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INTRODUCTION |
Stargardt disease (Mendelian Inheritance in Man number 248200;
also called fundus flavimaculatus) is an autosomal recessive disorder
that affects approximately one person in 10,000 (1). Affected
individuals experience central vision loss and a progressive bilateral
atrophy of the macular region of the retina and retinal pigment
epithelium (RPE).1 A
distinctive feature of Stargardt disease is the accumulation of
fluorescent, shortwave absorbing material in the RPE, which appears in
postmortem samples as lipofuscin-like deposits (2-4). These
clinical and histopathologic features resemble those seen in
the more common age-related macular degeneration, suggesting that
investigation of the pathophysiology of Stargardt disease may reveal
mechanisms relevant to age-related macular degeneration (5-8).
The gene responsible for Stargardt disease codes for a member of the
ABC transporter family, ABCR (9-11). The spectrum of mutations
identified thus far in Stargardt disease patients suggests that these
individuals carry at least one partially functional allele. By
contrast, some cases of autosomal recessive retinitis pigmentosa, a
disease characterized by a progressive degeneration of the rod-rich
peripheral retina, are associated with homozygosity of frameshift or
splice site mutations in the ABCR gene which are likely to
result in complete loss of ABCR function (12, 13). An association
between a subset of subjects with age-related macular degeneration and
variant alleles of the ABCR gene has been reported (14), but
interpretation of the data has been controversial (15).
Expression of the ABCR gene is confined exclusively to
retinal rod photoreceptors, and the ABCR protein is localized to the rim and incisures of rod outer segment discs, where it is present at a
molar ratio of approximately 1:120 with rhodopsin (16-19). This highly
specific localization suggests that ABCR may transport a molecule or
ion that plays a specialized role in photoreceptor homeostasis.
Although small quantities of ABCR may also be present in the rod outer
segment plasma membrane, the localization of the majority of ABCR to
the internal disc membranes suggests that the relevant transport event
is between the lumenal and cytosolic faces of the disc membrane.
Because outer segment turnover occurs via RPE phagocytosis, a
derangement in this transport mechanism (with the resulting
accumulation of one or more compounds within the outer segment) could
plausibly explain the progressive accumulation of material within the
RPE in Stargardt disease.
From the preceding discussion it is clear that identifying the molecule
or ion transported by ABCR is a prerequisite to understanding the
normal role of ABCR and the pathogenesis of Stargardt disease. To date,
substrates have been identified for only a few of the over 30 known
mammalian ABC transporters. In these few cases, critical information
regarding the identity of the substrate(s) has often come from genetic
studies. For example, the defect in chloride resorption in humans with
cystic fibrosis provided the first indication that CFTR mediates
chloride transport (20), and mutations in the human canalicular organic
anion transporter, SPGP, have been found in a subset of
patients with progressive familial intrahepatic cholestasis, an
inherited disorder of hepatobiliary excretion (21). For P-glycoprotein
and its close relatives, overexpression of the corresponding genes or
cloned cDNAs in a variety of mammalian cells was found to confer
resistance to chemotherapeutic agents, and targeted disruption of these
genes in the mouse shows that MDR1A is responsible for extrusion of
hydrophobic compounds across the blood-brain barrier (22).
For a number of transporters, including CFTR, SPGP, and P-glycoprotein,
these observations have been extended by experiments in
vitro. Purified and reconstituted CFTR shows chloride channel activity (23), purified and reconstituted P-glycoprotein shows substrate-dependent ATP hydrolysis (24-27) and
ATP-dependent vectorial transport of substrates (28, 29),
and partially purified recombinant SPGP mediates taurocholate transport
and taurocholate-dependent ATP hydrolysis (30). In the
in vitro experiments with P-glycoprotein, ATP hydrolysis is
stimulated to different extents by compounds that are extruded by this
protein in living cells. A second class of compounds, called
chemosensitizers, increases the sensitivity of cells to
chemotherapeutic agents and also stimulates ATPase activity in
vitro, possibly by uncoupling ATP hydrolysis and substrate extrusion. This work suggests that it may be possible to identify substrates and allosteric regulators of other ABC transporters by
searching for compounds that stimulate the ATPase activity of the
purified and reconstituted transporters.
To characterize the biochemical properties of ABCR with the goal of
identifying its substrate, we report here a procedure for solubilizing
and functionally reconstituting the purified protein into lipid
membranes. Several compounds, including amiodarone, digitonin, and
various geometric isomers of retinal, produce a significant increase in
ATPase activity of this preparation. Interestingly, amiodarone and
digitonin greatly enhance the stimulatory activity of
all-trans-retinal but not of each other, suggesting that
they act by a similar mechanism which differs from the mechanism by which all-trans-retinal acts. These data, together with the
clinical and histological data summarized above, suggest a model in
which ABCR is involved in the recycling of retinoids that attends
photoactivation of rhodopsin (the visual cycle).
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MATERIALS AND METHODS |
Reagents--
Reagents and their sources are as follows:
Extracti-Gel detergent removal resin (Pierce); CNBr-activated Sepharose
4 Fast Flow beads (Amersham Pharmacia Biotech); dark adapted bovine
retinas (Schenk Packing Co., Stanwood, WA);
(R)-(+)-1-octyn-3-ol,
5-methoxy-1,3,3-trimethylspiro[indoline-2,3'-(3H)naphtho]2,1-b[pyran], metergoline, Meldola's blue, lasalocid, (
)-isoreserpine,
hydroquinine 4-methyl-2-quinolyl ether, (
)-hecogenin acetate,
coumarin 334, corynanthine hydrochloride hydrate,
2-tert-butylanthraquinone, Brilliant Crocein MOO, and
Brilliant Green (Aldrich); digitoxin, digoxigenin, and digoxin (Fluka);
activated charcoal, caprylic acid, crude sheep brain PE,
all-trans-retinal, all-trans-retinol, all-trans-retinoic acid,
-ionone, actinomycin D,
amiodarone, colchicine, nifedipine, trifluoperazine, verapamil,
vinblastine, vincristine, CHAPS, cholic acid, quercetin, progesterone,
ethidium bromide, and dehydroabietylamine acetate (Sigma); bromphenol
blue and bromcresol green (Bio-Rad); digitonin (Eastman Kodak Co.);
-octyl glucoside and
n-dodecyl-
-D-maltoside (Calbiochem); brain polar lipids and egg PC (Avanti Polar Lipids);
[
-32P]ATP (NEN Life Science Products).
11-cis-Retinal was a gift of Dr. Lubert Stryer. Lipid
sonication was performed using the G12SP1 Special Ultrasonic Cleaner
from Laboratory Supplies Co., Inc.
Preparation of ROS--
ROS were prepared from dark adapted and
frozen bovine retinas under dim red light as described (31). Aliquots
were stored at
80 °C in the dark.
Conjugation of Rim 3F4 Antibody to Sepharose
Beads--
Monoclonal antibody Rim 3F4, which recognizes a linear
epitope near the C terminus of bovine ABCR (16), was purified from ascites fluid by caprylic acid precipitation (32) and conjugated to
CNBr-activated Sepharose 4 Fast Flow beads (Amersham Pharmacia Biotech)
at a ratio of 1 mg of antibody per ml of resin. The resin was discarded
after each use to improve reproducibility in the purification of ABCR.
Purification of ABCR from Bovine ROS--
All purification
procedures prior to the elution step were performed under red dim light
at 4 °C to minimize aggregation of photobleached rhodopsin. Purified
ROS from 4-8 bovine retinas were diluted into greater than 10 volumes
1× phosphate-buffered saline, pelleted, and solubilized in 1.4 ml of
ROS/CHAPS buffer (10% glycerol, 0.75% CHAPS, 50 mM HEPES,
pH 7.0, 0.5 mg/ml crude brain PE, and 0.5 mg/ml egg PC, 140 mM NaCl, 3 mM MgCl2, 5 mM
-mercaptoethanol). The sample was rotated at 4 °C
in the dark for 1 h to completely solubilize the ROS membranes.
Insoluble material was removed by microcentrifugation at 16,000 rpm for 5 min, and the supernatant was added to 200 µl of Rim 3F4-Sepharose and rotated in the dark at 4 °C for 3 h. The resin was
recovered by microcentrifugation at 3000 rpm and washed three times for 1 min and then two times for 20 min (with gentle rotation) using 1 ml
of ice-cold ROS/CHAPS buffer. Three successive elutions were performed
at 4 °C using 80 µl each of 0.2 mg/ml of a peptide
(H2N-NETYDLPLHPRTAG-COOH) containing the Rim 3F4 epitope
(16) in ROS/CHAPS buffer for a total of 45 min. An irrelevant peptide,
JN50 (H2N-MLRNNLGNSSDC-CONH2), was used as a
control for the specificity of elution.
Reconstitution of ABCR into Proteoliposomes--
240 µl of
immunoaffinity purified ABCR in ROS/CHAPS buffer was added to a
premixed solution containing 30 µl of 15% octyl glucoside and 90 µl 50 mg/ml sonicated lipid in 25 mM HEPES, 140 mM NaCl, 10% glycerol. The sample was mixed, incubated on
ice for 30 min, and then rapidly diluted into 2 ml of reconstitution buffer (25 mM HEPES, pH 7.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol) at room temperature and incubated
at room temperature for an additional 2 min. To remove detergent, the
sample was passed (at room temperature) over a 2-ml Extracti-Gel
(Pierce) column that had been thoroughly prewashed with the
reconstitution buffer. The flow-through was stored on ice. Light
microscopic examination of the reconstituted preparation stained with
1,1'-dioctadecyl-3,3,3',3'-tetraethylindocarbocyanine shows a
heterogeneous distribution of vesicle sizes. Unless noted otherwise all
reconstitutions reported here were performed with swine brain polar lipids.
ATPase Assays--
ATPase assays were performed using ABCR that
had been purified from ROS and reconstituted into liposomes on the same
day. ATPase assays were performed in 300 µl of reconstitution buffer supplemented with MgCl2 to a final concentration of 3 mM. Appropriate dilutions and/or mixtures of test compounds
were prepared in ethanol and were added to 0.1% of the volume of the
ATPase reaction; control reactions received the same volume of ethanol
alone. The reactions were started by the addition of 30 µl of 10×
ATP mix containing 50 mM
-mercaptoethanol, 500 µM ATP with 50 µCi/ml of 3000 Ci/mmol of
[
-32P]ATP, and incubated at 37 °C for 2 h. For
experiments in which the ATP concentration varied, the
[32P]ATP was held constant at 5 µCi/ml. At the starting
and final time points, triplicate samples of 50 µl were added to 200 µl of 10% activated charcoal in 10 mM HCl, vortexed
vigorously for 2 min, and microcentrifuged to pellet the charcoal. 50 µl of the resulting supernatant was counted. For experiments to
differentiate the effects of retinal isomers, the ATPase assay was
manipulated under dim red light and incubated in the dark. All other
ATPase assays are performed in room light. Data points in
Lineweaver-Burk plots were fitted to a straight line by a least squares
criterion; no attempt was made to to fit the data to more complex curves.
Handling and Storage of Retinoids--
All procedures were
performed under dim red light or in the dark. Retinal, retinol, and
retinoic acid were dissolved in ethanol, and for each experiment their
integrity was monitored by determining an absorption spectrum.
Retinoids were handled in glass vials to minimize absorption to
plastic. For storage, aliquots were dried under argon and maintained in
the dark at
80 °C. Oxidized all-trans-retinal was
prepared by exposing a sample of all-trans-retinal in
ethanol to air at 37 °C overnight in a sealed glass vial.
SDS-PAGE, Western Blots, and Determination of ABCR
Concentration--
These were performed as described (18). Western
blots were performed with affinity purified rabbit anti-human ABCR
antibodies and were standardized using bovine ROS.
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RESULTS |
Purification and Reconstitution of ABCR--
The purification
strategy employed in this work is essentially that of Illing et
al. (16) modified by the use of a mixture of CHAPS, PC, and PE
(ROS/CHAPS buffer) for the solubilization and chromatography steps.
Mixtures of CHAPS/PE or CHAPS/PC have been used previously to
solubilize and purify several photoreceptor membrane proteins in native
form, including cone visual pigments (33), the cGMP-gated channel (34),
and the Na/Ca-K exchanger (35). In preliminary experiments, we observed
that the ATPase activity from the ABCR preparation (see below) was
higher if solubilization and purification were performed with CHAPS
than with either octyl glucoside or dodecyl maltoside. In brief, the
ABCR purification involves (a) purifying bovine ROS on a
sucrose step gradient, (b) solubilizing the ROS in a mixture
of CHAPS, PC, and PE (ROS/CHAPS buffer), (c) selectively
binding ABCR to monoclonal antibody Rim 3F4-conjugated Sepharose, and
(d) selectively eluting ABCR with a synthetic peptide
corresponding to the Rim 3F4 epitope. All but the last of these steps
are carried out under dim red light to minimize denaturation and
nonspecific trapping of rhodopsin.
Fig. 1A shows the results of
the purification procedure. The specificity of the immunoaffinity
purification step was assessed by halving a sample of Rim 3F4-Sepharose
to which ABCR had been bound and carrying out parallel elutions with
either the Rim 3F4 peptide or an irrelevant peptide, JN50. The material
eluted with the JN50 peptide will be referred to throughout this paper
as "mock-eluted."

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Fig. 1.
Purification of ABCR and identification of an
associated ATPase activity. A, proteins were resolved
by SDS-PAGE (10% acrylamide) and stained with Coomassie Blue
(left) or immunoblotted and probed with affinity purified
rabbit anti-ABCR antibodies (right). The ROS lane
shows the starting material for immunoaffinity purification. Proteins
eluted from the Rim 3F4 affinity column by the JN50 or Rim 3F4 peptides
are shown in the right pair of panels.
ABCR migrates in SDS-PAGE with an apparent molecular mass of
approximately 250 kDa. B, ATPase assays in ROS/CHAPS buffer
were performed with the material eluted by the JN50 or Rim 3F4 peptides
or with the pure peptides in ROS/CHAPS buffer as controls. The basal
activity level refers to the ATPase activity of material eluted with
Rim 3F4.
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Coomassie staining and Western blotting show that the ABCR polypeptide,
with an apparent molecular mass of approximately 250 kDa, is eluted
with the Rim 3F4 peptide but is barely detectable following elution
with the JN50 peptide (Fig. 1A). Although we cannot rule out
the presence of minor ROS-derived contaminants in the final
immunoaffinity purified preparation, they are likely to be considerably
less abundant than ABCR as judged by the apparent homogeneity of the
ABCR preparation. The extensive washing of the column-bound material
should remove all low molecular weight compounds initially present in
the ROS membrane sample.
Reconstitution of ABCR into brain lipid membranes was accomplished by
gently mixing immunoaffinity purified ABCR in ROS/CHAPS buffer with an
excess of sonicated swine brain polar lipids (referred to hereafter as
"brain lipid"). Efficient removal of CHAPS with a high yield of
ABCR ATPase activity was most effectively achieved by absorption to
Extracti-Gel resin (Pierce). This method of detergent removal is rapid
and efficient and results in minimal losses of ABCR, most likely
because the proteolipid complexes are effectively excluded by the low
molecular weight pore size of the Extracti-Gel resin. In developing
this protocol we have avoided ultracentrifugation of the reconstituted
ABCR proteolipid. This circumvents problems associated with inefficient
recovery of membranes in the presence of 10% glycerol, used here as a
protein stabilizer, and with dispersing a compact proteolipid pellet.
Several other methods for detergent removal and proteolipid formation
were found to be less effective for reconstitution of ABCR as follows:
rapid dilution (36) followed by ultracentrifugation resulted in
inefficient removal of detergent and poor recovery of proteoliposomes;
gel filtration followed by ultracentrifugation (26, 37) resulted in
loss of ABCR ATPase activity; and detergent removal by binding to
Bio-Beads (Bio-Rad) followed by ultracentrifugation (38) efficiently
removed detergent but was accompanied by significant absorption of ABCR
protein to the beads.
Purified ABCR solubilized in ROS/CHAPS buffer or reconstituted in brain
lipid possesses considerable ATPase activity (Fig. 1B and
see Fig. 7 below). We ascribe this activity to ABCR because it is far
lower in the mock-eluted sample. In the experiments described below in
which various compounds were used to stimulate ATPase activity, we
observed variation in ATPase stimulation relative to the basal ATPase
activity of up to 1.5-fold for identical experimental protocols carried
out on different ABCR preparations. This variation may reflect
differences between ABCR preparations in the level of minor
contaminating ATPases, which would presumably contribute to the basal
activity but not the stimulated activity, and/or differences in the
absolute amount of lipid present in the sample which therefore changes
the ratio of lipid to the added compounds, most of which are
hydrophobic. To avoid normalizing and averaging data from different
ABCR preparations, all of the data presented in the figures show the
results of single representative experiments.
Stimulation of ABCR ATPase Activity by Low Molecular Weight
Compounds--
As one approach to identifying substrates or allosteric
regulators of ABCR, we surveyed a group of structurally diverse
compounds for their ability to stimulate or inhibit the ATPase activity of purified and reconstituted ABCR. Our earlier localization of ABCR to
ROS disc membranes suggested the possibility that it might be involved
in the movement of retinal or retinol (9, 16, 18), and therefore the
collection includes a variety of retinoids. The structures of these
compounds are shown in Fig. 2, and they fall into four groups as follows: retinoids (compounds 1-5), known P-glycoprotein substrates or sensitizers (compounds 6-14), detergents and digitonin-related (compounds 15-22), and compounds selected for
their structural diversity (compounds 23-43). The compounds were
initially tested at 20 µM (Fig.
3), a concentration at which many
P-glycoprotein substrates and sensitizers enhance ATP hydrolysis by
P-glycoprotein (24-27). Most of the compounds had little or no effect
on ATPase activity, and none significantly inhibited ATPase activity.
However, seven compounds, three retinal isomers (all-trans-retinal, 11-cis-retinal, and
13-cis-retinal), amiodarone, digitonin, dehydroabietylamine,
and 2-tert-butylanthroquinone, reproducibly activated the
ATPase activity 2-5-fold (the fold activation depending on the
compound), a degree of activation similar to that reported for purified
and reconstituted P-glycoprotein in the presence of the most potent
P-glycoprotein substrates and sensitizers (26, 27, 39). Most of the
experiments involving retinal were carried out with the
all-trans-isomer. Except where noted below, these
experiments were carried out under room lights and therefore some
degree of photoisomerization would have occurred during the 2-h
incubation.

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Fig. 2.
Chemical structures of compounds tested for
stimulation or inhibition of reconstituted ATPase activity.
A, retinoids: 1, 11-cis-retinal;
2, 13-cis-retinal; 3, all-trans-retinal; 4, all-trans-retinoic acid; and 5, all-trans-retinol. B, P-glycoprotein substrates
and sensitizers: 6, nifedipine; 7, actinomycin D
(Sar, sarcosine; Meval, N-methylvaline);
8, vinblastine; 9, trifluoperazine;
10, vincristine; 11, colchicine; 12, progesterone; 13, amiodarone; and 14, verapamil.
C, detergents and digitonin-related compounds:
15, ( )-hecogenin acetate; 16, CHAPS;
17, cholic acid; 18, n-octyl- -D-glycopyranoside; 19, digitonin; 20, digitoxin; 21, digoxin; and
22, digoxigenin. D, other structurally diverse
compounds: 23, ethidium bromide; 24, metergoline;
25, brilliant green; 26, hydroquinine
4-methyl-2-quinolyl ether; 27, bromcresol green;
28, 6-bromo-1',3'-dihydro-1',3',3'-trimethyl-8-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indole];
29, Medola's blue; 30, coumarin 334;
31, dehydroabietylamine acetate; 32, 2-tert-butylanthraquinone; 33, isoproterenol;
34, lasalocid; 35, ( )-isoreserpine; 36, brilliant crocein MOO; 37, beta-ionone; 38, bromphenol blue; 39, 1-octyn-3-ol; 40, hexanal;
41, corynanthine; 42, 5-methoxy-1,3,3-trimethylspiro[indoline-2,3'-(3H)naphtho[2,1-b]pyran];
and 43, xylene cyanol FF.
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Fig. 3.
Effect of various compounds at 20 µM on the ATPase activity of purified
and reconstituted ABCR. Each panel includes a basal reaction and
an all-trans-retinal-stimulated reaction, and ATPase
activity is shown as a percent of basal activity. In this figure and in
all subsequent figures, the basal ATPase is the ATPase level observed
in the absence of activators for Rim 3F4-eluted and reconstituted ABCR
or, for these panels showing Rim 3F4-eluted ABCR in ROS/CHAPS buffer,
the basal ATPase is the level in ROS/CHAPS buffer. A, nine
P-glycoprotein substrates and sensitizers. B, eight
detergents and compounds related to digitonin and digoxigenin.
C, 20 chemically diverse compounds. See Fig. 2 for chemical
structures.
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One example of the specificity of ABCR can be seen in comparing the
stimulatory effects of digitonin-like compounds (Fig. 3B).
Within this group only digitonin (compound 19 in Fig. 2C) shows activity at 20 µM, whereas the closely related
compounds (
)-hecogenin acetate, digitoxin, digoxin, and digoxigenin
(compounds 15 and 20-22) show no activity. Likewise, the other three
detergents tested,
n-octyl-
-D-glucopyranoside, CHAPS, and cholic
acid, show no activity at 20 µM.
The specificity of ATPase stimulation was assessed by comparing
preparations that had been identically reconstituted following elution
from the Rim 3F4 immunoaffinity column with either the Rim 3F4 peptide
or the irrelevant peptide JN50 (Fig. 4,
A-E). In each case, ATPase stimulation is observed only in
the sample that was eluted with the Rim 3F4 peptide. The concentration
dependence of ATPase stimulation by each of the stimulatory compounds
was determined and compared with that of verapamil, a P-glycoprotein sensitizer and a potent activator of P-glycoprotein ATPase in vitro (26). Verapamil had no effect on ATPase activity at any concentration up to 200 µM (Fig. 4F). Each of
the stimulatory compounds has a characteristic concentration dependence
of activation as follows: all-trans-retinal produces a
dose-dependent ATPase activation that is half-maximal at
10-15 µM and shows a simple Michaelis-Menten dose
dependence (Fig. 4, A and F; and see Figs. 7, 10,
and 11 below); amiodarone, dehydroabietylamine, and
2-tert-butylanthroquinone activate the ATPase beginning at
approximately 10-20 µM and show no evidence of
saturation up to 50 µM (Fig. 4, B, D and
E); and digitonin activates the ATPase with a sharp optimum
at approximately 20 µM, indicative of a high affinity
stimulatory interaction and a lower affinity inhibitory interaction
(Fig. 4C). The dose-response curves suggest a mechanistic
distinction between all-trans-retinal, which appears to act
at a single site, and amiodarone, dehydroabietylamine, and
2-tert-butylanthroquinone which appear to act through a
cooperative mechanism that involves two or more sites.

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Fig. 4.
Concentration dependence and specificity of
selected compounds in stimulating the ATPase activity of purified and
reconstituted ABCR. To assess the specificity of the ATPase
reaction in A E, parallel elution and reconstitution
procedures were performed using either the Rim 3F4 peptide or the JN50
peptide as indicated. ATPase stimulation by
all-trans-retinal (A), amiodarone (B),
digitonin (C), dehydroabietylamine (D), and
2-tert-butylanthroquinone (E) is specific to ABCR
as determined by comparing elution with the Rim 3F4 peptide and the
JN50 peptide. F, ABCR ATPase is stimulated by
all-trans-retinal but not by verapamil, one of the most
potent stimulators of P-glycoprotein ATPase.
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As an additional method for assessing the origin of the ATPase activity
in the reconstituted ABCR preparation, we examined the sensitivity of
basal and all-trans-retinal-stimulated ATPase activity to 1 mM sodium azide, 1 mM ouabain, and a range of
sodium vanadate concentrations. Sodium azide and ouabain at 1 mM had no effect, and sodium vanadate showed a very modest
concentration-dependent inhibition (Fig.
5). These experiments rule out
contamination by two of the most abundant membrane ATPases, the
mitochondrial F1F0-ATPase which is sensitive to
azide, and the Na/K-ATPase which is sensitive to ouabain. Moreover,
all-trans-retinal does not appear to be a general activator
of membrane ATPases as it had no effect on the ATPase activity of crude
mouse brain or liver membranes at concentrations of 100, 50, 25, 12, or
6 µM. Based on (a) the high purity of the ABCR
protein in this preparation, (b) the low ATPase activity and
the lack of ATPase stimulation by all-trans-retinal in
mock-eluted controls, (c) the insensitivity of the basal and
all-trans-retinal-stimulated ATPase activity to azide or
ouabain, and (d) the lack of stimulation of other membrane
ATPases by all-trans-retinal, we believe that both the basal
and all-trans-retinal-stimulated ATPase activities observed here are derived from ABCR and not from contaminating ATPases. In the
text below we will assume that this is the case.

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Fig. 5.
Effect of vanadate, azide, and ouabain on the
ATPase activity of purified and reconstituted ABCR. A,
titration curve of basal and all-trans-retinal-stimulated
ATPase activity at sodium vanadate concentrations up to 250 µM. B, effect of 1 mM sodium azide
or 1 mM ouabain on basal and
all-trans-retinal-stimulated ATPase activity. Both basal and
stimulated ATPase activities are relatively insensitive to the three
inhibitors.
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Factors Affecting the All-trans-retinal-dependent
Stimulation of ATP Hydrolysis--
The ATPase activity of purified and
reconstituted ABCR in the presence or absence of
all-trans-retinal exhibits a broad pH optimum, and the
reaction continues linearly for at least 180 min at 37 °C (Fig.
6, A and B). The
basal and all-trans-retinal-stimulated ATPase activities of
ABCR are sensitive to lipid environment, a property shared with
P-glycoprotein (40). Crude sheep brain PE, brain polar lipid, and
Escherichia coli polar lipid support all-trans-retinal-stimulated ATPase, but egg PC and soybean
PC do not (Fig. 6C and data not shown). In contrast to the
reconstituted sample, purified ABCR solubilized in ROS/CHAPS buffer
shows no stimulation by all-trans-retinal (data not shown),
suggesting that the precise arrangement of protein-lipid contacts is a
critical factor in the ability of retinal to activate the ATPase
activity. The lack of all-trans-retinal stimulation may be
related to the high basal Vmax observed for ABCR
in a mixed CHAPS/lipid environment relative to that in lipid. A similar
difference has been observed between detergent solubilized and
reconstituted P-glycoprotein in ATPase stimulation by both substrates
and sensitizers (26).

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Fig. 6.
Factors affecting basal and
retinal-stimulated ATPase activity of purified and reconstituted
ABCR. A, pH dependence of ATPase in the presence or
absence of 80 µM all-trans-retinal. Sheep
brain PE rather than brain polar lipid was used in the pH titration.
ABCR has a shallow pH dependence in the range pH = 6.0-8.0.
B, time course of ATP hydrolysis in the presence or absence
of 40 µM all-trans-retinal at 37 °C. The
linear time course of ATP hydrolysis over a 3-h incubation implies that
the standard 2-h reaction time used to quantitate ATPase activity is
within the linear range. C, comparison of retinal stimulated
ATPase following ABCR reconstitution with brain polar lipid, brain PE,
or egg PC.
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An analysis of the ATPase activity of ABCR in the presence of varying
ATP concentration shows simple Michaelis-Menten behavior for ATP
regardless of whether ABCR is solubilized in ROS/CHAPS buffer (Fig.
7, A and C) or
reconstituted in brain lipid (Fig. 7, B and D).
In ROS/CHAPS buffer, the Km for ATP is 278 µM, and the Vmax is 27 nmol of
ATP/min/mg ABCR, assuming that all of the ABCR is functional. For
comparison, highly purified P-glycoprotein in detergent has a
Km for ATP of 940 µM and a
Vmax of 321 nmol of ATP/min/mg (26).
Reconstituted ABCR in the absence or presence of 80 µM
all-trans-retinal shows a Km for ATP of
33 or 725 µM and a Vmax of 1.3 or
29 nmol of ATP/min/mg of ABCR, respectively. These data suggest that
one effect of a lipid environment is to constrain the ATP hydrolytic activity of ABCR and that this constraint is lost when the protein is
solubilized in a CHAPS/lipid mixture. In these calculations we assume
that all of the ABCR present in the reaction (and quantitated by
Western blotting) is functionally reconstituted and has equivalent access to ATP. As these assumptions are unlikely to apply, the calculated Vmax represents a lower limit of the
true Vmax, a consideration that makes any
quantitative comparison between solubilized and reconstituted
preparations problematic. In the Lineweaver-Burk plot shown in Fig.
7D the parallel shift of the curve of 1/V
versus 1/[ATP] upon addition of
all-trans-retinal indicates that
all-trans-retinal acts as an "uncompetitive activator"
analogous in effect but opposite in sign to the action of a classical
uncompetitive enzyme inhibitor (41, 42). The simplest interpretation of
these kinetic data is that all-trans-retinal binds to and
alters the ABCR-ATP intermediate at the rate-limiting step in the ATP
hydrolytic pathway.

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Fig. 7.
Km for ATP and
Vmax for ATP hydrolysis determined for
purified ABCR in ROS/CHAPS buffer or following reconstitution in the
presence or absence of all-trans-retinal.
A and C, the ATP concentration dependence of ATP
hydrolysis by purified ABCR in ROS/CHAPS buffer shows simple
Michaelis-Menten behavior. B and D, the ATP
concentration dependence of ATP hydrolysis by purified and
reconstituted ABCR in the absence or presence of 80 µM
all-trans-retinal. C and D show the
Lineweaver-Burk plots derived from the data shown in A and
B, respectively. Based only on the intercepts of the fitted
lines, the Km for ATP and the
Vmax of ATP hydrolysis are as follows: 278 µM and 27 nmol/min/mg (ROS/CHAPS buffer), 33 µM and 1.3 nmol/min/mg (reconstituted, no addition), and
725 µM and 29 nmol/min/mg (reconstituted, plus 80 µM all-trans-retinal).
All-trans-retinal addition therefore increases both the
Km for ATP and the Vmax of
ATP hydrolysis more than 20-fold. In these calculations of reaction
velocity, we assume that all of the ABCR present in the reaction is
functional and has access to ATP. As this assumption is unlikely to
apply, any quantitative comparison between solubilized and
reconstituted preparations is problematic. The calculated
Vmax values therefore represent a lower limit of
the true Vmax. At the 1-3 mM ATP
concentration present in the outer segment the reaction velocity will
be close to Vmax.
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Comparison of the Stimulatory Activities of Different
Retinoids--
To explore the stereochemical specificity of ABCR for
all-trans-retinal, we compared the stimulatory activity of
various retinoids (Fig. 8). The three
geometric isomers of retinal and a photoisomeric mixture of retinal
isomers (containing all-trans-, 7-cis-,
9-cis-, 11-cis-, 13-cis-, and various
di-cis-retinal isomers) are all equally active in ATPase
stimulation (Fig. 8A and data not shown). All-trans-retinol, all-trans-retinoic acid, and
-ionone have stimulatory activity, but in each case it is lower than
that seen with all-trans-retinal (Fig. 8, C and
D). Like amiodarone,
-ionone shows little activity below
20 µM and a progressive increase in activity at least up
to 100 µM. Partially oxidized
all-trans-retinal has a dose-response curve that is
indistinguishable from that of pure all-trans-retinal (Fig.
8B), suggesting that ABCR does not specifically recognize
oxidized retinoids preferentially over all-trans-retinal,
and therefore the activity of all-trans-retinal in this
assay is not due to small amounts of contaminating oxidation products.
We note that ABCR is not efficiently stimulated by the two hydrophobic
nonretinoid aldehydes tested,
-ionone and hexanal (compounds 37 and
40 in Fig. 2), which show little or no activity at 20 µM.

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Fig. 8.
Concentration dependence of stimulation by
various retinoids of the ATPase activity of purified and
reconstituted ABCR. A, all-trans-,
11-cis-, and 13-cis-retinal are equally effective
stimulators of ATPase activity. B,
all-trans-retinal and partially oxidized
all-trans-retinal are equally effective stimulators of
ATPase activity. The inset shows the absorption spectra of
all-trans-retinal before and after overnight oxidation, a
treatment that oxidizes an estimated 10-30% of the
all-trans-retinal. C,
all-trans-retinal, all-trans-retinol, and
all-trans-retinoic acid. D,
all-trans-retinal and -ionone. The various isomers of
retinal are significantly more potent stimulators of ATPase than are
retinol, retinoic acid, or -ionone.
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Synergistic Activation of ABCR by All-trans-retinal, Amiodarone,
and Digitonin--
The different shapes of the dose-response curves
for all-trans-retinal, amiodarone, and digitonin suggest
that these compounds may interact with ABCR in different ways. To
examine this possibility we asked whether mixtures of these compounds
activate ABCR in an additive or nonadditive manner. Fig.
9 shows an experiment in which these and
various other compounds, each at a fixed concentration, were assayed
singly or in combination for their effects on ABCR ATPase. In this
experiment, verapamil and progesterone were included as negative
controls.
-Ionone was included because it produced significant
ATPase activation when present at concentrations equal to or greater
than 50 µM (Fig. 8C).

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Fig. 9.
Synergistic stimulation ATPase activity of
purified and reconstituted ABCR by all-trans-retinal
and a subset of low molecular weight compounds. The bar
graph shows the relative ATPase activity in the absence of added
compounds (basal) or in the presence of 30 µM
all-trans-retinal, 40 µM amiodarone, 50 µM -ionone, 20 µM digitonin, 30 µM progesterone, 30 µM verapamil either
singly or in the indicated combinations. Progesterone and verapamil,
two stimulators of P-glycoprotein ATPase, serve as controls. The
filled regions indicate that part of the ATPase activity
that is in excess of the sum of the activities of the individual
compounds.
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As described above and shown in Figs. 3, 4, and 8, Fig. 9 shows that 30 µM all-trans-retinal, 20 µM
digitonin, and 40 µM amiodarone each activate the ABCR
ATPase 2-5-fold over the basal level, 50 µM
-ionone
induces an activation of less than 1.5-fold, and 30 µM
progesterone or 30 µM verapamil neither activates nor
inhibits the ATPase. Incubation with a combination of 30 µM all-trans-retinal and either 30 µM progesterone or 30 µM verapamil results
in a slight reduction in the ATPase activity relative to retinal alone, whereas incubation with a combination of 30 µM
all-trans-retinal and 50 µM
-ionone results
in a level of activation that is slightly greater than that predicted
by adding the two individual levels of activation. Interestingly,
incubation with 30 µM all-trans-retinal with
either 40 µM amiodarone or 20 µM digitonin
or with a combination of 40 µM amiodarone and 20 µM digitonin produces an increase in ATPase activity
significantly above that predicted for simple additivity. The synergy
obtained with 30 µM all-trans-retinal and a
combination of 40 µM amiodarone and 20 µM
digitonin is no greater than that produced by either 40 µM amiodarone or 20 µM digitonin alone,
suggesting that the latter two compounds synergize with
all-trans-retinal by the same mechanism and that this
synergistic effect is saturated when either amiodarone or digitonin is
present at 40 or 20 µM, respectively. In this experiment,
-ionone at 50 µM is unable to substitute for
all-trans-retinal and produces, at most, a small enhancement
in the ATPase activation when added to reactions containing 30 µM all-trans-retinal or a combination of 30 µM all-trans-retinal and either 40 µM amiodarone or 20 µM digitonin or both.
The combination of 40 µM amiodarone, 20 µM digitonin, and 50 µM
-ionone corresponds approximately
to the sum of the activation levels obtained for the three compounds individually. These data are reminiscent of the synergistic effects on
transport recently reported for some pairs of P-glycoprotein substrates
(43, 44).
To quantitate the concentration dependence of the synergistic
interaction between all-trans-retinal and amiodarone,
digitonin, or
-ionone, experiments were conducted in which one
compound was added at a fixed concentration, and a second compound was added at a variety of concentrations (Fig.
10). In Fig. 10, the degree of synergy
can be assessed by comparing the experimental curve in the presence of
both compounds with the curve calculated for simple additivity. In one
set of experiments the dose-response curve for ATPase activation by
all-trans-retinal was determined in the presence or absence
of 40 µM amiodarone (Fig. 10A), 20 µM digitonin (Fig. 10B), or 50 µM
-ionone (Fig. 10C). In this experiment, a somewhat greater synergy was observed with
all-trans-retinal and
-ionone compared with the
experiment shown in Fig. 9. In a second set of experiments, the
dose-response curve for ATPase activation by amiodarone was determined
in the presence or absence of 100 µM
all-trans-retinal (Fig. 10D) or 20 µM digitonin (Fig. 10E). At this
concentration, all-trans-retinal shows appreciable synergy
with 40 µM amiodarone (Fig. 10A). This
experiment shows that the synergy between all-trans-retinal
and amiodarone is minimal below 20 µM amiodarone and then
rises steeply at amiodarone concentrations greater than 30 µM (Fig. 10D). In Fig. 10E, the
combination of amiodarone and digitonin shows greater than additive
activation of ATPase below 40 µM amiodarone, but this
effect is absent above 40-50 µM amiodarone. The greater
than additive behavior of digitonin and amiodarone at low amiodarone
concentration may simply reflect the nonlinear concentration dependence
of ATPase stimulation by amiodarone alone, as can be seen from the
shape of the amiodarone dose-response curve in Fig. 10E
(e.g. the degree of stimulation by 50 µM
amiodarone is greater than the sum of that produced individually by 20 and 30 µM amiodarone). If the stimulatory mechanism(s) of digitonin and amiodarone are similar, as suggested by their ability to
synergize at higher concentrations with all-trans-retinal
but not with each other, then this could account for the greater than additive behavior of these two compounds at low amiodarone
concentration.

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Fig. 10.
Dose dependence of the synergistic
stimulation of the ATPase activity of purified and reconstituted ABCR
by all-trans-retinal, amiodarone,
-ionone, and digitonin. A-C,
dose-response curves for all-trans-retinal stimulation of
ATP hydrolysis with or without 40 µM amiodarone
(A), 20 µM digitonin (B), or 50 µM -ionone (C). D, dose-response
curve for amiodarone stimulation of ATP hydrolysis with or without 100 µM retinal. E, dose-response curve for
amiodarone stimulation of ATP hydrolysis with or without 20 µM digitonin. Dashed lines indicate the ATPase
activity predicted by simple additivity. The cooperative, multisite
mode of ATPase activation shown by amiodarone alone predicts that
addition of subsaturating concentrations of a second compound with the
same mechanism of action will lead to a greater than additive
stimulation of ATPase at low amiodarone concentration. This effect is
seen upon addition of digitonin (E), with saturation
apparent at higher amiodarone concentration. By contrast, an increasing
synergy in ATPase stimulation is seen with all-trans-retinal
at increasing amiodarone concentrations up to 50 µM, the
highest concentration tested (D). F,
Lineweaver-Burk plot of the data from A showing that, in the
presence of 50 µM ATP, 40 µM amiodarone
increases the Vmax for ATP by 1.7-fold and
decreases the Kapp for
all-trans-retinal from 9.6 to 4.6 µM. The
vertical axis is shown with arbitrary units because the
efficiency of reconstitution and the fraction of ABCR proteins oriented
with the ATPase sites facing the extravesicular space is unknown. For
this analysis, the value of the basal ATPase activity has been
subtracted from the values in the presence of
all-trans-retinal values, and the ATPase activity referable
to amiodarone alone has been subtracted from the values in the presence
of all-trans-retinal plus amiodarone.
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Kinetic Analysis of ATPase Stimulation by All-trans-retinal and
Amiodarone--
To explore the mechanism by which amiodarone, retinal,
and the combination of the two compounds stimulate ATP hydrolysis by ABCR, we measured the effect of amiodarone on the apparent affinity for
all-trans-retinal and the effect of
all-trans-retinal and amiodarone individually and in
combination on the Km for ATP and the
Vmax for ATP hydrolysis (Figs. 10F
and 11). A Lineweaver-Burk plot of the relationship between the rate of
ATP hydrolysis at 50 µM ATP and the concentration of
all-trans-retinal (Fig. 10F) shows by its fit to
a straight line that all-trans-retinal acts without
cooperativity at a single class of binding site. The 1.7-fold increase
in Vmax produced by the addition of 40 µM amiodarone is associated with a 2-fold increase in the
apparent affinity (a decrease in Kapp from 9.6 to 4.6 µM) of all-trans-retinal for this site
or sites. (We refer to this value as an apparent affinity or
Kapp rather than a true Km
because the binding and release of all-trans-retinal may be
complex and the reaction was not performed at saturating ATP
concentration.)
As revealed by a Lineweaver-Burk analysis, amiodarone and
all-trans-retinal alter the Km of ATP and
the Vmax for ATP hydrolysis via distinct
mechanisms (Fig. 11). The principal effect of amiodarone (at 40 µM) is to increase
Vmax with only a modest change in
Km for ATP, as measured from the intercept of the
best fitting straight line and the horizontal axis, which corresponds
to
1/Km. Amiodarone addition in the absence of
all-trans-retinal lowers the Km for ATP
by approximately 2-fold (Fig. 11B), whereas amiodarone
addition in the presence of 100 µM
all-trans-retinal raises the Km for ATP
approximately 2-fold (Fig. 11C). Importantly, these changes
are accompanied by a 2-fold decrease in the ratio
Km/Vmax (the slope of the
best fitting straight line), an effect that is analogous, but opposite
in sign, to that of classical noncompetitive enzyme inhibitors. We will
therefore refer to amiodarone as a "noncompetitive activator". The
simplest model to account for the kinetic behavior of amiodarone is one
in which ABCR is assumed to exist in an equilibrium mixture of
enzymatically active and inactive species. By analogy with the
diminution in the number of active enzymes resulting from the action of
a noncompetitive inhibitor, we propose that amiodarone binding shifts
the equilibrium between active and inactive enzymes in favor of the
active species.

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Fig. 11.
The effect of
all-trans-retinal, amiodarone, or a combination of the
two on the Km for ATP and
Vmax of ATP hydrolysis for purified and
reconstituted ABCR. A, ATP hydrolysis at different ATP
concentrations. In these calculations of reaction velocity we assume
that all of the ABCR present in the reaction is functional and has
access to ATP. B-D, Lineweaver-Burk plots comparing the
effect of no addition versus amiodarone (B),
all-trans-retinal versus
all-trans-retinal plus amiodarone (C), and
amiodarone versus amiodarone plus
all-trans-retinal (D). Concentrations of
amiodarone and all-trans-retinal are indicated in each
panel. It is apparent that amiodarone addition produces a shallower
best-fitting line to the data points, i.e. it lowers the
slope (Km/Vmax), whereas
all-trans-retinal addition produces a downward shift of the
best-fitting line, i.e. the slope
(Km/Vmax) is unchanged.
Comparison of B with C and D with Fig.
7D indicates that amiodarone and
all-trans-retinal act independently. Based only on the
intercepts of the fitted lines, the Km for ATP and
the Vmax for ATP hydrolysis in the presence of
the indicated compounds are as follows: 33 µM and 1.3 nmol ATP/min/mg (no addition), 18 µM and 2 nmol
ATP/min/mg (amiodarone), 400 µM and 20 nmol ATP/min/mg
(all-trans-retinal), 666 µM and 50 nmol
ATP/min/mg (all-trans-retinal plus amiodarone). In this
experiment and the one shown in Fig. 7D, the
Km and Vmax values should be
considered less reliable when obtained only from the intercepts of
those fitted lines that lie close to the origin. More reliable values
are obtained from the intercepts of the fitted lines that lie further
from the origin and from the slope
(Km/Vmax) of the fitted
lines.
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In contrast to amiodarone, the effect of all-trans-retinal,
either alone or in combination with amiodarone, is to increase the
Vmax for ATP hydrolysis and the
Km for ATP by the same factor, thereby leaving the
ratio Km/Vmax (the slope of
the best fitting straight line) unchanged (Figs. 7D and
11D). This effect on kinetic parameters is analogous but
opposite in sign to that displayed by classical uncompetitive enzyme
inhibitors, and we will therefore refer to all-trans-retinal
as an uncompetitive activator. The simplest model that accounts for
this kinetic behavior is one in which all-trans-retinal
interacts specifically with an intermediate in the ATPase reaction
pathway. By analogy with the specific trapping of enzyme-substrate
intermediates produced by the action of a classical uncompetitive
enzyme inhibitor, we propose that the binding of
all-trans-retinal creates an accelerated pathway for ATP
hydrolysis. The data can also be formalized along the lines of a
double-displacement ("ping pong") kinetic scheme in which the
progress of one substrate, ATP, through the reaction pathway is
accelerated by the binding of the second component, all-trans-retinal, at an intermediate point in the pathway
(41, 42).
The two different modes of interaction between ABCR and amiodarone or
all-trans-retinal predicts that their combined effects will
by multiplicative rather than additive, and therefore accounts in a
simple way for their ability to act simultaneously and synergistically on ABCR. The simple Michaelis-Menton behavior and uncompetitive mode of
ATPase activation exhibited by all-trans-retinal strongly suggest that this compound is a transport substrate. By contrast, the
cooperative behavior and the noncompetitive mode of ATPase activation
exhibited by amiodarone implicate this compound as an allosteric
effector. These observations suggest that the division of ATPase
activators into noncompetitive and uncompetitive classes may prove to
be generally useful in the study of other ABC transporters.
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DISCUSSION |
Purification and Reconstitution of ABCR--
The experiments
reported here define a purification procedure and a set of lipid,
detergent, and buffer conditions that permit the isolation of nearly
homogenous ABCR and its reconstitution into lipid membranes in
functional form. The purified and reconstituted preparation has ATPase
activity that appears to derive from ABCR rather than from a
contaminating ATPase. The stimulated ATPase activity of ABCR is highly
sensitive to its local membrane environment, as has been observed for
P-glycoprotein (40). PE supports functional reconstitution as do lipid
mixtures containing PE but PC does not. When ABCR is solubilized in a
mixture of PE, PC, and CHAPS (ROS/CHAPS buffer) it retains ATPase
activity, but this ATPase is not stimulated by
all-trans-retinal. This effect of CHAPS is unlikely to
reflect denaturation of ABCR, since sensitivity to all-trans-retinal appears upon transfer of ABCR from this
CHAPS/lipid mixture to brain lipid alone. These observations suggest
instead that the mixed CHAPS/lipid micelle permits cycles of ATP
hydrolysis that are uncoupled from conformational events at the site to
which all-trans-retinal binds.
Retinal Stimulation of ABCR ATPase--
The finding that
all-trans-retinal (and other geometric isomers of retinal)
stimulates the ATPase activity of ABCR can be explained by any of three
mechanisms as follows: (a) all-trans-retinal exerts a general effect on the lipid environment that alters the conformation of ABCR; (b) all-trans-retinal binds
to ABCR as an allosteric effector of the ATPase cycle but is not itself
a substrate for transport; and (c)
all-trans-retinal is a substrate for transport, and its
presence accelerates ATP hydrolysis by a conformational coupling
between the transport domain and one or both ATP binding domains.
The first mechanism is unlikely because (a) it does not
account for the specificity of ATPase stimulation by
all-trans-retinal relative to the other compounds tested,
(b) it seems implausible that retinal should exert an effect
on bulk membrane properties in a reaction in which lipid is present at
1-1.5 mg/ml and retinal is present at 10 µM (equivalent
to 3.3 µg/ml), and (c) a quantitative analysis of the
concentration dependence of all-trans-retinal stimulation
indicates that it acts with simple Michaelis-Menten behavior.
The second mechanism, if correct, would imply a novel strategy for
modulating the activity of a rod outer segment protein, i.e.
sensing the concentration of free retinal or a related retinoid. Under
this assumption, ABCR activity might be modulated by the level of
all-trans-retinal released by photoactivated rhodopsin or of
free 11-cis-retinal imported from the RPE for rhodopsin regeneration. This mechanism would be in contrast to the regulatory mechanisms identified thus far among outer segment proteins that involve phosphorylation or binding to calcium, GTP, or cGMP. Arguing against a simple allosteric mechanism is the effect of
all-trans-retinal on the Km for ATP and
the Vmax for ATP hydrolysis which indicates that
all-trans-retinal acts via an uncompetitive interaction with ABCR.
The third possible mechanism, that various geometric isomers of retinal
and/or other retinoids are transported by ABCR in a reaction that is
coupled to ATP hydrolysis, can only be definitively distinguished from
an allosteric mechanism by directly demonstrating ATP-dependent vectorial transport of these compounds in a
reconstituted membrane system. At present, the strongest evidence in
favor of this hypothesis is the observation that
all-trans-retinal acts without cooperativity at a single
class of binding site(s) on ABCR and exhibits uncompetitive stimulation
of ATPase activity. This kinetic behavior implies that
all-trans-retinal binds to an intermediate in the ATPase
reaction pathway and that this binding accelerates a rate-limiting step
in ATP hydrolysis and/or release of the hydrolysis products. This
behavior is precisely that predicted for a transported substrate, and
it was not observed with any of the 37 nonretinoid compounds tested in
this study. The localization of ABCR to the rod disc membrane also
favors retinal (and/or retinol) as the natural ligand since it predicts
that the endogenous ligand should be present within ROS, a property
fulfilled by all-trans-retinal, 11-cis-retinal,
and all-trans-retinol. Given the relatively simple chemical
composition of ROS (45) and the absence, at present, of any data
implicating other transport processes involving the photoreceptor disc
membrane, there are few competing candidate substrates for
ABCR-mediated transport. Finally, we also favor this hypothesis
because, as described below, various pieces of circumstantial evidence
suggest that retinal may require active transport across or extraction
from the disc membrane and that Stargardt disease is associated with an
accumulation of retinal or its derivatives in ROS.
Retinoid Metabolism in the Rod Outer Segment--
Photoactivation
of rhodopsin results in the hydrolysis and release of
all-trans-retinal which is reduced within the outer segment
to all-trans-retinol by all-trans-retinol
dehydrogenase (named for the reverse reaction).
All-trans-retinol is transported to the RPE where it is
esterified, isomerized to the 11-cis-configuration, released
from the ester linkage, and returned to the outer segment (46). Many of
the steps in this process are still poorly understood. In particular,
most of the enzymes involved have yet to be purified, and it is not
known which steps involve active transport or passive diffusion. The
flux of retinal through this pathway can be extremely high under
physiological conditions: viewing the blue sky on a cloudless day
produces 20,000 photoisomerizations per rod/s in the human retina
(47).
Analysis of mice that were exposed to physiological light levels
indicates that reduction of all-trans-retinal is the
rate-limiting step in the visual cycle (48); following a light flash or
under constant illumination, all-trans-retinal accumulates
in the retina without a concomitant accumulation of other retinoids.
In vitro experiments suggest that this accumulation may
increase photoreceptor noise because the combination of free
all-trans-retinal and opsin produces a partially active
species (49-52). By contrast, electrophysiological analyses of single
photoreceptor cells have failed to reveal a desensitizing effect of
exogenously applied all-trans-retinal (reviewed in 53),
suggesting either that the in vitro effect is of little
physiological relevance, that exogenous all-trans-retinal cannot readily gain access to opsin, or that there are mechanisms in
the intact cell that minimize the desensitizing effect of
all-trans-retinal. If ABCR facilitates the reduction of
all-trans-retinal by all-trans-retinol dehydrogenase, then one might predict that ABCR dysfunction would lead
to an increase in photoreceptor noise. In this regard, it is
interesting that individuals with Stargardt disease show an elevated
rod threshold and a delayed recovery of rod sensitivity following light
exposure (54).
When all-trans-retinal is released from photoactivated
rhodopsin, it presumably enters the hydrophobic environment of the disc
membrane. In the disc membrane, free all-trans-retinal can react to form a Schiff base with the amine present in PE (55, 56),
which constitutes 40% of ROS lipid (45). If we assume that Schiff base
adducts between all-trans-retinal and PE can form on either
the luminal or the cytosolic face of the disc membrane then it seems
plausible to suppose that those adducts on the cytosolic face will be
available for reduction by all-trans-retinol dehydrogenase, whereas those on the lumenal face will be sequestered from the enzyme
active site. This line of reasoning assumes that the active site of
all-trans-retinol dehydrogenase has access to substrates associated with only one side of the disc membrane, consistent with the
known asymmetric disposition of membrane-associated proteins. In the
absence of an active mechanism for transmembrane flipping of the
PE-all-trans-retinal Schiff base adduct, we would predict that this adduct would accumulate on the luminal face of the disc membrane. Its abundance there would presumably reflect the rate of
spontaneous hydrolysis of the PE-all-trans-retinal Schiff
base and subsequent equilibration of all-trans-retinal to
the cytosolic leaflet of the disc membrane.
A Model for ABCR in the Visual Cycle--
The considerations
outlined in the preceding three paragraphs, together with the
localization of ABCR to the disc membrane and the
retinal-dependent stimulation of ATP hydrolysis by ABCR, suggest that ABCR acts in the visual cycle to flip
PE-all-trans-retinal adducts from the lumenal to the
cytosolic face of the disc membrane, move free
all-trans-retinal from the lipid phase of the disc membrane to a juxtamembrane location, or possibly reorient
all-trans-retinal in the bilayer (Fig.
12A). In the first case,
ABCR activity would resemble the PC flippase activity of mouse MDR-2
(57) and human MDR3 (58), whereas in the latter cases, ABCR activity
would resemble the drug extrusion activity of P-glycoprotein,
especially as envisioned by the "hydrophobic vacuum cleaner model"
(59) in which P-glycoprotein is proposed to extract hydrophobic
compounds from the lipid phase and deliver them to the extracellular
space. In each case, we hypothesize that the effect of ABCR activity would be to deliver more efficiently all-trans-retinal to
all-trans-retinol dehydrogenase, leading to lower
photoreceptor noise, more rapid recovery following illumination, and
decreased accumulation of all-trans-retinal or its adducts
within the disc membrane. In these models of ABCR action, the proposed
vectorial movement is topologically equivalent to importing a substrate
from the extracellular space, a direction of transport opposite to the
direction of lipid flippase activity demonstrated for mouse MDR2 (57)
and the human MDR1 and MDR3 P-glycoproteins (58) and opposite to the
direction of drug extrusion mediated by P-glycoprotein. Substrate
import by ABC transporters is commonly observed in bacteria but has
thus far not been reported in multicellular organisms (60).

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Fig. 12.
Possible models for ABCR action in the
visual cycle and for the pathogenesis of Stargardt disease.
A, model of the rod outer segment showing the edge of one
outer segment disc and the adjacent plasma membrane. A hypothetical
path of all-trans-retinal and all-trans-retinol
movement following rhodopsin photobleaching is indicated by
arrows in the upper half of the figure. In this
model all-trans-retinal is released into the disc membrane
and is transported or presented to all-trans-retinol
dehydrogenase on the cytosolic face of the disc by ABCR. ABCR may also
facilitate the export of all-trans-retinol by extracting it
from the disc membrane as postulated for the action of P-glycoprotein
and its hydrophobic substrates by the hydrophobic vacuum cleaner model
(59). Ultimately, all-trans-retinol is exported across the
plasma membrane to the adjacent RPE. A hypothetical path of
11-cis-retinal movement is indicated by arrows in
the lower half of A. If 11-cis-retinal
partitions into the disc membrane, ABCR may facilitate its removal from
the lumen of the disc and/or its binding to opsin to regenerate
rhodopsin. Whether retinal is released from or attached to opsin with a
specific orientation or location relative to the membrane is currently
unknown. B, model for the accumulation of shortwave
absorbing material in Stargardt disease. The distal region of a rod
outer segment and the adjacent RPE cell is shown. Left, in
the normal retina there is minimal accumulation of retinoid derivatives
in the disc membrane and therefore only a slow accumulation of these or
related derivatives in the RPE. Right, in the Stargardt
disease retina, retinoids accumulate in the disc membrane (represented
by a heavy outline of the disc membrane), and these or
related derivatives accumulate in the RPE in phagolysosomes containing
ingested ROS (represented by filled cytoplasmic
inclusions).
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These models may account for the presence of ABCR in rods but not
cones, a difference that may be related to differences in the visual
cycle between these cell types (61). Since the lumenal face of the cone
disc membrane remains contiguous with the plasma membrane, free
all-trans-retinal within the cone disc membrane or
all-trans-retinal-PE adducts formed on the lumenal face of the cone disc could react with other amines in the extracellular space
and/or bind to extracellular proteins such as interphotoreceptor retinol-binding protein.
The stimulation of ATPase activity by 11-cis-retinal and to
a lesser extent by all-trans-retinol suggests that ABCR may
also play a role in the movement of these compounds (Fig.
12A). The facile reaction of 11-cis-retinal and
opsin in vitro has generally been interpreted to mean that
this reaction does not require additional cofactors or catalysts
in vivo. However, it is possible that in vivo the
selective pressure for rapid and complete regeneration, which is
thought to be one of the rate-limiting steps in dark adaptation, has
led to the evolution of enzymes that accelerate this reaction. The
relative abundance of ABCR together with the rapid lateral diffusion of
opsin within the disc membrane (62) should permit frequent encounters
between these proteins. Whereas any models for the role of ABCR in this
reaction are necessarily speculative, if ABCR facilitates the binding
of 11-cis-retinal to opsin, an ABCR-dependent
acceleration in regeneration rate should be demonstrable in
vitro.
Implications for the Pathogenesis of Stargardt Disease and Other
Retinopathies--
The work presented here supports the idea that
Stargardt disease and other retinopathies caused by mutations in ABCR
arise from defects in retinoid metabolism (Fig. 12B). This
idea was originally proposed as an explanation for the delayed recovery
of rod sensitivity observed in Stargardt disease patients (54). The
persistence of this delay in rod recovery despite high levels of
dietary vitamin A (63) distinguishes it from classical night blindness
of dietary origin (64), and more specifically suggests a model in which the defect in Stargardt disease is not related to the availability of
11-cis-retinal but rather to a defect in removing the
product of photobleaching, all-trans-retinal.
The model outlined above may also be relevant to recent structural
analyses of the fluorescent, shortwave absorbing compounds that
accumulate with age in the human RPE. These analyses show that the
major component is a diretinal-ethanolamine derivative, A2-E (65, 66).
A2-E has been hypothesized to form from the condensation of
all-trans-retinal and PE in the outer segment and then to
accumulate over time in the RPE as a residual product of ROS digestion
(65). Although it is not known definitively whether A2-E comprises the
fluorescent, shortwave absorbing material that accumulates to high
levels in the RPE of Stargardt disease patients, one in vivo
study suggests that this compound or a similar one is present at high
concentration in these patients (67, 68).
The severe defect in rod vision characterized by homozygosity for ABCR
null mutations is in contrast to the milder impairment of rod vision
experienced by many patients with Stargardt disease. If ABCR is
important only as a transporter and not as a structural protein, then
the clinical data would suggest that a complete absence of
ABCR-mediated transport leads to the death of rod photoreceptors, the
final common event in retinitis pigmentosa.
Implications for Pharmacologic Intervention in
Retinopathies--
The in vitro assay described here may
lend itself to the identification of compounds that either enhance or
diminish the activity of ABCR. Those that enhance ATPase activity might
represent lead compounds for drug development with the goal of
enhancing ABCR activity in patients with Stargardt disease or in a
subset of individuals at risk for age-related macular degeneration.
In vitro, such compounds might increase the ATPase activity
of ABCR when added alone or in combination with a transported compound.
We note, however, that this assay represents only a screening tool; any
interpretation of altered ATPase activity will require a detailed understanding of the mechanism of action of the test compound because,
in theory, ATPase activity could be increased by either promoting or
circumventing the normal transport cycle. Although we cannot make any
firm predictions regarding the in vivo effect on ABCR
function of compounds like amiodarone and digitonin, the existence of
such compounds suggests that environmental or drug effects may be
relevant to degenerative retinal diseases. In this context, it may be
of interest to study visual function in patients receiving amiodarone,
a drug that has been widely used in the treatment of cardiac
arrhythmias at tissue concentrations at or above 40 µM
(69).