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

Abstract

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background 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 intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the 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 average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.

Figure 1

History of the responses of the principal rods of the experiments. Each point represents the response to a saturating flash presented to a rod after two or more minutes of dark adaptation. (A) Histories of rods a and c of Table presented separately from those of the other rods (B) for clarity. The gaps in the response histories represent periods when the rods were exposed to background lights or to IBMX. (B) Histories of the seven additional principal rods.

Figure 2

Photocurrent response families of a salamander rod, obtained under four conditions of adaptation: in darkness (A) and in the presence of steady background lights of the indicated intensities (I) in photoisomerizations per second (B–D). Each of the four families presents averaged responses r(t) to the same sequence of flashes, estimated to produce Φ = 260, 830, 2,600, 8,300, 26,000, 83,000, and 260,000 photoisomerizations per flash. Each trace is averaged from at least 5 responses, but up to 30 responses were averaged to produce the responses with smallest amplitudes. Rod a of Table . The additional scales at the right of B provide graphical definitions of the fractional cGMP-activated current J _cG(t) and response R _cG(t), according to and .

Figure 3

Fractional responses of a rod under six conditions of adaptation: dark adapted, and in the presence of steady backgrounds producing I = 19, 60, 190, 600, and 1,900 photoisomerizations per second. The responses are expressed as a fraction of the maximal cGMP-activated current in each adaptational condition; i.e., as R _cG(t) ≈ r _tot(t) / j _cG(I), estimated according to and the scale in Fig. 2 B. Each panel illustrates responses to flashes of a single intensity, at the indicated value of Φ, in photoisomerizations; responses to the more intense flashes (right column) are presented on a shorter time scale, and the responses to Φ = 830 photoisomerizations are repeated on both scales (E and E′). Each trace shown is the average from at least 5 trials, with up to 30 trials being averaged for the smallest amplitude traces. Rod b of Table .

Figure 4

Fractional response per photoisomerization, R(t)/Φ, for dim flashes presented in darkness and on five backgrounds, for the rod of Fig. 3 (Table , rod b). In each adaptational state, the mean dim flash response per photoisomerization has been calculated, using only those flash intensities that elicited a relatively small signal; i.e., those with R(t _peak) ≤ 30%. The traces have broadly the same form as those in Fig. 3 B, but have been averaged from a larger number of trials, and from test flashes of >1 intensity. The background intensities were I = 0, 19, 60, 190, 600, and 1,900 photoisomerizations per second, and the respective numbers of flash responses averaged were 51, 37, 30, 30, 43, and 20. The smooth trace (gray) was computed with the pure activation model (Lamb and Pugh 1992, modified to include the membrane time constant, τ_m according to of Smith and Lamb 1997), with A = 0.063 s⁻², τ_m = 20 ms, and t_eff = 20 ms.

Figure 5

Effects of light adaptation on fractional response amplitude and recovery time for saturating flashes. (A) The cell of Fig. 2 (Table , rod a); and (B) the cell of Fig. 3 (Table , rod b). For each cell, a family of responses was obtained for flash intensities ranging from 10 to at least 10,000 photoisomerizations, presented in darkness or on backgrounds of at least three intensities. From these families, two parameters were measured and plotted: (1) the fractional response amplitude, R(t _peak) = r(t _peak)/j(I), measured at the peak, is plotted in the top left of both panels, using the left ordinate scale; and (2) the time to 50% recovery, T ₅₀, (for each flash sufficiently bright to saturate the response) is plotted in the bottom right of both panels, using the right ordinate. Each symbol shape represents a different background intensity; closed symbols are for 20 μs xenon flashes; open symbols are for 22 ms flashes from the shuttered beam. (A) •, ○, darkness; ▴, ▾, and ♦, I = 260, 810, and 2,600 photoisomerizations per second, respectively; (B) ▪, darkness; ▴, ▾, ♦, ▪, and ▴, I = 19, 60, 190, 600, and 1,900 photoisomerizations per second, respectively, from the top down. All points in the four plots represent averages derived from 2–15 individual responses; error bars have been omitted for clarity. For the points in the recovery half-time plots, the average SDs for the four adaptation conditions of A are 0.15, 0.28, 0.09, and 0.13 s; for the six adaptation conditions of B, they are 0.18, 0.17, 0.13, 0.12, and 0.08 s.

Figure 6

Dependence of steady circulating current and flash sensitivity of salamander rods on background intensity. (A) Relative circulating current in the steady state, J _rel(I) = j(I)/j _Dark, where j(I) is the steady current and j _Dark is the dark current. (B) Relative sensitivity, defined as s _rel(I) = s(I)/s _Dark, where s(I) is the absolute sensitivity and s _Dark is its dark-adapted value. (C) Relative fractional sensitivity, defined as S _rel(I) = (s(I)/s _Dark)/J _rel(I); see . Symbols from the present investigation are identified in Table . Symbols from three previous studies are: ○, Hodgkin and Nunn, 1988; □, Matthews et al. 1988, average of seven cells; ⋄, Koutalos et al. 1995b, average of six cells. The curves are the predictions of the model set out in the , using the parameters of the “standard” rod listed in Table . The curve in A was obtained from Eq. B7 in . The curves in B and C were obtained by simulating (at a range of background intensities) the response to a dim flash, and determining its peak amplitude.

Figure 8

Estimation of the steady rate constant β(I) of cGMP turnover, by the fitting of simulated responses to the IBMX-jump experiments. The top row (A and B) presents the averaged traces from Fig. 7 B (Table , rod d); the middle row (C and D) presents equivalent results from the rod of Fig. 2 (Table , rod a). The left and right columns show simulations for n _cG = 2 and 3, respectively. The procedure for fitting is described in the materials and methods. In brief, the response of the model in the was evaluated, in response to a sudden reduction in PDE activity from its initial steady level. This calculation was repeated over a range of values of steady intensity (I) to find the intensity and, hence, the value of β(I), that minimized the sum-of-squares error from each experimental trace over the time window 0–200 ms. The parameters of the model were set to those measured for the respective cells (Table , rods d and a), together with the remaining parameters for the “standard” rod in Table (except that Rec _tot was set to 20 μM for rod a, in C and D). The value of β_Dark in each panel was determined by applying the above fitting to the IBMX-jump data obtained under dark-adapted conditions. The dotted curves show the predictions of the model when the Ca²⁺ concentration was clamped at the level predicted by the model for the respective light-adapted state; these curves are well approximated by J@t≈1+It ^ncG. (E) Dependence of the RMS error on β(I), for the two cells, calculated with n _cG = 2, denoted with the same symbols as in Table : •, for A; , for C. This plot gives an indication of the precision with which β(I) is determined by this method.

Figure 9

Steady-state rate constant, β(I), of cGMP hydrolysis as a function of background intensity. (A) Estimates of β(I) obtained using the derivative method of Fig. 7 and are plotted (mean ± SD of repeated measurements). In addition, estimates obtained by the “fitting” method of Fig. 8 are also shown at intensities of I > 800 photoisomerizations per second, linked by vertical arrows to the corresponding points obtained with the derivative method. In all cases, it has been assumed that n _cG = 2. Closed symbols are from our own experiments and are identified in Table . Open symbols are from two other studies that used the IBMX-jump or Li⁺-jump methods. ȯ, Hodgkin and Nunn 1988 Li⁺-jump experiments: single cell from their Figure 2, plus β_Dark from Table , with mean ± SD for n = 17 rods. ▿, Cornwall and Fain 1994 IBMX-jump for a single cell, their Figure 6. ▵, Cornwall and Fain 1994 Li⁺-jump for a single rod, their Figure 2. Cornwall and Fain assumed n _cG = 3 in their analysis, and we have adjusted their data for n _cG = 2. The solid curve plots the steady-state expression for β(I) as a function of I given by Eq. B7, with β_Dark = 1.0 s⁻¹, A = 0.08 s⁻², n _cG = 2, τ_E = 1.6 s, and k _R,max = 12 s⁻¹ (which yields τ_{R, Dark} = 0.35 s). The two dashed curves are the same, except for β_Dark = 0.5 and 1.5 s⁻¹. The two dotted curves were derived with of Koutalos et al. 1995b for Ca²⁺ _{i, Dark} = 200 nM (bottom dotted trace) and 500 nM (top dotted trace).

Figure 7

Method of estimation of the steady-state PDE rate constant of cGMP hydrolysis. (A) Exposure to 500 μM IBMX, on a slow time-base, showing seven repeated trials under dark-adapted conditions. In A and B, the fractional circulating current, J(t) = j(t)/j(I), has been plotted, determined by the following protocol. The steady circulating current j(I) was measured, by delivering an intense flash (Φ = 8,600 photoisomerizations) at the first arrow. For this rod, we obtained j _Dark = 34 pA under the dark-adapted conditions of A. After a delay of 90 s, to allow complete recovery, a rapid translation of the chamber moved the laminar boundary of the flowing solutions across the rod (defined as time zero), exposing the outer segment to Ringer's solution containing 500 μM IBMX, and eliciting a rapid increase in circulating current because of inhibition of phosphodiesterase activity. A flash of the same intensity as before was then delivered under manual control (arrows), and ∼1 s later the rod was returned to control Ringer's solution. The rod was allowed to recover for several minutes, and the IBMX exposure and flash were repeated for a total of seven times. After the series of jumps, the response amplitude was again measured, and found to be 32.5 pA. Two of the seven records were obtained in the absence of infrared illumination, and are indistinguishable from the other five. The small “bumps” in the response tails occur at the time of return to control Ringer's solution, and are due to the extrusion of Ca²⁺ by the Na⁺/Ca²⁺-K⁺ exchanger. (B) Fractional circulating current J(t) on a faster time-base, for jumps into IBMX in the dark, or in the presence of steady illumination producing I = 15, 48, or 480 photoisomerizations per second, that reduced the relative circulating current to J _rel(I) = 0.90, 0.79, and 0.42 (n = 7, 3, 3, and 2 traces, respectively). The traces in darkness are the same as in A. (C) Derivatives of J(t)^1/2, estimated by fitting each trace with a running 61-point parabola, and evaluating the derivative at the midpoint. At the sampling rate of 300 Hz, this parabola covered 0.10 s before and after the midpoint; the apparent rise of the derivatives before time zero is an artifact of using this finite width. Derivatives estimated with narrower windows gave similar maxima, but greater noise. Rod d of Table .

Figure 10

Step/flash experiment used as the basis for determining the decrease in R* lifetime. (A) Responses of rod f of Table , stimulated with flashes producing Φ = 5,100 (a), 16,000 (b), or 51,000 (c) photoisomerizations, either in darkness (top set), or synchronously with the termination of a step of light delivering I = 370, 1,200, or 3,700 photoisomerizations per second, that had been applied for 20 s. The effect of the backgrounds in shortening the duration of the response is indicated diagrammatically for flash c by the leftward pointing arrows, which originate from a line coinciding with the time of 50% recovery for the flash delivered in darkness; the magnitude of this leftward shift in time to 50% recovery is denoted ΔT ₅₀. (The traces were digitally filtered at 50 Hz with the Matlab^TM routine “filtfilt”.) (B) Dependence of ΔT ₅₀ on step intensity for six rods from this study (closed symbols, identified in Table ), and for the rod in Figure 8 of Fain et al. 1989, □, which was exposed to steps lasting 7 s. When more than 1 flash intensity was used (as in A), ΔT ₅₀ was determined as the mean shift for the different intensities, and the bars indicate ± SD. The two curves plot predictions of the model in the using the parameters of the standard rod, except for τ_E, which is 2.4 s for the top curve and 1.6 s for the bottom curve.

Figure 11

Dependence of the three main time constants of rod phototransduction on the intensity of steady adapting light. (A) τ_R/τ_{R, Dark}, the effective lifetime of R* relative to its dark-adapted lifetime, inferred as described in the text () from the step/flash experiments of Fig. 10. (B) τ_E/τ_{E, Dark}, the effective lifetime of activated PDE, E*, relative to its dark-adapted lifetime, extracted by measuring the dominant time constant of recovery for saturating flashes (Fig. 5). (C) Time constant of cGMP hydrolysis, 1/β(I), determined as the reciprocal of the steady-state rate constant of hydrolysis as measured by the methods of Fig. 7 and Fig. 8, and summarized in Fig. 9. The error bars (Fig. 9) have been removed for simplicity, and only the estimates obtained with the fitting method (Fig. 8) have been shown in cases in which the two methods yielded different estimates of β.

Figure 14

Analysis of the contributions of different molecular mechanisms to light adaptation. All traces (except the dashed traces in C) were computed with the model salamander rod presented in the . The color coding identifies the eight combinations of the three calcium-dependent feedback mechanisms. (Blue) The calmodulin-dependent shift in K _cG of the cGMP-activated channels (“CaM mechanism”) alone. (Green) The recoverin-dependent inactivation of rhodopsin kinase (“Rec mechanism”) alone. (Red) The GCAP-dependent activation of guanylyl cyclase (“GCAP mechanism”) alone. (Black) All mechanisms inactivated. (Cyan) CaM + Rec mechanisms; (magenta) CaM + GCAP mechanisms; and (yellow) Rec + GCAP mechanisms. Dark gray (rather than white): all mechanisms active. (Light gray) Weber's Law, S _rel = I ₀/(I+I ₀), with I ₀ = 40 photoisomerizations per second. (A) Steady circulating current, j(I); this is as in Fig. 6 A, except that the scale is in absolute units of pA. (B) Relative sensitivity, s _rel = s(I)/s _Dark. The dotted curve shows the prediction of the model for the experimental condition illustrated in Fig. 13, in which Ca²⁺ _i is clamped to the level set by the steady background to which the rod is exposed in Ringer's. (C) Relative fractional sensitivity, S _rel = s _rel/J _rel. To avoid crowding, the traces for pairs of mechanisms (cyan, magenta, and yellow) have not been shown. Two additional curves are shown dashed, that were computed using a three time constant (τ_R, τ_E, and 1/β) model of calcium-clamped rods (Equation 19 of Nikonov et al. 1998). (Dashed trace) β was varied according to the full model, while τ_R was held at its dark level. (Dot-dash trace) β was held at its dark level, while τ_R was varied according to the full model.

Figure 12

Comparison between experiment and theory, for fractional responses R(t) to families of flashes presented in darkness or on backgrounds. Same cell and traces as in Fig. 2 (rod a of Table ). A–D each depict responses to the same set of flash intensities, but presented on steady backgrounds delivering I = 0 (A), 260 (B), 810 (C), or 2,600 (D) photoisomerizations per second. Each set of raw responses has been normalized by the saturating response amplitude at 200–250 ms for that background condition (see Fig. 2), to extract the fractional response. In each adaptation condition the flashes delivered Φ = 260, 830, 2,600, 8,300, 26,000, 83,000 and 260,000 photoisomerizations. The smooth traces were computed with the model in the , under calcium-clamp conditions; i.e., with Ca²⁺ _i held at the level calculated for each steady background. The parameters of the model were those for the standard rod, given in Table (including the value of β_Dark = 1.0 s, which was measured for the individual rod, see Table ). In the presence of the three backgrounds, the model gave the following steady state values: β(I) = 3.2, 6.7, and 15.9 s⁻¹; τ_R(I) = 0.26, 0.21, and 0.18 s. (E) All the traces from A–D have been superimposed, with the same color-coding according to flash intensity as in the individual panels. (F) The theory traces from A (dark-adapted, solid lines) and D (the brightest background, dashed lines) are superimposed to illustrate the predicted differences for calcium-clamped responses between the two extremes of adaptation.

Figure 13

“Calcium-clamped” dim-flash responses, reproduced from the data of Figure 10 of Fain et al. 1989, compared with the predictions of our model. In the three panels, the backgrounds delivered: I = 0 (dark-adapted), 96, and 2,100 photoisomerizations per second, and the test flashes delivered Φ = 23, 87, and 620 photoisomerizations (assuming a collecting area of 40 μm², for light polarized in the preferred orientation). In each case, the rod had been allowed to equilibrate to the adaptational state for several minutes. It was then exposed to “low-Ca²⁺, 0-Na⁺ solution” designed to minimize flash-induced changes in Ca²⁺ _i (see Fain et al. 1989, for further details) and, after 5 s, a dim test flash was delivered; 9 s later, a bright flash was given, to measure the circulating current, and then the bathing solution was returned to Ringer's. The solution change with flash delivery was repeated at least four times for each panel. The theory traces have been simulated with the model in the , using the standard parameter values in Table , with the following exceptions: A = 0.045 s^-2, τ_R,Dark = 0.4 s, τ_E = 1.5 s, and β_Dark = 0.8 s⁻¹. In addition, the following parameter values were specific to the three panels: β(I) = 0.8, 1.7, and 10.1 s⁻¹, and τ_R(I) = 0.95, 0.27, and 0.13 s. These values are determined by application of the steady-state model, with the exception of the value τ_R = 0.95 s used for the dark-adapted response (I = 0) in A. A possible rationale for this relatively high value is that Ca²⁺ _i may have risen during the “clamping” exposure, reducing the free RK to ∼0.44 of the level it would normally be in the dark in Ringer's. The traces in B and C have been corrected for slight baseline drifts, of 0.01 and 0.027 pA s^-1, respectively.

Scheme S1