 10‐20 ms. 4. We derive approximate analytical solutions for the electrical response of the photoreceptor to light, both for bright flashes (isotropic conditions) and for single photons (involving longitudinal diffusion of cyclic GMP in the outer segment). The response to a brief flash is predicted to follow a delayed Gaussian function of time, i.e. after an initial short delay the response should begin rising in proportion to t2. Further, the response‐intensity relation is predicted to obey an exponential saturation. 5. These predictions are compared with experiment, and it is shown that the rising phase of the flash response is accurately described over a very wide range of intensities. We conclude that the model provides a comprehensive description of the activation steps of phototransduction at a molecular level.

The electrical response of turtle cones to flashes and steps of light

1. The linear response of turtle cones to weak flashes or steps of light was usually well fitted by equations based on a chain of six or seven reactions with time constants varying over about a 6‐fold range.

Molecular mechanisms of vertebrate photoreceptor light adaptation

An important recent advance in the understanding of vertebrate photoreceptor light adaptation has come from the discovery that as many as eight distinct molecular mechanisms may be involved, and the realization that one of the principal mechanisms is not dependent on calcium. Quantitative analysis of these mechanisms is providing new insights into the nature of rod photoreceptor light adaptation.

Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup.

Charles Darwin appreciated the conceptual difficulty in accepting that an organ as wonderful as the vertebrate eye could have evolved through natural selection. He reasoned that if appropriate gradations could be found that were useful to the animal and were inherited, then the apparent difficulty would be overcome. Here, we review a wide range of findings that capture glimpses of the gradations that appear to have occurred during eye evolution, and provide a scenario for the unseen steps that have led to the emergence of the vertebrate eye.

Spatial spread of activation and background desensitization in toad rod outer segments.

1. The spread of activation and background desensitization in rods was studied by recording membrane current from single outer segments in pieces of isolated toad retina.

Reconstruction of the electrical responses of turtle cones to flashes and steps of light

1. Theoretical equations which predict the electrical response of turtle cones to a wide range of light stimuli are developed from the experiments described in previous papers.

The Gain of Rod Phototransduction: Reconciliation of Biochemical and Electrophysiological Measurements

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.

Cytoplasmic calcium as the messenger for light adaptation in salamander rods.

1. In order to study the role of cytoplasmic calcium concentration (Ca2+i) in rod photoreceptor light adaptation, we have attempted to prevent light‐induced changes in Ca2+i by minimizing calcium fluxes across the outer segment plasma membrane. This was achieved by exposing the outer segment to a low‐Ca2+, 0‐Na+ solution, in which sodium was replaced with either guanidinium or lithium and the external calcium concentration (Ca2+o) was reduced to micromolar levels. 2. With guanidinium and 1‐3 microM‐Ca2+o, the circulating current in darkness was maintained for a period of at least 15 s, consistent with approximate stability of Ca2+i. With Li+ rather than guanidinium most of the initial current was suppressed, but the residual current was again relatively stable. 3. During prolonged exposures (greater than 30 s) to low‐Ca2+, 0‐Na+ solution followed by dim illumination, the circulating current did not remain constant but slowly increased. Incorporation of calcium buffer into the cytoplasm greatly reduced the rate of change of current, consistent with the idea that the increase arose from a gradual decrease in Ca2+i. 4. Light responses of rods exposed to low‐Ca2+, 0‐Na+ solution in darkness were altered in a characteristic manner. Although the initial rising phase of the light response was little changed, the peak amplitude of the response was larger and occurred later, and the response decayed more slowly than in control. The response‐intensity relation was steepened and was shifted towards lower intensities both for flashes and for steps of light. The normal sag in the response to steps disappeared, and the waveform of the step response could be predicted to a close approximation from the integral of the dim flash response. 5. Presentation of background illumination in Ringer solution produced a marked acceleration of the response to a subsequent bright flash. No such acceleration was observed if the background was given in low‐Ca2+, 0‐Na+ solution. 6. The results described in paragraphs 4 and 5 indicate that, under conditions expected to minimize changes in Ca2+i, all manifestations of light adaptation disappear, and the rod simply sums the effects of incident photons with an invariant integration time. 7. Exposure of a light‐adapted rod to low‐Ca2+, 0‐Na+ solution altered the responses to superimposed test flashes in much the same way as for rods in darkness. The initial rising phases in low‐Ca2+, 0‐Na+ solution were unchanged, but the responses were larger, reached peak later and decayed more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of temperature changes on toad rod photocurrents.

Rod current responses were measured over the range 5‐30 degrees C. Following a rapid decrease in temperature the amplitude of the dark current decreased without detectable delay (less than 3 s). Over a period of several minutes the amplitude of the dark current sometimes relaxed slightly towards its previous value. The rapid change cannot be accounted for simply by altered activity of the sodium pump and instead indicates that the conductance of the outer segment in darkness changes with temperature. Over the range 10‐30 degrees C the amplitude of the dark current increased approximately linearly with temperature, and the straight line of best fit extrapolated to zero current at about 5 degrees C. The few points available below 10 degrees C indicated that the relationship flattened out, but this could not be investigated properly. The kinetics of responses to dim flashes accelerated with a Q10 of about 2.2, and were well described by an Arrhenius plot with an activation energy of 13.8 kcal mol‐1 (HEPES Ringer solution). The time course of recovery of dark current following a saturating flash showed a similar temperature dependence to that of the dim flash kinetics. A simple explanation of the previous two findings is that the delays determining the time course of responses to both dim and bright flashes are largely determined by the fluidity of the disk membrane. The sensitivity to dim flashes had a broad peak at about 22 degrees C, decreasing at both lower and higher temperatures. The relative sensitivity to long wave‐length light increased slightly with temperature. The sensitivity at 700 nm relative to that at 500 nm increased by 0.225 log10 units (1.68 times) upon a temperature increase from 11.5 to 29.3 degrees C (from approximately ‐5.0 log10 units to approximately ‐4.8 log10 units). This change appears to be approximately what would be expected theoretically.