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The Journal of Neuroscience, March 15, 2000, 20(6):2209-2217
Mice Lacking G-Protein Receptor Kinase 1 Have Profoundly Slowed
Recovery of Cone-Driven Retinal Responses
A. L.
Lyubarsky1,
C.-K.
Chen2,
M. I.
Simon2, and
E. N.
Pugh Jr1
1 Department of Ophthalmology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, and
2 Division of Biology, California Institute of Technology,
Pasadena, California 91125
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ABSTRACT |
G-Protein receptor kinase 1 (GRK1) ("rhodopsin kinase") is
necessary for the inactivation of photoactivated rhodopsin, the light
receptor of the G-protein transduction cascade of rod photoreceptors. GRK1 has also been reported to be present in retinal cones in which its
function is unknown. To examine the role of GRK1 in retinal cone
signaling pathways, we measured in mice having null mutations of GRK1
(GRK1  /) cone-driven electroretinographic (ERG) responses,
including an a-wave component identified as the field potential
generated by suppression of the circulating current of the cone
photoreceptors. Dark-adapted GRK1 / animals generated cone-driven
ERGs having saturating amplitudes and sensitivities in both visible and
UV spectral regions similar to those of wild-type (WT) mice. However,
after exposure to a bright conditioning flash, the cone-driven ERGs of
GRK1 / animals recovered 30-50 times more slowly than those of WT
mice and similarly slower than the cone-driven ERGs of mice
homozygously null for arrestin (Arrestin /), whose cone (but not
rod) response recoveries were found to be as rapid as those of WT. Our
observations argue that GRK1 is essential for normal deactivation of
murine cone phototransduction and provide the first functional evidence
for a major role of a specific GRK in the inactivation of vertebrate
cone phototransduction.
Key words:
G-protein coupled receptor; G-protein coupled
receptor kinase; phototransduction; cone photoreceptors; photoresponse recovery kinetics; mouse genetics; electroretinography
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INTRODUCTION |
The initial amplifier in visual
transduction is a photoactivated state (R*) of a photopigment
G-protein-coupled receptor (GPCR); in vertebrate rod photoreceptors,
the GPCR is rhodopsin. Biochemical evidence has long supported the
conclusion that inactivation of R* in vertebrate rod photoreceptors
involves phosphorylation by the G-protein-coupled receptor kinase GRK1
("rhodopsin kinase") of one or more serine residues on the
rhodopsin C terminus (Wilden et al., 1986 ; Kuhn
and Wilden, 1987; Ohguro et al., 1996).
Proof that GRK1 binding to and/or phosphorylation of R* is required for
normal inactivation of R* in rods in vivo has come from
recordings of photocurrent responses of single rods of mice expressing
mutant rhodopsin lacking the C terminus (Chen et al., 1995) and more recently of responses of rods from mice having null mutations in GRK1 (CK Chen et al., 1999).
The expression of GRK1 in human rod and cone photoreceptors has been
documented (Zhao et al., 1998). Lack of functionally active GRK1 in humans has been shown to cause congenital night blindness (Oguchi disease) characterized by profoundly slowed rod dark
adaptation (Yamamoto et al., 1997). A human patient with a homozygous null mutation in GRK1 has been shown to have a reliable but "slight slowing of cone deactivation kinetics"
(Cideciyan et al., 1998) and, in evaluating the latter
evidence, the hypothesis has been proposed that human cones might rely
mainly on pigment regeneration for deactivation of the photoactivated
pigment (Cideciyan et al., 1998).
To further examine the possible role of GRK1 in mammalian cone
function, we have investigated the cone-driven responses of mice with
the GRK1 gene inactivated (CK Chen et al., 1999).
Wild-type (WT) mice and mice with the arrestin gene inactivated
(Arrestin /) (Xu et al., 1997) were also
investigated. Arrestin / mice serve as controls not only for the
genetic inactivation of a retina-specific (and rod-specific)
protein involved in R* inactivation but also for the adaptational state
produced by the very strong and prolonged activation of the rods in the
GRK1 / mice (relative to WT) during the experiments.
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MATERIALS AND METHODS |
All experimental procedures were done in compliance with
National Institutes of Health guidelines, as approved by the respective Institutional Animal Care and Use Committees of the University of
Pennsylvania and the California Institute of Technology. GRK1 /
mice and Arrestin / were derived at the California Institute of
Technology on C57BL/6-129/SvJ background as described previously (Xu et al., 1997; CK Chen et al., 1999).
C57BL/6 (Charles River Laboratories, Wilmington, MA) mice were used to
derive WT controls. All animals used for electroretinographic (ERG)
recordings were born and maintained under controlled ambient
illumination on a 12 hr light/dark cycle with the illumination level at
2.5 photopic lux as described previously (Pugh et al.,
1998; Lyubarsky et al., 1999). ERG recordings
were made when animals were between 8 and 12 weeks of age. Mice used
for histological analysis were raised in complete darkness (CK
Chen et al., 1999).
In situ hybridization. Eyes of killed mice were
enucleated, and eyecups were prepared by removal of the cornea and
lens. The eyecups were fixed overnight at 4°C with 4%
paraformaldehyde in PBS, pH 7.4, and then impregnated with 30%
sucrose in PBS at 4°C for 8 hr. They were then embedded and frozen in
OCT (Sakura Finetek, Torrance, CA). Frozen sections 15-µm-thick were
cut and mounted on precleaned superfrost slides (Fisher Scientific,
Houston, TX). The first 660 nucleotides of the murine GRK1 coding
sequences was cloned into pGEM-T vector (Promega, Madison, WI)
and served as a template for in vitro transcription. An
SP6/T7 transcription kit and DIG RNA Labeling Mix (Boehringer Mannheim,
Indianapolis, IN) were used to produce digoxigenin-labeled sense and
antisense riboprobes of GRK1 according to the manufacturer's
instructions. Sections were washed three times with PBS, 5 min
each, before prehybridization in Hyb buffer [50% formamide, 5× SSC
(0.9 M NaCl and 90 mM sodium citrate, pH 7.0),
1 mg/ml yeast RNA, 100 gm/ml heparin, 1× Denhardt's solution (0.1%
w/v each Ficoll, polyvinylpyrrolidone, and bovine serum albumin), 0.1%
Tween 20, 0.1% CHAPS, and 5 mM EDTA] at 65°C for
4-8 hours. Probes were used at a concentration of 100 ng/ml in Hyb
buffer. Hybridization was performed at 65°C for 36-48 hr. After
hybridization, the sections were washed in 5× SSC at 60°C for 5 min
and then washed three times in 0.2× SSC at 60°C for a total of 60 min. After cooling to room temperature, the sections were washed with
MAB buffer (0.1 M maleic acid, pH 7.5, and 0.15 M NaCl) for 5 min and then blocked by 1% BLOCK (Boehringer Mannheim) in MAB buffer at room temperature for 2 hr. To detect the
hybridized probes, alkaline phosphatase-conjugated anti-digoxigenin antibody (used at 1:5000 dilution; Boehringer Mannheim) was added to
the sections and incubated at room temperature for 2 hr. The sections
were then washed twice with MAB buffer, 30 min each, and washed once
for 5 min with AP buffer (0.1 M Tris-HCl, pH 9.5, 50 mM MgCl2, and 100 mM NaCl).
Sixty-seven microliters of nitroblue-tetrazolium-chloride (50 mg/ml;
Promega) and 35 µl of 5-bromo-4-chloro-indolyl phosphate (50 mg/ml; Promega) were added to 10 ml of AP buffer, and the resulting
solution was used to develop the sections immediately after the washes.
Typical developing time for GRK1 was 12-24 hr. To stop the
development, the sections were washed in TE buffer (10 mM
Tris-HCl, pH 7.5, and 10 mM EDTA) and sealed in 50%
glycerol with coverslips.
Immunocytochemistry. Eyecups of mice raised in complete
darkness were fixed with 4% paraformaldehyde at 4°C for 1 hr,
impregnated with 30% sucrose in PBS, embedded, and frozen in OCT.
Sections were cut 15-µm-thick and mounted on precleaned superfrost
slides. The sections were washed once with PBS for 5 min before
blocking by 1% BLOCK in MAB buffer for 1 hr at room temperature.
GRK1-specific polyclonal antibody 8585 (generously provided by Dr.
Robert Lefkowitz, Duke University, Durham, NC) was used at 1:100
dilution and biotin-labeled peanut agglutinin (PNA) (Vector
Laboratories, Burlingame, CA) was used at 1:200 dilution in 1% BLOCK
in MAB buffer. After blocking, the sections were incubated with 8585, PNA, or both at room temperature for 1 hr and then washed with PBS
three times for a total of 15 min. Sections stained with 8585 were then
incubated in dark at room temperature for 1 hr with
fluorescein-conjugated goat antibody against rabbit IgG (1:100
dilution; Vector Laboratories). Sections stained with biotin-labeled
PNA were incubated in dark at room temperature for 1 hr with Texas
Red-conjugated streptavidin (used at 1:100 dilution; Life
Technologies, Gaithersburg, MD). Sections stained with both 8585 and biotin-labeled PNA were incubated in darkness at room temperature
with both the fluorescein-conjugated goat antibody against rabbit IgG
and Texas Red-conjugated streptavidin. After incubation, sections were
then washed three times with PBS for a total of 30 min and sealed in
VECTASHIELD (Vector Laboratories) with coverslips. Micrographs were
taken with an Axiovert 35 microscope (Zeiss, Oberkochen, Germany)
equipped with incident-light fluorescence.
Electroretinography. The experimental apparatus, methods of
light stimulation and quantification, electroretinogram (ERG) recording, and cone signal isolation have been described in detail previously (Lyubarsky and Pugh, 1996; Lyubarsky
et al., 1999). In brief, mice that had been dark adapted
overnight were anesthetized under dim red light with an intraperitoneal
injection of a solution containing (in µg/gm body weight): 25 ketamine (KetaJect; Phoenix Pharmaceutical, Mountain View, CA), 10 xylazine (XylaJect; Phoenix Pharmaceutical), and 1000 urethane (Sigma,
St. Louis, MO). The anesthetized animal was immobilized in a holder so
that the right eye was pointing upward; the pupil of the right eye was
then dilated with 1% tropicamide solution (Mydriacil; Alconox, New
York, NY). Next, a drop of methylcellulose solution (Goniosol; Iolab
Pharmaceutical, Indianapolis, IN) was placed on the eye for protection
and electrical contact, and a recording platinum wire electrode was put
into electrical contact with the cornea. A tungsten needle reference electrode was next inserted subcutaneously on the forehead. The holder
with the animal was then placed inside a light-proof aluminum Faraday
cage whose interior was lined with aluminum foil to maximize UV
reflectivity (Lyubarsky et al., 1999). Light stimuli
were delivered through several ports in the walls of the cage. The
intensity of stimulation and its spectral composition were controlled
with neutral density and bandpass interference filters. For the latter, the wavelength cited corresponds to the median of the filter spectral transmission function (Lyubarsky et al., 1999). Baffles
against the ports were used to avoid direct illumination of the
tested eye. At the position occupied by the mouse eye during
experiments, directional variations of light intensity did not
exceed ±15% of the average intensity.
ERGs were amplified, bandpass filtered at 0.1-1000 Hz, sampled at 5 kHz, averaged, and stored on a personal computer using a Digidata 1200 acquisition board and Axotape2 software (Axon Instruments, Foster City,
CA). For some analyses, oscillatory potentials (Gorgels and
Norren, 1992; Peachey et al., 1993;
Lyubarsky et al., 1999) were removed by digital
filtering with a gaussian filter having a bandwidth of 11 Hz (3 dB). We
refer to the peak amplitude of the response filtered in this manner as
the cone b-wave magnitude (bmax). Before
experiments, mice were dark-adapted overnight and all the preparations
were performed under dim red light. Before electrical recordings
commenced animals were kept in the Faraday cage in complete darkness
for at least 15 min. The overall duration of a recording session was
80-100 min.
Stimulus quantification: conversion of light stimuli to numbers
of photoisomerizations and relative amounts of visual pigment isomerized in rods and cones. Previous work has established that, in addition to rhodopsin in rods, the mouse retina expresses two different cone visual pigments with  max values near 360 and 510 nm in two types of cones having distinctive regional
distributions over the retina (Jacobs et al.,
1991; Szel et al., 1992; Calderone and Jacobs, 1995; Lyubarsky et al., 1999). We
identify the two types of cones as the "M cones" and the "UV
cones" after the dominant species of pigment in each.
For a monochromatic light stimulus isomerizing a small fraction ( 1)
of pigment in a specific class of photoreceptor, the number ( ) of
photoisomerizations per photoreceptor produced by a light flash in a
ganzfeld is proportional to the quantal flux density Q()
(photons µm 2, at the cornea):
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(1)
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where Apupil and
Aretina are the areas of the mouse pupil and
retina, respectively,  SC is a scale factor (<1)
introduced to account for the Stiles-Crawford effect of the first kind
(Stiles and Crawford, 1933; Snyder and Pask,
1973), aC() is the collecting area at
the retina for the specific photoreceptor class, and  () is the
transmissivity of the prereceptor eye media. A standard expression for
aC() has been developed by Baylor et
al. (1979, their Eq. 14) and was adopted previously with
appropriate parameter values for the mouse (Lyubarsky and Pugh,
1996; Lyubarsky et al., 1999, their Eq. 5). In
our previous presentation of Equation 1, the factor SC
for the Stiles-Crawford effect was not made explicit but was discussed
as modifying the effective pupil area (Lyubarsky et al.,
1999).
One can collapse the eye- and wavelength-dependent terms of Equation 1
into two parameters: one for the efficacy of light at the wavelength of
maximum absorbance (max) and a second accounting for the spectral sensitivity of the receptor, i.e., the sensitivity at
the variable wavelength . Thus,
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(2)
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where
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(3)
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is the apparent collecting area of the specific photoreceptor
type at the cornea in a ganzfeld for light of wavelength , and
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(4)
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is the normalized spectral sensitivity at the cornea of the
photoreceptor under consideration. A potential problem with these formulas is the neglect of the Stiles-Crawford effect of the second kind, i.e., the wavelength dependence of SC. This
dependence is not known for mouse, but in experiments using very
broadband flashes it can be expected that such dependence will have
only a second-order effect on our calculations, relative to the SC-I effect for wavelengths near the cone max.
Because we used broadband ("white") flash stimulation, the
calculation of the number of photoisomerizations per flash must sum the
effects of different wavelengths. Thus, Equation 2 must be modified as
follows:
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(5)
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where q() is spectral density of quantal flux
(photons µm2 nm1 at the
cornea), measured as described by Lyubarsky et al.
(1999) at the position of the mouse cornea in the recording
chamber. We used Equation 5 with the measured values of the spectral
flux density and with spectral sensitivities and parameter values as described in the succeeding paragraph to estimate for all flashes used in the experiments reported here.
We will assume the following values and conventions for parameters in
Equations 1-5, as explained previously (Lyubarsky and Pugh,
1996): collecting areas at the visual pigment
max values at the retina
aC(max), 1.3 µm2 for rods and 2.4 µm2 for
cones;
Apupil/Aretina = 0.22 (fully dilated pupil) (Pennesi et al., 1998);
SC = 1 for rods and 0.33 for cones;
max of 498 nm for rods, and for cones, 355 and 508 nm,
and normalized cone spectral sensitivities,  (), as
described previously (Lyubarsky et al., 1999, their Fig.
6B). The prereceptor spectral transmissivity ()
of the eye media for the mouse has not been measured. As an improved
approximation for the transmissivity, we have used data on the spectral
transmittance of the rat lens in the spectral range of 330-700 nm
reported by Gorgels and Norren (1992) assuming that (1)
the lens, which occupies most of the preretinal optical path, is the
major prereceptor light absorber and (2) absorption by the lens in the
mouse eye is half of its value in rat, because the size of the mouse
eye is about half that of the rat (Remtulla and Hallett,
1985). With these assumptions, (max)
is 0.78 at the peak of the UV pigment and 0.96 at the peak of the M
pigment, and
aC,cornea(max) are 0.14 and 0.17 µm2 for the UV and M cones, respectively.
The fraction of visual pigment isomerized,
fisomerized, in each receptor class by a
specified flash was computed from the estimated number of
photoisomerizations as follows:
|
(6)
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where Ntotal is the total number of
pigment molecules per photoreceptor, C the is molar
concentration of visual pigment in the outer segment (~3.5
mM; see Harosi, 1975),
NA is Avogadro's number (6.02 × 1023) and VOS is the envelope
volume of the outer segment (liters). The volume used for mouse cone
outer segments was based on the anatomical investigation of
Carter-Dawson and LaVail (1979): rod outer segments were
assumed to be 25 µm long with uniform diameter of 1.8 µm; cone
outer segments were assumed to have length of 13 µm and diameter of
1.5 µm at their base, tapering to 1.0 µm at their tips. Combining
these factors, one obtains for rods VOS = 64 µm3, Ntotal = 1.3 × 108 and
fisomerized = 7.7 × 109 , and for cones,
VOS = 16 µm3,
Ntotal = 3.5 × 107
and fisomerized = 2.9 × 108 .
Recent histological evidence has been presented that both mouse cone
visual pigments are coexpressed in many of the cones, in a manner that
may vary with retinal location (Gloesman and Ahnelt,
1998), and electrophysiological data have provided support for
the hypothesis that the M cones coexpress a small amount of UV pigment
(Lyubarsky et al., 1999). Although coexpression of pigments presents a serious complication for stimulus quantification in
experiments using monochromatic stimulation with wavelengths far from
the pigment max, it is unlikely to greatly alter
the estimation of the number of photoisomerizations per cone produced by the flashes used in the experiments reported here and will be
neglected. The justifications for such neglect are that results to date
indicate that the level of coexpression is relatively low, ~3% for
the UV pigment in the M cones (Lyubarsky et al., 1999)
and that we used either narrowband stimulation near the max values of the cone pigments or a broadband
white flash. The relative number of isomerizations from the
coexpressed pigment are estimated to be negligible for either of these
flash stimuli. Moreover, any error in estimation will be such that more
pigment will be isomerized per cone per flash than we compute, and this will not differentially affect our comparison between the recoveries of
the cone-driven ERGs of WT and mutant animals.
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RESULTS |
GRK1 is present in rods and cones of WT mice but absent in
GRK1 /
Previous investigations have shown that GRK1 is present in both
rod and cone photoreceptors of human, bovine, and chicken retinas
(Palczewski et al., 1993; Zhao et al.,
1998). Consistent with these previous results, we found GRK1
mRNA to be present in the photoreceptor inner segment layer in the
retina of WT mice, demonstrated by the strong binding of an
antisense probe (Fig. 1,
middle); as expected, GRK1 mRNA was not detectable in GRK1 / retina (Fig. 1, right) nor was GRK1 message detectable
in any other layer of WT retina (middle). Also, consistent
with observations made in retinas of other species, we found GRK1 to be
expressed in murine cones (Fig. 2). The
localization of GRK1 in murine cones led us to examine the hypothesis
that GRK1 plays a role in cone-driven signaling similar to that which
it plays in rod signaling.
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Figure 1.
Localization of GRK1 mRNA expression in the
retina. In situ hybridization was performed as described in
Materials and Methods. Each panel is from a different retinal section
of a mouse with genotype as labeled above the panel. The GRK1 antisense
probe, which binds to the complementary message, strongly labels the
layer corresponding to the photoreceptor inner segments of the WT mouse
retina (GRK1 +/+, middle); antisense labeling is missing in
the GRK1 / retina (right). The GRK1 sense probe serves
as a control for nonspecific labeling (left). Scale bar, 30 µm. OS, Outer segment; IS, inner segment;
ONL, outer nuclear layer; OPL, outer plexiform
layer; INL, inner nuclear layer.
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Figure 2.
Localization GRK1 in rod and cone photoreceptor
outer segments. Immunocytochemistry was performed as described in
Materials and Methods on retinal sections from WT mice; 1-3
show images made from a single section of WT retina and 4-6
from a single section of GRK1 / retina. GRK1-specific antibody 8585 was raised in rabbit against the first 50 amino acids of bovine GRK1;
its binding was visualized with fluorescein-conjugated goat anti-rabbit
antibody (1, 3, 4, 6). Biotin-conjugated PNA, which
binds to cone membranes, was used to localize cone photoreceptors; its
binding was visualized by conjugation with Texas Red-conjugated
streptavidin (2, 3, 5, 6). The outer segment layer of the
retina of WT mice is strongly labeled with 8585 antibody
(1); WT cones seen to be labeled with PNA in 2 are also clearly labeled with 8585 (arrows in
1-3). Both rod and cone outer segments of GRK1 / mouse
are negative for GRK1 (4). Scale bar, 30 µm. Layer
abbreviations are given in Figure 1.
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Rods of fully dark-adapted GRK1 / mice generate nearly normal
circulating currents in situ but are very slow to recover
from strong light stimuli
Figure 3A shows ERGs
elicited from a WT, a GRK1 /, and an Arrestin / mouse with a
flash estimated to isomerize ~1% of the rhodopsin. Traces
a-c were obtained when the animals were fully dark-adapted.
The initial corneal-negative component of these traces is the a-wave;
the immediately following positive-going potential is a mixture of the
b-wave and oscillatory potentials. As illustrated schematically in
Figure 3B, the a-wave is the transient field potential
generated by suppression of photoreceptor circulating currents: the
amplitude of the a-wave in response to such intense flashes is directly
proportional to the photoreceptor circulating current present
immediately before the flash, and in WT mice, more than 95% of this
amplitude is from suppression of rod circulating current
(Lyubarsky and Pugh, 1996; Pugh et al.,
1998; Lyubarsky et al., 1999). The amplitude of
the initial a-wave of the GRK1 / animal (215 µV) is 63% that of
WT (342 µV) and 77% of that of the Arrestin / mouse (280 µV),
indicating somewhat diminished rod circulating current for both mutant
strains under our experimental conditions. Nonetheless, these a-wave
responses show that the rods of dark-adapted GRK1 / and Arrestin
/ animals generate circulating currents of near normal magnitude
and that the activation phase of the rod phototransduction cascade in
them is unaffected by the null mutation (Xu et al.,
1997; CK Chen et al., 1999). The diminished
a-wave magnitudes of the GRK1 / and Arrestin / mice in Figure 3
may have arisen from shortened outer segments (J Chen et
al., 1999) or from incomplete recovery of the rod circulating current after the exposure to red light during the anesthetization and
placement of the corneal electrode. (The necessarily limited period of
anesthesia, coupled with the experimental design of the present
experiments, aimed at testing cone function, precluded examination of
the nature of the differences between animals in the amplitudes of the
rod a-wave to the initial saturating flash delivered in this
investigation.)
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Figure 3.
ERGs (transretinal field potentials) from
WT, GRK1 /, and Arrestin / mice, and cellular basis of the a-
and b-waves of the ERG. A, A white flash isomerizing ~1%
of the rhodopsin in the retina was delivered in a ganzfeld to each
dark-adapted animal, generating responses a-c; the response
to the same flash was then recorded again after 2 min in darkness for
the WT (d), after 15 min for the GRK1 / (e),
and after 20 min for the Arrestin / (f)
mouse. The initial corneal-negative component clearly seen in
a-d is the a-wave, and the corneal-positive deflections
that follow (and truncate) the a-wave are a mixture of rod- and
cone-driven b-waves and the so-called oscillatory potentials
(Gorgels and Norren, 1992). B, In the dark,
the circulating currents of the rods and cones (arrows at
bottom left) flow in the extracellular spaces of the
outer nuclear layer (ONL), inner segment (IS),
and outer segment (OS) layers toward the receptor outer
segment tips, creating a vitreal- (and thus corneal-) positive
transretinal field potential (Hagins et al., 1970)
represented by the + and symbols near the word
dark. An intense ganzfeld flash of light initially
completely suppresses the receptor circulating currents; the consequent
collapse of their field potential generates the vitreal-negative-going
a-wave, recordable in diminished magnitude but unaltered kinetics at
the cornea (Hagins et al., 1970; Hood and Birch,
1995; Cideciyan and Jacobson, 1996; Smith
and Lamb, 1997; Pugh et al., 1998). The
suppression of their circulating currents hyperpolarizes the
photoreceptors, diminishing their glutamate release at their synapses,
leading to the opening of nonspecific cation channels in the dendrites
of ON bipolar cells [two of which are shown spanning the inner nuclear
layer (INL)]. Thus, a strong light exposure causes ON bipolar cells to
generate circulating currents that flow in the inner plexiform layer
(IPL) and inner nuclear layer toward cationic sinks in the
outer plexiform layer (OPL); the consequent,
vitreal-positive field potentials (symbolized by the + and symbols near the word light) are now understood
to underlie the b-waves (for review, see Pugh et al.,
1998). The oscillatory potentials have been hypothesized to
originate in a feedback circuit that involves certain amacrine cells
(Wachmeister, 1998). The retinal schematic is modified
from Dowling and Boycott (1966); the proper layer
thicknesses of the mouse retina are seen in Figure 1.
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In contrast to the modest diminution of its saturated, dark-adapted
amplitude, the recovery time course of the a-wave amplitude of GRK1
/ and Arrestin / mice was greatly retarded relative to that in
WT. In the experiments of Figure 3, after exposure to the initial
flash, the mice were left to dark adapt and then restimulated with the
same flash (traces d-f). Whereas the ERG from the WT
mouse shows a completely recovered a-wave (and thus rod circulating
current) after 2 min, those of the GRK1 / and Arrestin / mice
exhibit only very small a-waves (~10 µV) and a substantially
reduced-amplitude b-wave after 15 and 20 min of dark adaptation,
respectively. The absence of all but a small fraction of the initial
a-wave in the GRK1 / and Arrestin / mutants shows that the
recovery phase of the rod transduction cascade has been greatly slowed,
consistent with the hypotheses that GRK1 and Arrestin are essential for
normal inactivation of photoactivated rhodopsin (Wilden et al.,
1986; Kuhn and Wilden, 1987; Palczewski
et al., 1993; Chen et al., 1995; Ohguro
et al., 1996; Xu et al., 1997; Yamamoto
et al., 1997; Zhao et al., 1998; Cideciyan et al., 1998; CK Chen et al.,
1999).
Previous investigations of murine ERG responses obtained when the
a-wave is strongly suppressed by intense flashes or steady "rod-saturating" backgrounds has led to the conclusion that the responses obtained under such conditions are driven by signals originating in cones (Peachey et al., 1993; Pugh
et al., 1998; Lyubarsky et al., 1999). Based on
these previous observations, we attribute responses of the GRK1 /
and Arrestin / mice in Figure 3A (e,
f) to the activity of cone-driven retinal neurons and
proceed to characterize these responses.
Both UV and M cone-driven retinal responses are functional in GRK1
/ and Arrestin / mice
WT mice have both midwave (M)-sensitive (max  510 nm) and UV-sensitive cones (max 355 nm)
(Jacobs et al., 1991; Calderone and Jacobs,
1995; Lyubarsky et al., 1999), and so we
inquired whether the sensitivity of the cone-driven responses of GRK1
/ and Arrestin / animals are comparable with those of WT mice. Figure 4A presents
families of ERGs elicited from a GRK1 / mouse by monochromatic 361 and 513 nm flashes of increasing intensity. The saturating magnitudes
of the cone-driven response components were similar for WT, GRK /,
and Arrestin / mice (Table 1), indicating that cones and cone-driven secondary neurons (Fig. 2B) are present in normal numbers and are functional.
Intensity-response relationships derived from the data of Figure
4A are shown in 4B
(filled circles), together with data from three
additional GRK1 / and four Arrestin / mice; from such data, the
ratio of peak spectral sensitivities,
UV/M,
of the two cone types at the cornea can be derived (Lyubarsky et
al., 1999). The sensitivity ratios so derived were also similar
among the different animals (Fig. 4C; Table 1). Generally,
the absolute sensitivities exhibited greater variability than their
ratios; possible reasons for this include inter-animal variability in
transparency of eye tissues and pupil size.
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Figure 4.
A, Response families of cone-driven
b-waves for 361 and 513 nm flashes for a GRK1 / mouse; each trace
is the average of three to five individual responses. The 361 nm flash
intensities were (from lowest to highest intensity) 740, 1400, 4300, 7200, and 13,500 photons µm2 at the cornea
(estimated to produce from 104 to 1900 photoisomerizations in the UV
cones), and the 513 nm intensities were 2100, 4200, 8800, 21,500, and
90,000 photons µm2 at the cornea (estimated to
produce 360 to 15,000 photoisomerizations in the M cones). The
topmost trace in the 361 nm column is the
response to a "white" saturating flash isomerizing ~1.2% of the
"green" and 0.09% of the UV cone pigment. B, Amplitude
versus intensity data for GRK1 / mice (filled
symbols, n = 4) and Arrestin / mice
(open symbols, n = 4) obtained with flashes
of 361 nm (symbols including dots) and 513 nm (not
dotted), under cone-isolation conditions, as in A. Each
symbol represents the normalized peak amplitude of a
cone-driven b-wave response (points derived from the responses
illustrated in A are shown as filled or
filled + dotted circles); peak amplitudes were measured
after filtering responses at 11 Hz to remove oscillations (see
Materials and Methods). The peak amplitudes were normalized by
dividing them by the saturating amplitude, obtained in response to the
white flash (W on abscissa). The flash intensities for each
animal's data were scaled by a single, common factor; this factor was
the intensity at 513 nm (I513) estimated
by linear interpolation to produce a response of 20% saturated
amplitude (dashed line). Two saturation functions
(unbroken lines), having the form
rpeak/rmax = 1 exp(  )
have been plotted through the data, where
 is the scaled flash intensity
and is a wavelength-dependent
sensitivity factor; the black curve was arranged to
intercept the dashed line at the abscissa value 1.0; the
gray curve is shifted left by the average relative
sensitivity of cone-driven responses of WT mice to these two
wavelengths, i.e., by the factor
UV/M = 5.2 (Table 1). C, Spectral sensitivity of
cone-isolated b-wave responses of GRK1 / (filled
circles) and Arrestin / (open circles) compared
with WT. For each GRK1 / and Arrestin / animal, the lateral
shift (in logarithmic units) between the two saturation functions best
fitting the 513 and 361 nm data in B was measured; the
points plotted at ~361 nm are the mean ± SD of these shifts
(the open symbols have been shifted laterally for clarity).
The theoretical spectra give the spectral sensitivities of the UV and M
cone-driven b-wave responses of WT mice and are replotted without
alteration from Figure 6 of Lyubarsky et al.
(1999).
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A distinctive feature of cone-driven ERGs in GRK1 / mice was the
exaggeration and tight synchronization of the oscillatory potentials.
In some records after a single saturating flash, we observed hundreds
of oscillations, even after averaging; in WT mice, the oscillations in
the responses to comparable flashes typically damp out after no more
than a half of a dozen cycles (Peachey et al., 1993;
Lyubarsky et al., 1999). The oscillatory potentials are
generally associated (but not exclusively) with the light-adapted,
cone-driven retina (Peachey et al., 1993) and are
thought to be generated by a type of inner retinal neurons, such as
amacrines with dendritic fields radially extended in the retina or by a
multicellular feedback loop (Wachmeister, 1998). We
suggest that the most likely basis of the exaggerated phenotype in the
GRK1 / mice is the strongly and persistently adapted state of the
retina during the experiments rather than a "loss of function" of
GRK1 in an inner retinal cell, because no GRK1 message or protein has
been identified outside the photoreceptor layer (Figs. 1, 2). An
interesting alternative hypothesis is that some "miswiring" of the
retina may have occurred during development (Banin et al.,
1999).
Cone-driven responses of GRK1 / animals have profoundly slowed
recovery from strong stimulation
Because GRK1 is involved in the inactivation of R* in rods but is
also expressed in cones, we investigated the possibility that
cone-driven responses recover from strong stimulation more slowly in
GRK1 / mice than in WT or Arrestin / controls. The paradigm
used to investigate cone-driven response recovery is illustrated in
Figure 5. A conditioning flash
sufficiently intense to temporarily suppress all cone-driven responses
was followed at different interstimulus intervals (ISIs) by a probe
flash of the same intensity. Recovery of the cone-driven responses of
the WT and Arrestin / mice was complete in about 1 sec (Fig.
5A,C); in contrast, for the GRK1 / mouse, the first sign
of recovery appeared only after 5 sec, and complete recovery required
more than 50 sec (Fig. 5B).
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Figure 5.
Recovery of cone-driven ERGs after a
conditioning flash in WT, GRK1 /, and Arrestin / mice. White
probe flashes isomerizing ~1.2% of the M cone pigment and ~0.09%
of the UV cone pigment were delivered after a conditioning flash
isomerizing ~1% of the M and ~0.06% of the UV pigment at ISIs
specified in seconds to the left of the traces.
Responses obtained without immediately preceding conditioning flashes
are marked as Control. The upward pointing arrows
for the bottommost traces in each panel show the time of the
flash (a time gap of 3-5 msec containing a flash artifact has been
omitted from some traces); the downward pointing arrows on
the topmost traces indicate the times when the first four
test flashes were delivered for the WT and Arrestin / mice.
A, Recovery in WT mouse. The control record was obtained
with an orange ( > 530 nm) steady background that produced
~6000 photoisomerizations rod1
sec1, suppressing rod signals (Lyubarsky et
al., 1999); for all other recordings, rod activity was
suppressed with the conditioning flash. Each trace is the
average of 10 records. B, Recovery in GRK1 / mouse; each
trace is the average of five measurements. C,
Recovery in Arrestin / mouse; each trace is the average
of 15 records.
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Because the corneal-positive component of the cone-isolated ERG
represents field potentials generated mostly by second-order neurons
(Fig. 3B) and because GRK1 is expressed in cones, a question of special interest is whether the slowed recovery was caused by
prolonged activation of the cones themselves or only by prolonged activity in neurons downstream from the photoreceptors. To address this
question, we examined the behavior of the initial corneal-negative component of the ERG observed under cone-isolation conditions (Fig. 5);
this small component exhibits properties, including its electrical
sign, its magnitude, and its activation kinetics in response to intense
flashes, consistent with origination in the suppression of cone
photoreceptor circulating current (Lyubarsky et al.,
1999). Indeed, in human subjects, the homologous but
appropriately larger ERG component has been identified as originating
in the suppression of cone circulating current (Hood and Birch,
1995; Cideciyan and Jacobson, 1996; Smith
and Lamb, 1997). Thus, in Figure
6, the initial portions of records from
mice stimulated as in Figure 5 have been replotted on expanded scales
to facilitate examination of the cone a-wave; here, the differences
between the animals in the recoveries of the cone a-waves are seen to parallel the recoveries of the cone-driven b-wave (although the oscillatory potentials interfere with extraction of the cone a-wave from records of WT and Arrestin / mice in the first 200-300 msec
after the conditioning flash).
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Figure 6.
Recovery of the a-wave component of the
mouse ERG under cone isolation conditions for WT, GRK1 /, and
Arrestin / mice. The format of presentation is the same as in
Figure 5, except that the time base and amplitude scales have been
expanded to reveal the initial 25 msec of the records. In each panel,
the portion of the traces identified with the suppression of
the cone circulating current has been emphasized by
thickening of the trace. The traces of
the WT and Arrestin / mice are the same as those shown in Figure 5;
the data of the GRK1 / mouse were taken from a different animal
than those of Figure 5B, obtained in an experiment
engineered to minimize the flash artifact. Nonetheless, for the larger
artifact, the slowed recovery of the cone a-wave of the GRK1 /
mouse can also be seen in Figure 5B. (In this and in other
figures, a 3.5 msec segment of the recorded trace
immediately after the flash trigger has been excised; this segment
contains the flash artifact, which is caused largely by a
difficult-to-eliminate magnetic interference effect.)
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In Figure 7, we summarize the results of
the experiment of Figures 5 and 6 and present data obtained with the
same experimental protocol from additional WT, GRK1 /, and Arrestin
/ mice. Figure 7A plots the saturated amplitude of the
cone-driven b-wave response relative to its baseline amplitude, as a
function of time after the conditioning flash. Taking the average time
to 50% b-wave recovery for WT and Arrestin / mice to be 0.4 sec, the cone b-wave of the GRK1 / mice is seen to recover 20-70 times
more slowly. Figure 7B plots the recovery of the saturating cone a-wave on a common abscissa with A. Again, using the
recovery to 50% amplitude as a benchmark, the cone a-waves of the GRK1 / mice are seen to recover 40-100 times more slowly than those of
WT and Arrestin / mice.
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Figure 7.
Time course of recovery of cone b-waves and cone
a-waves from the conditioning flash. A, Normalized
amplitudes of cone-driven b-waves for WT (open symbols,
n = 5), Arrestin / (gray-filled
symbols, n = 4), and GRK1 / mice
(filled symbols, n = 5) plotted as
function of the time (interstimulus interval) between the conditioning
and probe flashes. B, Normalized amplitudes of cone a-waves
plotted versus the interstimulus interval. The points extracted from
the experiment of Figure 6 are plotted as circles in each
case. All data were obtained with the experimental protocol illustrated
in Figures 5 and 6; the same symbols are used in
A and B for the data of the same animal. The
curves plotted through the cone a-wave data have the form
amax(t)/amax( ) = 1/[1 + exp((t t0)/], where amax is
the saturated cone a-wave amplitude, and t0 = 0.3 sec and = 0.15 sec for the WT and Arrestin /
data, and t0 = 19 sec and = 6.2 sec for the GRK1 / data; these curves are equivalent in form to
that used by Thomas and Lamb (1999) to characterize the
recovery of the human rod a-wave after a bleaching exposure. The
normalization in each panel was based on the amplitude of the responses
at ISIs of 2 or 3 sec for WT and Arrestin / and at 300 sec for GRK1
/. Note that the time axis is in logarithmic units.
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DISCUSSION |
Observations made with the rod a-wave in this investigation (Fig.
3A) confirm previous findings (Yamamoto et al.,
1997; Cideciyan et al., 1998; CK Chen et
al., 1999) demonstrating that lack of GRK1 causes severe
retardation of the recovery of retinal rod responses after intense
stimulation, consistent with the hypothesis that GRK1 is essential for
the normal inactivation of photoactivated rhodopsin (Wilden et
al., 1986; Kuhn and Wilden, 1987;
Palczewski et al., 1993; Chen et al.,
1995; Ohguro et al., 1996; Xu et al., 1997; Yamamoto et al., 1997; Cideciyan et
al., 1998; Zhao et al., 1998; CK Chen et
al., 1999).
Our results make a strong case that GRK1 / mice have a specific
defect in the inactivation of cone phototransduction, as follows.
First, besides its expression in mammalian rods, GRK1 has only been
found to be present in cones (Zhao et al., 1998); consistent with this general finding, the antisense probe generates no
signal in the inner nuclear layer (Fig. 1), as would be expected, for
example, were GRK1 present in cone ON bipolars and playing a role in
their mGluR6 cascade (Masu et al., 1995). Thus, the cone
b-wave recoveries of the GRK1 / mutants are not retarded because of
a defect in the G-protein signaling cascade of the cone ON bipolars,
but rather these slowed recoveries point to a defect in the cone
photoreceptors that drive them. Second, the greatly slowed recoveries
of the a-wave component recorded under cone isolation conditions
provide specific functional evidence for a defect in the inactivation
of cone transduction. The evidence is compelling because the ERG
component isolated in Figure 6 (thickened portion of traces)
is the murine cone a-wave, i.e., represents the suppression of the
circulating current of the cones. Specifically, as presented previously
(Lyubarsky et al., 1999), this component has the sign,
magnitude, and activation kinetics expected from human ERG studies
(Hood and Birch, 1995; Cideciyan and Jacobson, 1996; Smith and Lamb, 1997) and from
microelectrode recordings from single mammalian cones
(Schneeweis and Schnapf, 1995) for generation in the
suppression of cone circulating current. Thus, the greatly slowed
recovery of the cone-isolated a-wave in GRK1 / mutants (Figs. 6,
7B) shows that the circulating currents of the cones of the
mutant mice recover much more slowly from the conditioning flash than
do the cone circulating currents of WT mice or those of mice lacking
arrestin, which is necessary for normal rod inactivation (Xu et
al., 1997) (Fig. 3A, c, f).
The very rapid recovery of the murine cone circulating current in WT
and Arrestin / mice (Figs. 6, 7B) after a flash
estimated to isomerize a few percent of the cone photopigments serves
to underscore a feature of cones that differentiates them fundamentally from rods; assuming that the isomerized cone pigment is not regenerated in 1 sec, these results confirm that cones function well with amounts
of bleached pigment that can suppress the circulating currents of rods.
Moreover, the rapid recoveries in WT and Arrestin / mice also show
that the flash intensities used in the experiments reported here are by
no means nonphysiological for cones. Similarly rapid recoveries of
human cone a-waves under comparable stimulation conditions have been
reported recently (Mahroo et al., 1999).
Although the conclusion that GRK1 plays a critical role in the
inactivation of murine cone phototransduction may seem surprising in
view of evidence for cone-specific GRKs (Weiss et al.,
1998), it bears emphasis that in only one previous
investigation (Cideciyan et al., 1998) has any
functional evidence been presented that any GRK plays a role in cone
phototransduction. Although our results are thus not in qualitative
conflict with the histological evidence for cone-specific GRKs, they
nonetheless appear at odds quantitatively with the report of
Cideciyan et al. (1998), who found a reliable, but only
"slight slowing of cone deactivation kinetics" (measured with
cone-isolated a-waves) in a human patient with a deletion of exon 5 of
the GRK1 gene. What might account for the quantitative discrepancy
between the human data and those reported here from mice? As possible
explanations, we offer the following three hypotheses. First, the loss
of exon 5 of human GRK1 gene may not constitute a functionally null
mutation. Although it was demonstrated that GRK1 with an exon 5 deletion does not phosphorylate rhodopsin (Cideciyan et al.,
1998), the data also show that a truncated GRK1 protein is
expressed and that it remains in the cell without rapid degradation.
Thus, it remains possible that a functional domain encoded in exons
1-4 can fold properly and participate in the recovery of cone
phototransduction, perhaps by binding to cone-opsin rather than
phosphorylating it. It has been shown in vitro that binding
of GRK1 to photoactivated rhodopsin competitively blocks the activation
of transducin (Pulvermuller et al., 1993). Second, it is
possible that the loss of GRK1 in human cones can be compensated for by
another GRK (Hisatomi et al., 1998; Weiss et al.,
1998). Third, the recovery of cone phototransduction in human
cones could depend primarily on the regeneration of cone pigments and
not on interaction with GRK1. Clearly, our results reject the third
hypothesis as it applies to mice, i.e., reject the hypothesis that
independent of GRK1, regeneration of isomerized pigments in mouse cones
under our stimulation conditions is sufficient to rapidly inactivate
cone-opsin. Additional experiments are being planned to test the first
and second hypotheses.
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FOOTNOTES |
Received Sept. 23, 1999; revised Dec. 21, 1999; accepted Dec. 29, 1999.
Correspondence should be addressed to Prof. Edward N. Pugh Jr., F. M. Kirby Center for Molecular Ophthalmology, Department of
Ophthalmology, University of Pennsylvania School of Medicine, Stellar-Chance Building, Room 309B, 422 Curie Boulevard, Philadelphia, PA 19104-6069. E-mail: pugh{at}mail.med.upenn.edu.
Drs. Lyubarsky and Chen contributed equally to this work.
This work was supported by grants from the National Eye Institute.
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TOP
ABSTRACT
INTRODUCTION
