1. The Gain of Rod Phototransduction
Ilya B. Leskov, Vadim A. Klenchin, Jason W. Handy, Gary G. Whitlock, Viktor I. Govardovskii, M.Deric Bownds, Trevor D. Lamb, Edward N. Pugh, Vadim Y. Arshavsky 
Neuron 
Volume 27, Issue 3, Pages (September 2000)
DOI: /S (00)

2. Figure 1
Molecular Mechanisms of Activation and Their Contributions to the Gain of Phototransduction
(A) Schematic of the molecular mechanisms underlying activation (modified from Pugh 1999). Absorption of a photon (hν) activates a rhodopsin molecule in the disk membrane to R*. Each R* sequentially contacts numerous molecules of transducin (G), catalyzing their activation to G* through exchange of a bound GDP for a GTP from the cytoplasm. Two G*s can bind to each phosphodiesterase holomer, activating the PDE to PDE**. The activated PDE** hydrolyzes cGMP, reducing its cytoplasmic concentration, and thereby causing closure of cGMP-gated channels in the plasma membrane. The rate constant of cGMP hydrolysis by PDE** is indicated by β, and the rate of cGMP synthesis by guanylyl cyclase (GC) is indicated by α.
(B) Predicted kinetics of protein activation, of cGMP concentration, and of electrical current in response to a single photoisomerization when all inactivation reactions are ignored. Isomerization activates a single R*, which (as inactivation reactions are ignored) simply remains present. Transducin is activated to G* at a constant rate, denoted νG, and G* in turn activates PDE at a rate that we denote as νE in terms of PDE* subunits (solid trace). The cGMP concentration and the circulating electrical current decline from their respective dark levels along trajectories that are approximately parabolic at early times. Both variables are plotted normalized to their dark levels, and the symbol F denotes the fraction of current remaining. For a single photoisomerization, the decline of F(t), which constitutes the response, at early times is given by Rt ≡ 1 − Ft = 1 2A t2, where A is defined as the amplification constant of phototransduction (see text). For purposes of illustration, the parameters employed in this panel were rounded to νG = 160 G* s−1 per R*, νE = 150 PDE* s−1 per R*, A = 0.1 s−2, and ncG = 3.
(C) Estimation of amplification constant from single-photon responses. The continuous trace reproduces the mean single-photon response for the cell in Figure 1C of Whitlock and Lamb 1999 after normalization to the circulating dark current of 42 pA. The dashed parabola plots the predicted “pure activation” single-photon response, Rt = 1 2A t2, using A = 0.08 s−2. The dotted trace allows for inactivation reactions. It plots the mean of the theory curves that were fitted to the individual single-photon responses, according to the method described in fd2 and fd3of Whitlock and Lamb 1999 and yields A = 0.11 s−2.
(D) Estimation of amplification constant from a family of flash responses. Continuous traces are experiments from another toad rod. Dotted curves plot predictions of the “pure activation” LP model (Smith and Lamb 1997, fd5), with a membrane time constant of τm = 20 ms and filtering delay of teff = 28 ms. All seven curves use A = 0.16 s−2, with individual values of Φ ranging from 3 to 18,000 photoisomerizations, determined from the measured intensities and a collecting area of Ac = 22 μm2.
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3. Figure 2
Measurements of Transducin Activation Rate in Suspensions of Frog ROS
Light dependent GTPγS binding to transducin was measured as described in Experimental Procedures. In different experiments, the rhodopsin concentration was varied between 15 and 100 μM, the fraction of rhodopsin isomerized was varied from % to %; the GTPγS concentration in experiments of (A), (B), and (D) was 100 μM. The dark level of GTPγS binding has been subtracted from each data point. Error bars in all panels represent SEM.
(A) The structure of ROS membranes does not influence the rate of transducin activation. The time course of GTPγS binding was monitored in electropermeabilized ROS (black circles, n = 7), ROS passed through a 28G needle (gray circles, n = 5), and homogenized ROS (open circles, n = 2). The lines are linear regressions fitted to the data, whose slopes yield rates of transducin activation (νG) of 72, 71, and 81 G* s−1 per R* for electropermeabilized ROS (unbroken black line), ROS fragments (gray line), and homogenized ROS (dashed line), respectively.
(B) Rates of transducin activation νG in homogenized ROS suspensions containing 15 and 100 μM rhodopsin. The data are averaged from three independent experiments.
(C) Dependence of transducin activation rate νG on GTPγS concentration in homogenized ROS. The data are fitted by a hyperbolic saturation function, yielding a half-saturating GTPγS concentration of 16 μM and a maximal rate of transducin activation of 82 G* s−1 per R*. The data are averaged from three independent experiments.
(D) Rates of transducin activation at 2 and 5 mM Mg2+. The data are averaged from two independent experiments with homogenized ROS.
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4. Figure 5
Determination of the Rate of PDE Activation after a Dim Flash of Light
The dark-adapted preparation of homogenized ROS contained 10 μM rhodopsin, and the reaction mixture contained 400 μM GTPγS and 2 mM [3H]cGMP. The dim flash isomerized % of the rhodopsin, the bright flash 2%. The rate of PDE activation per R* derived from the fit of the parabola (fd4) to the “dim flash” data, after correction for dark hydrolysis (see Experimental Procedures), was 116 PDE* s−1 per R*; ksub was calculated from the slope of the “bright flash” data fit after determining the total amount of PDE as described in Experimental Procedures. The data are from one of eleven similar experiments.
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5. Figure 3
One Activated Transducin Is Required for Activation of One PDE Catalytic Subunit
Light-dependent transducin activation (A) and PDE activation (B) were measured with homogenized ROS containing 21 μM rhodopsin, 1.36 μM transducin, and 60 nM PDE in the presence of 1 μM [35S]GTPγS (or 400 μM [35S]GTPγS in the “bright flash” experiment in [B]) and 4 mM [3H]cGMP. The bright flash isomerized 6.6% of the rhodopsin, causing complete cascade activation within a few milliseconds; the dim flash isomerized % of the rhodopsin. The rate νG of transducin activation derived from the linear regression line fitted to the data of (A) was 2.8 G* s−1 per R*. The maximal PDE activity derived from the line fitted to the “bright flash” data in (B) was 3300 s−1. The PDE* activation rate νE derived from the parabolic fit of the “dim flash” data in (B) (see Experimental Procedures) was 2.7 PDE* s−1 per R*. The data are taken from one of three similar experiments.
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6. Figure 4
Dependence of PDE Activation on Concentration of GTP and GTPγS
(A) PDE activation in ROS was initiated by a flash isomerizing % of the rhodopsin (10 μM) in the presence of 2 mM cGMP and various concentrations of GTP and GTPγS. The reaction was terminated after 3 s, and the amount of hydrolyzed cGMP was determined by the radiotracer assay. Each point has been performed in duplicate; the error bars plot the range of the data. Hyperbolic saturation (Michaelis) functions were fitted, giving the following parameters: for GTP, K1/2 = 24.5 ± 6 μM and Vmax = 144 ± 7 μM cGMP hydrolyzed over 3 s; for GTPγS, K1/2 = 24.8 ± 3.5 μM and Vmax = 172 ± 5 μM cGMP hydrolyzed over 3 s (mean ± SEM). The curves plot this function with the mean parameters.
(B) PDE activation was initiated by a 1 ms flash isomerizing % of the rhodopsin at time zero in the presence of 10 mM cGMP and 250 μM GTP or GTPγS, and cGMP hydrolysis was monitored with the pH assay. The total rhodopsin concentration was 3 μM; the reaction volume was 100 μl, temperature 24°C. Each trace is the average of at least five records. The maximal hydrolytic velocity, obtained with a flash that isomerized 10% of the rhodopsin, was 34 μM s−1. The thickened dark gray curve for the trace with GTPγS plots fd4, with the factor 1/2R*νE ksub set to 0.25 μM s−2. The light gray curve drawn through the trace obtained with GTP allows for first order inactivation of transducin at a rate of 0.09 s−1: this curve has the formula ΔcGt = R* νE ksubτE t − τE 1 − exp −t τE, with τE = 11.5 s = s and reduces to fd4 when t is small. Given R* = 3 × 10−6 μM and taking ksub = 2200 s−1, the relation 1 2R* νE ksub = 0.25 μM s−2 can be solved to obtain νE = 76 PDE* s−1 per R*. A series of ten such experiments using GTP, GTPγS, or GppNHp as cofactors yielded νE = 84 ± 40 PDE* s−1 per R* (mean ± SD).
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7. Figure 6
Dependence of the Apparent Km of the PDE for cGMP on the Fraction of Activated PDE in ROS Membranes
(A) Schematic of how diffusion with hydrolysis influences the cGMP concentration profile in the intradiscal space at a high (upper half) or low (lower half) degree of PDE activation. The red ellipsoids represent PDE**s, and the white ellipsoids inactive enzyme.
(B) Scanning electron micrograph of large frog ROS fragments with essentially unaltered macroscopic structure. The average ROS diameter is 8 μm.
(C) Scanning electron micrograph of maximally disrupted ROS preparation used in the experiments from (D).
(D) Measurements of apparent Km of the PDE at various levels of PDE activation. All experiments were performed in suspensions of completely disrupted ROS containing 6 μM rhodopsin. Each point on the graph is derived from a separate experiment in which a constant concentration of GTPγS (in the range 2 nM to 10 μM) was used to set the level of PDE activation; the time course of cGMP hydrolysis was measured at five to seven different concentrations of [3H]cGMP, as described in Experimental Procedures. The Km value was estimated by fitting the data with the Michaelis-Menten equation; error bars represent SEM. For each experiment, the estimated value of apparent Km is plotted as a function of the maximal hydrolytic activity observed at that concentration of GTPγS (PDE* ksub; see Appendix 1). The upper x axis represents the percentage of PDE activation in each experiment with respect to the maximum value obtained from an experiment where all the PDE was activated. For the three leftmost points, obtained under conditions where the basal PDE activity was comparable with the GTPγS-stimulated activity, we measured the time course of cGMP hydrolysis at each cGMP concentration with and without 2 nM GTPγS; we then performed point-by-point subtraction of the basal activity from the activity with GTPγS and used the resulting values (representing the activity of transducin-activated PDE only) in the fitting of the Michaelis function. The curves represent theoretical predictions of the “diffusion with hydrolysis” theory for three different hypotheses about the particles' structure (see Appendix 1 for details). The solid dark gray line shows the prediction for spherical particles containing non-oriented membrane structure and surrounded by an additional diffusion barrier. The fit was computed with fd5fd6fd7, assuming Dr = 225 μm2 s−1 and B = 170 μm s−1. The dashed line is the solution for the same particles but in the absence of a surrounding diffusion barrier B = ∞ and with Dr = 45 μm2 s−1. The light gray line is the solution for cylindrical particles without a diffusion barrier and with Dr = 50 μm2 s−1. The size distribution of the ROS particles used in the computations is shown in the inset.
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