The experiments presented here test the hypothesis that the passive transport of retinal through the cytoplasm between the plasma and disk membranes could constitute an important determinant of the rate of pigment regeneration. To do this, we have compared the rates of pigment regeneration and sensitivity recovery in salamander rod photoreceptors that have been bleached and subsequently exposed to exogenous solutions containing native 11-cis-retinal and 11-cis 4-OH retinal. 11-cis 4-OH retinal differs from 11-cis-retinal in that it has an additional hydroxyl group attached to position 4 on the β-ionone ring, thus increasing its aqueous solubility. The data presented here demonstrate the following: (a) The aqueous solubility of 11-cis 4-OH retinal is substantially greater than for 11-cis-retinal. (b) 11-cis-retinal has a partition coefficient that is roughly 100 times higher than that of 11-cis 4-OH retinal, although both species of retinal partition extremely well into lipid membranes. (c) When exogenously supplied to an extensively bleached vertebrate rod photoreceptor, 11-cis 4-OH retinal combines with opsin to form a fully functional blue-shifted visual pigment, whose physiological properties can be predicted from its spectral absorbance. (d) The rate of pigment regeneration and sensitivity recovery as well as the return of response kinetics to prebleach levels that occur when 11-cis 4-OH retinal is applied exogenously to bleached rods is over fivefold faster than occurs when similarly bleached rods are treated with 11-cis-retinal. These more rapid rates of pigment regeneration and sensitivity recovery occur even though the rate of pigment regeneration measured in solutions containing opsin and 11-cis 4-OH retinal is slower than in solutions containing 11-cis-retinal. (e) The sensitivity and response kinetics that are fully restored to dark-adapted values in rods containing 4-OH rhodopsin demonstrate that the photosensitivity of 4-OH rhodopsin is close to that of rhodopsin.
These results demonstrate that there exists a close correlation between the aqueous solubility of retinoids and the rate at which pigment regeneration and sensitivity recovery occur in bleached isolated rods. This is in spite of the fact that the intrinsic rate at which 4-OH rhodopsin forms is slower than the rate of rhodopsin formation, as we demonstrate in in vitro measurements of pigment regeneration, both with bovine opsin expressed in solution or salamander rod opsin expressed in nanodiscs ().
In order for the visual pigment to regenerate in a rod photoreceptor, 11-cis-retinal must be transported from the extracellular space to the internal membrane disks and bind to opsin. This process can be broken down into the following steps: (1) transfer of 11-cis-retinal from the extracellular space into the plasma membrane, (2) transfer of 11-cis-retinal from plasma membrane to the cytosolic aqueous phase, (3) diffusion of 11-cis-retinal within the cytosol, (4) transfer of 11-cis-retinal from the cytosolic aqueous phase into disk membrane, and (5) reaction of 11-cis-retinal with opsin resulting in regeneration of visual pigment.
The data summarized in show that both 11-cis-retinal and 4-OH retinal partition extremely well into lipid membranes. In effect, this means that the external plasma membrane is unlikely to act as a barrier for retinal movement, as the retinoid concentration in the cytosol next to the plasma membrane will quickly equalize to a concentration set by the aqueous solubility of retinal. This is supported by the fact that we observed no substantial differences in the rate of recovery of sensitivity when we used different delivery methods of retinal (). Thus, steps 1, 2, and 4 are expected to be very fast, consistent with the rapid rate for the transfer of retinol between membranes and aqueous solution of 0.64 s−1
as reported previously (Noy and Xu, 1990
). The high partition coefficient values together with a rapid equilibrium rate constant for retinoids eliminates the plasma membrane as a potential bottleneck for the rate of pigment regeneration and dark adaptation. It follows that the regeneration is limited either by step 3, the diffusion across the cytosol, or by step 5, the reaction rate of 11-cis-retinal with opsin.
We believe that the intrinsic rates of regeneration that we have measured in in vitro experiments (step 5) cannot explain the pigment regeneration rates that we observe in intact rod photoreceptors. Our argument is as follows: The in vitro regeneration rates we observed (R11-cis = 0.053 s−1 and R4-OH = 0.027 s−1) are much faster than those observed in intact cells (R11-cis = 0.0013 s−1 and R4-OH = 0.0064 s−1). These rates of pigment regeneration are given by
in which k
is the second order rate constant, and Cretinal
are the concentrations of retinal and opsin in the membrane, respectively. The in vitro regeneration experiments with nanodiscs were made using very low concentrations of opsin (0.5 µM) and retinal (3 µM). A naked nanodisc contains ~250 lipid molecules (Bayburt et al., 2007
). When containing opsin, a nanodisc contains ~200 lipid molecules (Bayburt et al., 2007
). Equal concentrations of naked nanodiscs and opsin-containing nanodiscs were mixed. Thus, the concentrations of retinal and opsin in the nanodiscs are Copsin
= 1 opsin/450 lipids and Cretinal
= 6 retinals/450 lipids. The corresponding opsin concentration in a rod outer segment is ~1 opsin per 100 molecules of lipid. At early times, in the linear range, the pseudo–first order rate constants for regeneration are proportional to the rates of pigment regeneration. Thus, using Eq. 2
, together with the experimentally obtained rates, R
, and the concentrations given in this paragraph, we can calculate the retinal concentrations, Cretinal
, in the rod outer segment disks from Cretinal-cell
) × (Cretinal-nanodisc
) and obtain C11-cis
= 7.4 × 10−5
(mole retinal/mole lipid) and C4-OH
= 7.0 × 10−4
(mole retinal/mole lipid). In the cytosolic aqueous phase next to the disk membrane, the retinal concentrations can be estimated from the known partition coefficients (Eq. 1
). For a water concentration of 55 M, we get C11-cis
= 2.5 × 10−11
M and C4-OH
= 1.3 × 10−8
M. These concentrations are several orders of magnitudes lower than the concentrations of retinal delivered at the plasma membrane, indicating that the rate of pigment regeneration would be higher if higher retinoid concentrations were achieved at the disk membrane. Thus, pigment regeneration (step 5) is not limiting.
Instead, the most likely bottleneck is at the cytosolic gap (step 3) between the plasma membrane and the intracellular disk membranes in which the bulk of the bleached opsin is located. This notion is consistent with a simple diffusional model of retinal translocation across the cytosolic gap based on Fick’s second law of diffusion (Carslaw and Jaeger, 1959
; Crank, 1975
, in which J
is the retinal flux per unit area, and D
is the diffusion coefficient for retinal taken to be 5 × 10−6
(Szuts and Harosi, 1991
). Given the high partition coefficient for retinoid in the membrane and its rapid equilibration between aqueous and lipid phases (Noy and Xu, 1990
), we set the boundary condition (concentration) in the cytosol next to the plasma membrane, Cpm
, to be equal to the aqueous solubility. In this case, we predict this to be the amount of retinoid that can be dissolved in aqueous solution (i.e., at 10 µM nominal concentration; ). The cytosolic concentration of retinoid at the disk membrane boundary, Cdm
, will be much smaller than that at the plasma membrane because of the presence of a large sink in the form of opsin on the disk membrane. This is indeed borne out by the calculations of the concentration for Cdm
from the pigment regeneration rates. If we view the rod outer segment as a cylinder with the same size as used in the calculation of the regeneration rate (radius [r
] = 5.7 µm and length [l
] = 29.8 µm; see Results), the flux, ψ
, becomes ψ
, in which δ
represents the cytosolic gap between the plasma and disk membrane of 0.05 µm (Mariani, 1986
). Because Cpm
initially, we can approximate the previous equation as ψ
It is apparent from this equation that the flux of retinoid across the cytosolic gap is, at least at initial times, directly proportional to the concentration in the cytosol just inside the plasma membrane, Cpm, which equals the solubility in the cytosol. Using values from (Cpm = 3.5 µM for 11-cis-retinal and Cpm = 10 µM for 11-cis 4-OH retinal), we determine the fluxes to be ψ11-cis = 3.6 × 10−17 mol s−1 and ψ4-OH = 1.10−16 mol s−1 for 11-cis-retinal and 11-cis 4-OH retinal, respectively. These values are reasonably close to the regeneration rates in intact cells, which we have measured: K11-cis = 1.4 × 10−17 mol s−1 for 11-cis-retinal and K4-OH = 6.8 × 10−17 mol s−1 for 11-cis 4-OH retinal, calculated from our MSP data (see Results). Thus, the model, at least partly, explains the five times faster regeneration rate of 4-OH rhodopsin compared with rhodopsin that we have observed in intact cells.
With the cytosolic gap acting as a barrier, the aqueous solubility of retinal seems to be an important factor setting the rate of regeneration of the visual pigment after a substantial bleach. The model suggests that the higher aqueous solubility of 11-cis 4-OH retinal is sufficient to drive a greater retinoid flux across the cytoplasmic gap in spite of its lower partition into the membrane and, thus, driving visual pigment regeneration at a higher rate. We believe that this is the first observation that solubility of cis-retinoids may play an important role in the rate of pigment regeneration and sensitivity recovery. This may have important pharmacological implications for the design of retinoid drugs that may be used in the treatment of disorders of visual pigment regeneration in human patients.