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The Journal of Neuroscience, February 15, 2003, 23(4):1287
GTPase Regulators and Photoresponses in Cones of the Eastern
Chipmunk
Xue
Zhang1,
Theodore G.
Wensel1, and
Timothy W.
Kraft2
1 Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, Texas 77030, and
2 Department of Physiological Optics, School of Optometry,
University of Alabama, Birmingham, Alabama 35294
 |
ABSTRACT |
Vertebrate cone and rod photoreceptor cells use similar mechanisms
to transduce light signals into electrical signals, but their responses
to light differ in sensitivity and kinetics. To assess the role of
G-protein GTP hydrolysis kinetics in mammalian cone photoresponses, we
have characterized photoresponses and GTPase regulatory components of
cones and rods from the cone-dominant retina of the eastern chipmunk.
Sensitivity, based on the stimulus strength required for a half-maximum
response, of the M-cone population was 38-fold lower than that of the
rods. The relatively lower cone sensitivity could be attributed in part
to lower amplification in the rising phase and in part to faster
recovery kinetics. At a molecular level, cloning of chipmunk cDNA and
expression of recombinant proteins provided standards for quantitative
immunoblot analysis of proteins involved in GTPase acceleration. The
ratio of the cGMP-phosphodiesterase inhibitory subunit 
to cone
pigment, 1:68, was similar to the levels observed for ratios to
rhodopsin in bovine retina, 1:76, or mouse retina, 1:65. In contrast,
the ratio to pigment of the GTPase-accelerating protein RGS9-1 was 1:62, more than 10 times higher than ratios observed in rod-dominant retinas. Immunoprecipitation experiments revealed that, in contrast to
rods, RGS9-1 in chipmunk retina is associated with both the short and
long isoforms of its partner subunit G
5. The much higher
levels of the GTPase-accelerating protein complex in cones, compared
with rods, suggest a role for GTPase acceleration in obtaining rapid
photoresponse kinetics.
Key words:
photoreceptors; cones; rods; G-proteins; phosphodiesterase; RGS proteins; PDE; vision; retina
| |
Introduction |
Humans and other diurnal animals
rely primarily on their cone photoreceptors for functional vision.
Daylight vision, color discrimination, rapid motion detection, and
finely focused images all rely on cones (for review, see Masland,
2001
). Humans lacking rod function but retaining cone function have the
relatively mild condition of night blindness. In contrast, those
retaining rod function but lacking cone responses have very severe
visual defects, (for review, see Birch, 1999; Hicks and Sahel, 1999).
Therefore, it is of great interest to understand the biochemical
properties of cones and the relationships between their molecular and
physiological properties. Cone photoresponses have been recorded from
some mammalian species (Kraft, 1988; Schnapf et al., 1990; Kraft et
al., 1998; Schneeweis and Schnapf, 1999), but biochemical data are, for
the most part, lacking, because no procedures have been developed for
isolation of cone outer segments in amounts comparable with those
obtainable from rod outer segments. Consequently we have a wealth of
biochemical information about rods but only rudimentary information on
the biochemistry of mammalian cones. In rod cells, quantification of
the amounts of phototransduction components and characterization of
their biochemical properties allow detailed modeling of the molecular
mechanisms of phototransduction (Detwiler et al., 2000; Hamer, 2000;
Leskov et al., 2000; Arshavsky et al., 2002). In cones, similar
biochemical components are present, but their concentrations are mostly unknown.
Retinas of cone-dominant animals represent useful starting material for
biochemical analysis of cone phototransduction as well as for
electrophysiological recording. Two readily available examples are
ground squirrels (Kraft, 1988) and chipmunks. We have analyzed
photoresponses of the eastern chipmunk Tamias striatus and
measured amounts of proteins essential for their inactivation phases.
Because these proteins are found almost exclusively in photoreceptor
outer segments, and because rods make a negligible contribution to
their amounts, this quantification can be performed on crude extracts
of total retina without purification of cone outer segments. Because
the detection method relies on recognition by antibodies raised against
orthologous proteins from other species, it was necessary to clone cDNA
encoding the chipmunk proteins and to express them for accurate
calibration of antibody sensitivity.
Our focus has been on proteins that determine the lifetime of
transducin activation through regulation of the kinetics of G-protein
GTP hydrolysis. The reason is that major differences between cones and
rods include much lower sensitivity and faster recovery kinetics in
cones. Faster recovery may contribute to lowering sensitivity. In both
cones and rods, recovery is the slow phase of photoresponses, and it
has been proposed that the rate-limiting step in rod transduction is
transducin GTP hydrolysis (Sagoo and Lagnado, 1997; Nikonov et al.,
1998). Immunostaining in bovine and human retina (Cowan et al., 1998;
Zhang et al., 1999) suggested that cones had higher RGS9-1 content than
rods but did not allow quantitative estimation of relative amounts.
We report here results from electrophysiological recordings of light
responses in chipmunk rods and cones, along with quantification of the
GTPase accelerating proteins RGS9-1, G5, and
cGMP phosphodiesterase inhibitory subunit (PDE) in chipmunk retina.
| |
Materials and Methods |
Buffers. Standard buffers were buffer A (in
mM: 10 3-(N-morpholino) propane sulfonic acid
(MOPS), pH 7.0, 30 NaCl, 60 KCl, 2 MgCl2,
and 1 DTT and ~20 mg/l phenylmethylsulfonyl fluoride), buffer B (in
mM: 25 Tris and 192 glycine, pH 8.3), buffer C (in mM: 10 MOPS, 1 MgCl2, 50 NaCl, and
0.1 EDTA, pH 8.0), and buffer D (in mM: 10 HEPES, 100 NaCl,
and 2 MgCl2). Other buffer components and
conditions were varied as indicated throughout.
Animal and tissue preparation. The eastern chipmunk is
common in rural and suburban neighborhoods of the southeastern United States. Animals were trapped by permit in Jefferson County Alabama. Animals were housed singly with a hiding tube and ad libitum
access to food and water; captivity was well tolerated, because some animals were kept for >4 years. All experimental protocols were approved by the University of Alabama Institutional Animal Care and Use
Committee. To start, a chipmunk was dark-adapted overnight and then
killed by carbon dioxide asphyxiation. Details of the tissue
preparations and electrolytes have been given previously (Kraft et al.,
1993). Briefly, the retina was isolated under infrared light in
Lebovitz's L-15 medium and stored at 4°C in the dark. Experiments
were performed on the same retina for 2-3 d; each tissue sample lasted
3-4 hr. For each experiment, one-third of a retina (~3 × 4 mm)
was removed from cold storage and chopped under infrared light to
produce small pieces of retina ~50-100 µm on a side and then
warmed to near body temperature in a perfusion chamber. The circulating
dark current of individual rods or cones was recorded by drawing the
outer segment into a suction electrode, whose inner diameter matched
the outer segment diameter of the cell. The photocurrent and stimulus
monitor signals were digitized with hardware (MIO16) and software
(LabView) from National Instruments (Austin, TX). A
stimulus set consisted of 5-30 responses to the same wavelength and
intensity of light. The light bench focused a 440-µm-diameter spot of
light at the plane of the cells. The wavelength was controlled with
three-cavity interference filters (Andover Corp., Salem, NH)
with an average bandwidth of 10 nm. Neutral density filters (Reynard
Corp., San Clemente, CA) attenuated the light. Calibration of
unattenuated light at each wavelength was performed daily with a
photometer (model 350; Graseby Optronics, Orlando, FL).
Measuring the action spectra. Spectral sensitivity of the
visual pigments was estimated by measuring the action spectra using the
criterion response method and the photocurrent responses of individual
cones. The action spectrum was determined for up to 20 wavelengths at
~20 nm intervals between 380 and 760 nm. Spectral sensitivity was
measured by adjusting the intensity of light at each wavelength to
produce a criterion response of ~25% of the maximum current. The
sensitivity at each wavelength was measured relative to a standard
wavelength, (500 nm). Initially, for each cell, the complete intensity
response function was determined at 500 nm. Subsequently, for each test
wavelength, two or three light intensities were used to
obtain current responses of 10-60% of maximum. Sensitivity
measures at the standard wavelength were repeated after every two or
three test wavelengths to avoid errors attributable to changes in the
physiologic state of the cell or electrode seal.
Calculations of phototransduction gain. The gain factor for
the rising phase of the photoreceptor response was estimated by fitting
Equation 22 from Pugh and Lamb (1993), given below as Equation 1, to
the initial portion of the rising phase of the photocurrent response.
The fitting was performed simultaneously to responses to two to five
intensities in the linear range (see Fig. 2C,D).
|
(1)
|
where Rmax is the
maximum response; td is a combined
delay factor for all the biochemical reactions, and A is the
gain factor for the activation phase of phototransduction (Lamb and
Pugh, 1992; Pugh and Lamb, 1993). The number of photoisomerizations, 
, is the product of the stimulus strength, i (photons per
square micrometer at 
max), and
Ac, the effective collecting area of the outer segment, calculated as by Baylor et al. (1984):
|
(2)
|
where VOS is the volume of the
outer segment; Qisom is the quantum
efficiency of photoisomerization (0.67)(Dartnall, 1972); f is a factor allowing for the use of unpolarized light
entering the outer segment perpendicular to its long axis
(f = 0.5); and 
is the specific pigment
density (0.016 µm
1) (Bowmaker et al.,
1980). Typically stimuli of the optimum wavelength were used; if not,
the stimulus strengths were converted to the equivalent number of
photons at the optimum wavelength, based on the spectral sensitivity
function (see Table 2). The signals from rods were digitized at 4 msec
intervals and typically low-pass-filtered at 50 Hz. The signals from
cones were digitized at 3 msec intervals and typically
low-pass-filtered at 100 Hz.
For seven cells, individual outer segment volume was calculated from
digital images of the cells taken during or after recording. The
average volume for rods (n = 4) was 11.9 µm3 and for cones (n = 3) was 14.3 µm3, corresponding to
collecting areas of 0.147 and 0.175 µm2 respectively. For nine other cells
(five rods and four cones), the outer segment volumes and collecting
areas were assumed to be similar to the measured means. The small size
of mammalian outer segment dimensions and the resolution of the light
microscope limit these volume estimates to an accuracy of ~20-30%,
based on a linear measurement error of 0.2 µm. The outer segment of cone photoreceptors is physically fragile but physiologically sturdy;
although only a small stub of the OS remained in some cases,
high-quality stable recordings were obtained.
For each cell, two to five rising phase responses were
simultaneously fit by Equation 1, where A and
td were allowed to vary to optimize
the fitting (Igor; Wavemetrics Inc.).
Rmax was fixed as the value measured
in each cell.
RNA isolation, reverse transcription-PCR, and cDNA cloning.
Total RNA was extracted from chipmunk whole retina with retina pigment
epithelium using Trizol reagent (Invitrogen, San Diego, CA) by following manufacturer's instructions. Chipmunk RGS9-1, G5S, and cone PDE cDNAs were cloned by
reverse transcription (RT)-PCR and rapid amplification of cDNA ends
(RACE) strategies as described previously (Davis et al., 1994). A cDNA
fragment encoding amino acids 327-394 within the conserved RGS domain
of RGS9-1 was amplified by RT-PCR using degenerate primers cRGS9a, 5'-GGNTT(C/T)TGGGA(A/G)GCNTG(C/T)GAGA-3'; and cRGS9b,
5'-CAT(A/G)TA(A/G/T)AT(A/G)TGNGT(C/T)TGNGCNGC(A/G)TC-3'. To obtain the
coding sequence on the 5' end of this fragment, PCR was performed using
a degenerate primer, cR5UTR,
5'-T(C/G/T)(A/C)(A/G)TCCAGG(A/G)(G/T)CCAG-3', corresponding to the
conserved sequence within the 5' untranslated regions (UTRs) of RGS9-1
from different species, and primer cRNon, 5'-GTAGCGGTGGGGGTG-3',
which is reverse complementary to nucleotides encoding amino acids
379-383. The rest of the coding sequence was amplified by RT-PCR using
primer cRCod, 5'-CACGGTGAAGGGGCTGAAG-3', encoding amino acids 373-378;
and degenerate primer cRCnon, 5'-TTA(C/T)TTNGGNGGNAG(C/T)TC(C/T)TT-3', designed to be the reverse complement of nucleotides encoding amino
acids 479 to stop code, assuming conservation of the last six amino
acids with bovine, human, and mouse sequences. A fragment of chipmunk
G5S cDNA encoding the first 338 amino acids
was cloned by RT-PCR using degenerate primers cGa,
5'-ATGGCNACNGANGGN(C/T)T-3'; and cGb, 5'AANACNGTNCC(A/G)TCNGG3'. To
obtain upstream sequence, RT-PCR was performed using degenerate primer
cG5UTR, 5'-CCG(C/G)(A/G)CGAAGATGGC-3', which is conserved in 5' UTRs of
G5S; and primer cGNon,
5'-GGTCTTCATGACAAACTG-3'. The rest of the coding sequence
was obtained by 3' RACE using primers cG3raceI,
5'-GAGTCTCCATCCTGTTTG-3'; cG3raceII, 5'-GTACTCTACGAGTCTC-3'; and
adaptor, 5'-GACTCGAGTCGACATCG-3'. Chipmunk cone PDE cDNA was
cloned by RT-PCR using degenerate primers cP5UTR,
5'-GCCG(A/C)CC(A/G)GGGG(A/C)AGT(C/T)AAAATG-3'; and cP3UTR,
5'-TGGCAGAACC(C/T)CTGG(C/T)(A/G)CT-3'. These sequences are
conserved in 5' and 3' UTRs of mammalian cone PDE.
Sequence data analyses. The chipmunk RGS9-1,
G5S, and cone-type PDE (GenBank accession
numbers AF480878, AF480879, and AF480880, respectively) were compared
with the corresponding sequences, which were taken from the GenBank or
National Center for Biotechnology Information database, with the
following accession numbers: mouse (Mus musculus) RGS9-1,
AAC99481; G5, P54314; cone PDE, BAB32255.1;
and rod PDE, CAA68714.1; rat (Rattus norveqicus) cone
PDE, AAG43400.1; human (Homo sapiens) RGS9-1, AAG09311;
G5, AAC63826; cone PDE, BAA08241.1; and rod
PDE, AAA03653.1; bovine (Bos taurus) RGS9-1, O46469; cone
PDE, AAA30689.1; and rod PDE, CAA28507.1; tiger salamander
(Ambystoma tigrinum) G5,
AAK52836.1; fruit fly (Drosophila melanogaster) G5, AAF46336; nematode (Caenorhabditis
elegans) G5, AC Q20636; 13-lined ground
squirrel (Spermophilus tridecemlineatus) cone PDE,
CAA04720.1; leopard frog (Rana pipiens) cone PDE,
AAK95403.1; and rod PDE, AAK95404.1; guinea pig (Cavia
porcellus) rod PDE, AAG43274.1; and dog (Canis familiaris) rod PDE, CAA93815.1. The sequences were aligned, and phylogenetic trees were constructed by CLUSTALW (Thompson et al.,
1994) based on the neighbor-joining method (Saitou and Nei, 1987).
Protein expression and purification. Chipmunk RGS9-1 and
PDE and mouse RGS9-1 cDNAs were subcloned into the pET-14b
expression vector (Novagen, Madison, WI) using
NdeI and BamHI restriction sites.
His6-tagged recombinant proteins were purified
using a Ni+-nitrilo triacetic acid
column (Qiagen, Hilden, Germany) by following the
manufacturer's instructions using denaturing conditions.
His6-tagged chipmunk PDE was further purified
by reverse-phase HPLC as described previously (Angleson and Wensel,
1994). Endogenous bovine rod PDE was purified from the bovine rod
outer segment (ROS) following procedures described previously (Wensel
and Stryer, 1990). In each case, the concentration of purified protein
was determined by absorbance at 280 nm in 6 M
guanidinium chloride using extinction coefficients calculated from the
sequence (Gill and von Hippel, 1989).
Immunofluorescence staining. Immunofluorescence staining of
mouse and chipmunk RGS9-1 and G5 was performed
according to the procedure described previously (Lyubarsky et al.,
2001). To stain chipmunk RGS9-1 and rhodopsin, chipmunk eyes were fixed in 4% paraformaldehyde and PBS, pH 7.2, for 10-16 hr at 4°C. After protection in 30% sucrose and PBS for 1 hr at 4°C, the eyes were embedded in OCT compound (Tissue-Tek), and frozen sections were cut at
16 µm. Tissue sections were postfixed in 1:1 methanol/acetone (v/v)
for 10 min at room temperature and rehydrated in PBS, pH 7.2, for 20 min at room temperature. Nonspecific binding was blocked by incubating
the sections for 1 hr at room temperature with 10% sheep serum
(Sigma, St. Louis, MO) and PBS. Then the sections were
incubated with primary antibody to RGS9, anti-RGS9-1c (He et al.,
1998), at a 1:200 dilution, and anti-rhodopsin monoclonal antibody, 1D4
(Wu et al., 1998), at a 1:500 dilution in 10% sheep serum and PBS. The
sections were incubated with primary antibodies at room temperature
overnight in a humidified atmosphere. After being washed three times
for 5 min in PBS at room temperature, sections were incubated with
secondary antibodies, fluorescein isothiocyanate-conjugated anti-rabbit
IgG (Vector Laboratories, Burlingame, CA) and rhodamine-conjugated
anti-mouse IgG (Vector Laboratories), at a 1:100 dilution, in 10%
sheep serum and PBS for 1 hr at room temperature in a humidified
atmosphere. Sections were washed three times for 10 min in PBS at room
temperature and mounted in aqueous mounting medium (Gel/Mount;
Biomeda, Foster City, CA). Sections were examined and
images recorded using a Zeiss (Thornwood, NY) 510 LSM
confocal microscope.
Preparation of retina extracts. All procedures were
performed in complete darkness or under infrared light. Because of the extracellular matrix structures known as cone sheaths, chipmunk retina
is not easily peeled from the underlying layer of retinal pigmented
epithelium (RPE) without substantial loss of cone outer segments. Also,
previous studies have shown that the proteins of interest are not
expressed at detectable levels in RPE; therefore, we homogenized a
sample of retina plus RPE to maximize the yield of cone-derived
material. One chipmunk retina with attached retinal pigmented
epithelium, two mouse retinas, and 100 µg of bovine retina were
homogenized in 600 µl of buffer A. After centrifugation for 15 min at
80,000 × g, the pellets were resuspended in 300 µl
of buffer A supplemented with 3 µl of 4 mM
ethanol solution of 11-cis-retinal and incubated at 4°C
for 2 hr. Retina pellets were recovered by centrifugation again for 15 min at 80,000 × g and incubated in buffer A
supplemented with 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate for 30 min
at 4°C. Supernatants containing extracted membrane and soluble
proteins were obtained by centrifugation for 15 min at 10,000 × g.
Spectroscopic measurements. The UV-visible absorption
spectra of detergent-solubilized retina extracts were recorded with an
HP 8452A spectrophotometer in cuvettes of 1.0 cm path length at room
temperature. Spectra were recorded before and after illumination of
samples for 3 min with a 150 W light bulb (Reflector Flood; Philips).
Hydroxylamine-hydrochloride, pH 7.0, was added to a final concentration
of 20 mM after the samples were illuminated, and the
spectra were recorded again. Difference spectra were obtained by
subtracting the spectrum after bleaching from that before illumination. Absorbance values were all within the linear response range of the
spectrophotometer. To generate the model spectra for rods and M-cones,
the Dawis (1981) polynomials were used in a two-parameter least squares
fit varying the log of maximum sensitivity,
bmax, and peak wavelength,
max. To generate estimates of extinction coefficients, the sensitivity values at each wavelength were normalized by dividing by 10bmax and then multiplied
by 40,000 M1 · cm1
(Vissers et al., 1998). For the S-cone sensitivity data and for the
M-cone data for wavelengths <450 nm, in which the Dawis polynomial fits poorly, the data were fit to Gaussian curves,
S() = ao + (S(c) 
ao)exp[(
c)2/2w2],
to provide a smooth curve declining monotonically with distance from
c and closely approximating the sensitivity
data (Ditchburn, 1963; Harris and Bertolucci, 1978), and the extinction
coefficients were calculated as 
() = S() · 40,000 M1 · cm1/S(c).
The assumptions are that the extinction coefficients () are
proportional to the sensitivities S() with a constant
proportionality constant for each cell type and pigment, and that the
extinction coefficient (max or
c) at the wavelength of maximal sensitivity (max or c) is equal
to 40,000 M1 · cm1.
Values found by fitting were as follows: rods,
Lmax = 502 nm; max = 500.36 nm; and
bmax = 0.0058 (bmax = 0 assigned to maximal measured
value tabulated in Table 2); M-cones,
Lmax = 562 nm; max = 537.48 nm;
bmax = 0.0066;
ao = 0.21384;
S(c) = 1.126; c = 538.78 nm; and w = 44.34 nm; and S-cone, ao = 0.0206;
S(c) = 1.324;
c = 452.8 nm; and w = 58.49 nm. The difference spectrum was fit to a linear combination of the
model spectra in the range of 400-600 nm using the
Levenberg-Marquardt (Levenberg, 1944; Marquardt, 1963) least squares
algorithm as implemented in the program Origin and the equation
A() = mm() + rr() + ss() (m + r + s)R*(). Here
m, r, and s are the molar
concentrations of M-cone pigment, rhodopsin, and S-cone pigment,
respectively; m(),
r(), and s()
are the corresponding model spectra from Figure 3A; and
R*() is the metarhodopsin II spectrum
shown in Figure 3A.
Immunoblotting and densitometry. After spectrophotometry to
quantify visual pigments, chipmunk, mouse, and bovine retina detergent extracts and bovine ROS were analyzed by SDS-PAGE, followed by immunoblotting and densitometry. Immunoblotting was performed according
to a standard protocol (Harlow and Lane, 1988) on proteins separated by
SDS-PAGE. Buffer B was used for electrophoretic transfer of PDE, and
buffer B supplemented with 0.1% SDS was used for transfer of RGS9-1
and G5. The membranes for immunoblotting were
supported nitrocellulose (NitroPure; Osmonics, Inc.).
After 60 min for RGS9-1 and G5 or 45 min for
PDE transfer at 350 mA at 4°C, membranes were blocked by 5%
nonfat dry milk and a solution of 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.1% (v/v) Tween 20 for 1 hr, followed by
incubation with primary antibody for 4 hr. Polyclonal antibodies
anti-RGS9-1c and anti-G5 (He et al., 2000)
were used at a 1:1000 dilution, and anti-PDE was used at a dilution
of 1:500. The secondary antibody used was horseradish
peroxidase-conjugated anti-rabbit IgG (Promega, Madison, WI), with detection by chemiluminescence using the ECL system (Amersham Biosciences, Arlington Heights, IL). For
densitometry of chemiluminescence signals on film, x-ray films were
scanned, and bands were quantified by software UN-SCAN-IT (Silk
Scientific Corp.). To quantify RGS9-1 and PDE or to calibrate
antibody specificity, purified recombinant proteins and highly purified
PDE from bovine retina were used as standards. The concentrations of
purified proteins were determined by spectrophotometry at 280 nm as
described above or by using densitometry of Coomassie blue-stained
bands on SDS-PAGE gels calibrated with standards whose concentrations were determined by 280 nm absorbance. On each gel, varying amounts of
standard protein were loaded next to different volumes of the retinal
extract. Films were exposed to the blots after processing for
chemiluminescence detection, with varying exposure times to ensure that
film optical density was linear with the protein amount for standards
whose values of optical density bracketed those of the corresponding
bands from the extracts. The optical density of the band in question
from each extract lane was then substituted into the linear function
(derived by least squares fitting) for density, to solve for the amount
of specific protein in each extract sample. Dividing this amount by the
volume loaded gave a value of concentration for each sample, and these
were averaged. In some cases, the blot was initially performed with a
standard from another species (e.g., bovine PDE), and then later,
the relative sensitivity of the antibody for the protein of the species
in question (e.g., chipmunk) was determined using blots with purified recombinant proteins from both species (e.g., bovine and chipmunk) to
obtain correction factors. The correction factors were determined as
the ratios of calculated amounts of protein from Western blots and
densitometry to the amounts of each purified protein (determined by
spectrophotometry) loaded on SDS-PAGE (see Figs. 6B,
7C,D). The correction factor for chipmunk cone PDE
detection by anti-PDE is 0.79. The correction factors of
anti-RGS9-1c for chipmunk and mouse RGS9-1 are 0.97 and 1.97, respectively.
Immunoprecipitation. Purified anti-RGS9 antibody
anti-RGS9-1c was covalently attached to cyanogen bromide-activated
Sepharose 4B-CL as described previously (Hu et al., 2001). For
immunoprecipitation, chipmunk or bovine retina was homogenized and
solubilized with 200 µl of buffer A supplemented with 1% Nonidet
P-40. The insoluble material was removed by centrifugation for 15 min
at 80,000 × g. The solubilized retina extracts were
incubated with 10 µl of anti-RGS9-1c IgG-coupled beads for 10-16 hr
at 4°C after mixing on a shaker. The beads were separated from
supernatant by a brief centrifugation and washed three times with the
solubilization buffer. Bound proteins were redissolved in the SDS-PAGE
sample buffer and separated from the beads by a brief centrifugation.
PDE assays. PDE catalytic activity was measured with the
pH-recording method (Liebman and Evanczuk, 1982) as modified previously (Malinski and Wensel, 1992). Specifically, assays were performed in
buffer C with initial cGMP concentration of 2 mM and a
total volume of 200 µl. Assays were performed in 96-well microtiter plates and monitored with MI-410 microelectrodes
(Microelectrodes, Inc.). To test chipmunk cone PDE and
bovine rod PDE activity, each assay was initiated by adding 15 µl of
chipmunk retina homogenate or 10 µl of bovine ROS, which had 0.4 pmol
of PDE calculated from PDE quantitative immunoblots, and maximal PDE
activation was obtained by adding 30 µg of trypsin
(Sigma) to remove inhibitory subunit PDE. After PDE was
fully activated, 300 µg of soybean trypsin inhibitor
(Sigma) was added to quench trypsin. Then PDE activity was
blocked by adding 0.2 nmol of His6-tagged bovine PDE and restored by adding 300 µg of trypsin again. To test
chipmunk cone PDE and bovine rod PDE inhibitory activity, bovine
PDE was purified and treated with trypsin as described previously (Wensel and Stryer, 1990). Each assay was performed in 200 µl of
buffer C with 2 mM cGMP, and the pH recordings were
initiated when trypsin-treated PDE was added to a final concentration
of 5 nM. After the reactions had proceeded ~1 min,
His6-tagged bovine rod or chipmunk cone PDE
was added to different concentrations of 0, 2, 4, and 8 nM.
To test chipmunk cone PDE inhibitory activity on chipmunk PDE, one
chipmunk retina was homogenized in 200 µl of buffer C, and 150 µl
of the homogenate was treated by 150 µg of trypsin at room
temperature for 1 min, followed by 1.5 mg of soybean trypsin inhibitor.
Each pH assay was performed in a final volume of 200 µl of buffer C
with 2 mM cGMP. The recordings started when 40 µl of
trypsin-treated chipmunk retina homogenate (~0.4 pmol of PDE) was
added. After the reactions had proceeded ~1 min, His6-tagged chipmunk cone PDE was added to
different concentrations of 1, 2, and 4 nM.
GTPase single turnover assays. GTPase single turnover assays
were performed to test the bovine rod or chipmunk cone PDE RGS9-1 GTPase-accelerating protein (GAP) enhancement effect, essentially as
described previously (He et al., 2000). Specifically, bovine rod outer
segments containing 10 µM rhodopsin were exposed to light
and mixed with different amounts (0, 10, 20, 50, or 100 nM)
of His6-tagged bovine rod or
His6-tagged chipmunk cone PDE in buffer D. Then GTP hydrolysis was initiated by adding 7 µl of
[-32P]GTP (Amersham
Biosciences) to 14 µl of the above mixture by vortexing. The
reaction was quenched by 100 µl of 5% trichloroacetic acid at
various times, and Pi (free phosphate ion) released
from hydrolyzed GTP was determined by activated charcoal assay. The first-order rate constants for GTP hydrolysis
(kinact) were obtained by
fitting data to single exponentials.
| |
Results |
Immunostaining of RGS9-1 and G5 in mouse and
chipmunk retinas
RGS9-1 immunofluorescence was observed primarily in outer segments
of photoreceptor cells (Fig.
1A,B,D,E). In
rod-dominant mouse retina, much brighter immunofluorescence was
observed in cone outer segments, identified by staining of cone sheaths
with rhodamine-conjugated peanut agglutinin. In cone-dominant chipmunk retina, cone outer segments were stained brightly, whereas RGS9-1 staining in rod outer segments, identified by staining with
anti-rhodopsin antibody 1D4, was barely detectable. These results,
together with previous results in bovine and human retinas (Cowan et
al., 1998; Zhang et al., 1999) confirm that in mammals, much more
RGS9-1 is present in cone outer segments than in rods, and that most RGS9-1 in chipmunk retina is in cone outer segments.
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Figure 1.
Immunofluorescence localization of RGS9-1
(A, B, D, E) and G5 (C,
F) in mouse (A-C) and chipmunk
(D-F) retinas. IS, Inner
segments; ONL, outer nuclear layer; OPL,
outer plexiform layer. Scale bars, 10 µm. RGS9-1 and
G5 staining are shown in green. Mouse
cone sheaths (peanut agglutinin staining; B) and
chipmunk rods (rhodopsin staining; E, same section as
D) are shown in red.
Arrowheads indicate chipmunk rods in
F.
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Strong G5 staining (Fig.
1C,F) was observed in photoreceptor outer segments.
The G5 staining of the multiple chipmunk rods
present in each field was so weak as to be undetectable, whereas the
cones stained brightly (Fig. 1F). There is also
staining in what appear to be the photoreceptor synaptic termini in the outer plexiform layer. The function of G5 in
this region of the cells remains to be determined. Because RGS9-1
staining is not observed in the outer plexiform layer, any
G5 there is unlikely to be associated with
RGS9-1.
Photoresponses of chipmunk cones
Suction electrode recordings were made from chipmunk rod and cone
cells to define the differences in their sensitivity and the kinetics
of the recovery phase of their light response as well as to determine
the action spectra. Figure 2 shows
families of photocurrent responses in a rod (Fig. 2A)
and M-cone (Fig. 2B) to brief light flashes of
increasing intensity. The average time to peak of the linear range
responses in the rods was 2.25 times slower than in M-cones (Table
1). The graphs on the
right of Figure 2, A and B, plot the
peak response amplitude versus the log-stimulus intensity. The
smooth curves are fits to the data with an exponential
saturation function of the type used by Lamb et al. (1981). There was a
38-fold difference in the sensitivity as measured by
I1/2 (the stimulus strength required
to produce a half-maximal response) or 140-fold difference as measured
by the flash sensitivity (Sf,
picoamperes per photon per square micrometer), which is measured for
linear range responses (Table 1).
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Figure 2.
Suction electrode recordings of the light-evoked
currents from chipmunk rod and cone photoreceptors. A,
Rods. A family of responses evoked by 2 msec flashes of light of
increasing strength. Each trace represents the averaged
response of 3-39 repeated stimuli (DC to 50 Hz, 37°C). The stimulus
strength varied from 0.73 to 3.97 log-photons/µm2
(or 5.4-9200 photons/µm2). On the
right, the peak amplitude of each response is plotted
against the log of the stimulus strength. The long thin
lines between the left and right
graphs connect selected data points with their respective
photocurrent traces. The smooth curve is an exponential
saturation function fit to the intensity response data (see
Results). The short vertical bar near the
middle of the intensity graph indicates
the mean value of I1/2 for 30 rods; the
short horizontal bar indicates ±1 SD. B,
Cones. Left, Family of responses recorded from a cone;
each trace is the average of 5-20 responses to stimuli
whose strength ranged from 3.38 to 4.83 log-photons/µm2 (or 2410-67,200
photons/µm2; DC to 100 Hz, 36°C).
Right, Intensity-versus-response plot for the cone data
fit by an exponential saturation function. The short vertical
bar near the middle of the intensity
graph indicates the mean value of
I1/2 for 11 M-cones; the short
horizontal bar indicates ±1 SD. C, Fitting of
the equation of Lamb and Pugh (1992) (Equation 1) to responses of a
typical rod, over the period from 0 to 85 msec after the flash.
Photoisomerizations for the four traces were = 1.5, 3.0, 5.6, and 11.1, and Rmax = 26 pA in this cell. Parameters
obtained from the fit were gain, A = 11.3 sec2; and delay,
td = 7.6 msec. D,
Fitting of the same equation to responses of a typical M-cone over the
period from 0 to 52 msec after the flash. Photoisomerizations for the
four traces were = 61.9, 108, 176, and 319, and
Rmax = 15 pA in this cell. Parameters obtained from
the fit were gain, A = 1.7 sec2; and delay,
td = 9.8 msec.
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Use of RGS9-1 staining to identify cones and 1D4 staining to identify
rods allowed counting of rods and cones in the chipmunk sections. In
eight fields counted, cones made up an average of 75.0 ± 2.6% of
the total photoreceptors; if we account for the slightly higher
probability of finding cones in an optical section because of their
~11% greater width, this value becomes 68%. Of the 15 cones studied
electrophysiologically, two were S-cones; the remainder were M-cones.
Eleven of 13 M-cones and 0 of 2 S-cones had an undershoot in the
recovery phase of the response.
The efficiencies of signal amplification in the transduction cascade in
different cell types can be compared by fitting the rising phases of
the responses to Equation 1, which models the activation reactions
only, and then comparing the values of A, or gain. Results
of the model fitting to multiple responses are shown for one rod (Fig.
2C) and one cone (Fig. 2D). The mean
values for 16 chipmunk rod and cone cells are given in Table 1. The gain for cones had a mean value of 1.0 sec2, and a range of 0.3-1.7
sec2. Similar results were obtained when
the same analysis was applied to previously published results of human
cone (Kraft et al., 1998, their Fig. 1; A = 0.9 sec2) and ground squirrel cone (Kraft,
1988, Fig. 3a,b;
A = 1.0 and 1.7 sec2,
respectively). Chipmunk rods had a mean gain of 10.4 sec2 with a range of 6.2-14.2
sec2. Although there is some variability
from cell to cell for both rods and cones, the average values indicate
an order of magnitude lower amplification, as reflected in the
parameter A, in cones of all three species compared with
chipmunk rods.
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Figure 3.
Estimation of pigment content by difference
spectrophotometry. A, Model absorbance spectra used for
estimates. Relative spectral sensitivity data, determined from suction
electrode current recordings as described in Results, are shown
along with curves representing the model spectra fit to the data as
described in Materials and Methods. Circles, M-cones;
squares, rods; triangles, S-cones. The
solid line labeled R* is a plot of
measured absorbance data for bovine rhodopsin after illumination.
B, Difference spectrum of detergent extract of chipmunk
retina-RPE. Circles, Difference between absorbance
before and after exposure to room light; line, fit of
linear combination of model spectra to difference spectrum. The
curve is the result predicted for a molar ratio of M
pigment/rhodopsin/S pigment of 1:0.30:0.05 and a total cone pigment
concentration of 0.60 µM.
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To achieve some appreciation for the difference in the recovery
kinetics of the photoresponses, a simple linear regression of the
photocurrent recovery was performed covering saturating and
semisaturating responses. The recovery kinetics were also significantly
faster in cones than in rods. As indicated in Table 1, the slopes of
the recovery phases (picoamperes per second) for the cones were 3.1- to
4.5-fold steeper than those of rods, with both normalized to the
maximum photocurrent in each cell.
The action spectra of the rod and cone visual pigments were measured by
determining the stimulus strength that was required to generate a
criterion linear range response. The results are presented in Table
2 and graphically in Fig. 3A.
S- and M-cones were encountered with peak sensitivities of ~450 and
540 nm, respectively. The rod peak sensitivity was 504 nm; no L-cones
were encountered. Behavioral testing of the cone-mediated vision found
the threshold difference between 539 and 580 nm of 0.231 log units (R. E. Van Arsdel and M. S. Loop, personal communication), an
excellent match of the action spectra sensitivity differences for the
M-cones from single-cell recordings at similar wavelengths (0.239 log units for 541 vs 580 nm; Table 2).
Cloning of chipmunk RGS9-1, G5, and cone
PDE cDNAs
Chipmunk RGS9-1, G5S, and PDE cDNAs
were cloned from chipmunk retina via RT-PCR and RACE; sequences are
available from GenBank with accession numbers listed in Materials and
Methods. The cloned chipmunk RGS9-1 cDNA includes part of the 5'
UTR and coding sequence corresponding to first 478 of 484 amino acids. The rest of the coding sequence was cloned by degenerate PCR on the
basis of the assumption that the last six amino acids are conserved
among mammalian RGS9-1. The deduced amino acid sequence of chipmunk
RGS9-1 shares >90% identity with human, mouse, and bovine RGS9-1
(Fig. 4A). By RT-PCR,
chipmunk cone PDE cDNA was cloned, using two degenerate primers
conserved in the 5' and 3' UTR of mammalian cone PDE. The deduced
amino acid sequence of chipmunk PDE has 95% identity to bovine,
mouse, ground squirrel, and human cone PDE and 78% identity to
bovine, human, mouse, and dog rod PDE (Fig. 4C). Chipmunk
G5S cDNA was also cloned from chipmunk retina
RNA by RT-PCR and RACE. The deduced amino acid sequence is 100%
identical to that of mouse G5S (Fig. 4B).
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Figure 4.
Phylogenetic trees calculated from the amino acid
sequences of RGS9 (A), G5
(B), and PDE (C) by the
neighbor-joining method. The horizontal distances are proportional to
the percent differences in amino acid sequences. Scale bar, 10%
replacement of an amino acid per site.
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Catalytic activity of chipmunk cone PDE
To measure the efficiencies of hydrolysis of cGMP by PDE in
mammalian cones and compare the results with those in rods, we performed pH-based PDE assays in homogenates from cone-dominant chipmunk retina and bovine ROS. Figure
5A shows pH recordings in
bovine or chipmunk samples containing 4 nM
endogenous PDE. Both chipmunk cone and bovine rod PDE displayed low
basal activity and were similarly activated by addition of trypsin to
remove the inhibitory subunit PDE, and the activation was reversed
by adding back His6-tagged bovine rod PDE
after the addition of soybean trypsin inhibitor. Basal and maximal PDE
activities in chipmunk retinal homogenates were similar to those in
bovine samples. The concentration of PDE in each of the samples was
calculated to be 2 nM from PDE immunoblots,
using the ratio of two PDE subunits per holoPDE heterotetramer. On
the basis of this concentration and the observed catalytic activity,
the calculated maximum turnover numbers for chipmunk and bovine PDE
were 3600 and 3900 mol of cGMP hydrolyzed per mole of PDE per second,
respectively. These numbers are consistent with a previously reported
rod PDE kcat value of ~4000 cGMP
hydrolyzed per PDE per second (Hurley and Stryer, 1982; Stryer et al.,
1983), indicating chipmunk cone PDE and bovine rod PDE have similar
cGMP hydrolytic activities when fully activated. Bovine cone PDE
has been reported to have a similar specific activity of 3500-4670
cGMP per second (Gillespie and Beavo, 1988).
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Figure 5.
Biochemical properties of chipmunk cone PDE and
PDE. A, pH recordings from chipmunk retina homogenate
(C-Ret.) and bovine ROS (B-ROS) to test
activity of bovine rod PDE and chipmunk cone PDE. Each sample contained
2 nM PDE, as estimated by immunoblots
(inset) and densitometry. PDE was activated by removal
of inhibitory subunit PDE using trypsin (T.). The
hydrolysis of cGMP by PDE leads to a decrease in pH, shown as positive
deflection on the y-axis. Inhibition of PDE was restored
by addition of bovine recombinant PDE. Left, Activity
of bovine PDE; right, activity of chipmunk PDE.
TI, Soybean trypsin inhibitor. The long
arrowheads indicate time 0, when bovine ROS or chipmunk retina
homogenate was added, and short arrowheads indicate the
addition of 2 mM cGMP. B, inhibition of PDE
activity followed by pH recording. Top, At the indicated
times, first trypsin-activated PDE and then indicated amounts of
His6-tagged PDE (chipmunk cone or bovine rod) were added
to a reaction vessel containing 2 mM cGMP. Hydrolytic
velocity (d[cGMP]/dt) is proportional
to the slope at each point along the
traces. Bottom, Inhibitory activities of
bovine rod (triangles) and chipmunk cone PDE
(squares) are plotted along with linear least squares
fits of decreases in cGMP hydrolytic velocity
(y-axis) as a function of added PDE. The
inhibitory activities calculated from the slopes are 1773 ± 207 mol of cGMP hydrolysis per second inhibited per mole of bovine rod
PDE and 1620 ± 275 mol of cGMP hydrolysis per second inhibited
per mole of chipmunk cone PDE. C, stimulation of GAP
activity. The first-order rate constants for GTP hydrolysis in bovine
rod outer segment membranes measured under single-turnover conditions
are plotted as a function of concentrations of added recombinant PDE
from bovine rods (squares) or chipmunk cones
(circles).
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Biochemical properties of chipmunk cone PDE
To test the potency of PDE inhibition by chipmunk cone PDE, pH
assays were performed using 5 nM trypsin-treated bovine PDE or 2 nM chipmunk PDE, and varying concentrations of bovine
(0, 2, 4, and 8 nM) or chipmunk (0, 1, 2, 4, and 8 nM) His6-PDE (Fig. 5B).
Hydrolysis of cGMP slowed dramatically within seconds of addition of
recombinant PDE. cGMP hydrolytic velocity,
d[cGMP]/dt, was determined from the slope at
each point along the pH-recording traces (Fig. 5B,
top). PDE inhibitory activity toward trypsin-activated PDE was determined by the difference in hydrolytic velocity before and
after addition of PDE. Figure 5B, bottom,
shows that chipmunk cone and bovine rod PDE have similar inhibitory
effects on bovine rod PDE. The PDE inhibitory activities were
calculated from linear least squares fits. When added to bovine rod
PDE, each mole of bovine rod PDE inhibited hydrolysis of 1773 ± 207 (mean ± SD) mol of cGMP per second, and each mole of
chipmunk cone PDE inhibited hydrolysis of 1620 ± 275 mol of
cGMP per second. When added to chipmunk PDE, each mole of recombinant
chipmunk cone PDE inhibited 1781 ± 109 mol of cGMP hydrolysis
per second, indicating similar potencies in PDE inhibition, as observed
previously for bovine cone PDE (Hamilton et al., 1993).
Rod PDE can enhance the GAP activity of
RGS9-1/G5L (He et al., 2000; Skiba et al.,
2000). To find out whether chipmunk cone PDE has similar RGS9-1 GAP
activity enhancement activity, GTPase single turnover assays were
performed in bovine ROS containing 10 µM rhodopsin (Fig.
5C). Chipmunk cone and bovine rod recombinant PDE
increased RGS9-1/G5L-mediated hydrolysis of
GTP by transducin with similar potencies; the same concentrations of
recombinant proteins led to similar GTP hydrolysis rates, with a
threefold acceleration above basal hydrolysis at a concentration of 50 nM in each case.
Quantification of proteins
To quantify the levels of phototransduction proteins that
determine the lifetime of effector activation through regulation of the
kinetics of G-protein GTP hydrolysis, we determined the molar ratios of
RGS9-1 and PDE to visual pigments in cone-dominant chipmunk and
rod-dominant mouse and bovine retinas. The amounts of visual pigments
were quantified by difference spectrophotometry. Figure 3B
shows the chipmunk retina-RPE extract difference spectrum, with a
maximal value at 534 nm. Because in chipmunk retina, there are three
kinds of visual pigments, rhodopsin, mid-wavelength-sensitive (M-cone)
pigment, and short-wavelength-sensitive (S-cone) pigment, the
difference spectrum is a linear combination of the difference spectra
of the two cone pigments and rhodopsin. To quantify cone pigments, we
generated model spectra (Fig. 3A) on the basis of our
spectral sensitivity data (Table 2) as described in Materials and
Methods and used a measured spectrum of bovine metarhodopsin II as an
approximation of the spectra for all three photoexcited chipmunk
pigments (Fig. 3A). Figure 3B shows a linear
combination of the model spectra from Figure 3A fit to the
observed difference spectrum in the range of 400-600 nm. The
smooth curve is the result predicted for a molar ratio of M
pigment/rhodopsin/S pigment of 1:0.3:0.05, assuming equal extinction
coefficients at the absorbance peak for each. The proportion of total
visual pigment contributed in this fit by rhodopsin, 22%, is
consistent with our immunofluorescence results indicating that rods
make up 25% of total photoreceptors. Our results do not provide an
accurate estimate of the relative numbers of S-cones or the amounts of
S pigments, although they are clearly much lower than the numbers for
M-cones and rods. By using the reasonable assumption that the cone
visual pigments' extinction coefficients are similar to those of other
visual pigments (~40,000
M1 · cm1;
Vissers et al., 1998), a total cone pigment concentration of 0.60 µM was determined for the sample shown. The
amounts of rhodopsin in rod-dominant bovine and mouse retina and
bovine ROS samples were quantified by difference spectrophotometry as well.
To determine the amounts of RGS9-1 and PDE from different
samples, Western blots, followed by densitometry, were performed, and
the results were compared with standard curves generated with purified
proteins as described in Materials and Methods.
Similar molar ratios of PDE to visual pigments were obtained
in chipmunk, 1:68, mouse, 1:65, and bovine retinas, 1:76 (Fig. 6D), indicating similar
relative concentrations of PDE in cones and rods. Bovine rod outer
segments, purified from frozen retinas, had a slightly lower ratio of
PDE to rhodopsin, 1:104, likely because of loss of the soluble form
of PDE associated with the PDE
subunit (Gillespie et al., 1989;
Florio et al., 1996). The molar ratio of RGS9-1 to cone pigments in
chipmunk retina was determined (Fig.
7E) to be ~1:62, >14-fold
higher than that in purified bovine ROS, 1:910, and ~10 times higher
than the ratio in bovine and mouse retina, 1:610. Because the molar
ratios of visual pigments to transducin are not very different between
rods and cones (Tachibanaki et al., 2001), these results indicate that there is a 10-fold higher ratio of RGS9-1 to transducin in cones than
in rods. The total concentrations of RGS9-1 and PDE, which work
together to achieve maximal transducin GTPase acceleration, are very
similar in cones, whereas in rods there is an almost 10-fold excess of
PDE over RGS9-1. The higher concentration of RGS9-1 in cones may be
important in the faster recovery of light responses in cones.
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Figure 6.
Determination of PDE levels by quantitative
immunoblots. A, Examples of immunoblots used to create
standard curves from PDE purified from bovine retina and to
determine amounts in each sample. B-Ret., Bovine retinal
extract; M-Ret., murine retinal extract;
C-Ret., chipmunk retinal extract; B-ROS,
bovine rod outer segment homogenate. B, Example of
immunoblots used to calibrate relative sensitivity of anti-PDE for
chipmunk cone PDE using purified his6-tagged recombinant
proteins. C, Example of a standard curve for purified
bovine PDE from the blot in A.
D, Ratios of PDE to rhodopsin or cone pigment in
chipmunk (C-ret.), mouse (M-ret.), and
bovine (B-ret.) retinas and in bovine rod outer segments
(B-ROS). The ratios are 0.0132 ± 0.007 in bovine
retina, 0.0143 ± 0.0015 in chipmunk retina, 0.0155 ± 0.0028 in mouse retina, and 0.0096 ± 0.0016 in bovine ROS.
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Figure 7.
Determination of RGS9-1 levels by quantitative
immunoblots. A, Examples of immunoblots used to generate
standard curves and to determine amounts in each sample.
B, Example of a standard curve for
his6-RGS9-1 (bovine). C, D, Examples of
immunoblots used to calibrate relative sensitivity of RGS9-1-specific
antibodies for RGS9-1 from bovine
(His6-bS9), chipmunk
(His6-cS9), and mouse
(His6-mS9).
E, Ratios of RGS9-1 to rhodopsin or cone pigment in
chipmunk retina (C-Ret.), 0.0157 ± 0.0016; mouse
retina (M-Ret.), 0.0015 ± 0.0006; and bovine
retina (B-Ret.), 0.0016 ± 0.0001; and in bovine
rod outer segments (B-ROS), 0.0011 ± 0.0004.
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Both G5L and G5S bind to RGS9-1 in
chipmunk cones
RGS9-1 forms a complex with G5L in
photoreceptor outer segments of rod-dominant mouse and bovine retinas,
and the proteins are mutually dependent on one another for expression
and stability (Makino et al., 1999; Chen et al., 2000; He et al.,
2000). From previous studies, the molar ratio of RGS9-1 to
G5L appears to be very close to 1:1 in rod
photoreceptors. To find out whether the molar ratio of RGS9-1 to
G5L is similar in cone outer segments, we
performed immunoblots of chipmunk retina using
G5 antibodies. Surprisingly, a much smaller
ratio of G5L to RGS9-1 immunoblot signal was
found in chipmunk retina homogenate compared with that in bovine retina
homogenate (Fig. 8) on the same blot. The
difference in immunoblot G5L signal is
unlikely to be attributable to differences in antibody sensitivity,
because the epitope recognized by our antipeptide antibodies is
identical in bovine and chipmunk G5L. To find
out whether some RGS9-1 is associated with the short form,
G5S, in chipmunk cones, RGS9-1 GAP complexes
were immunoprecipitated from chipmunk retina homogenate using
anti-RGS9-1c antibody. In the precipitated pellets, G5L and G5S were
found in similar amounts (Fig. 8). Because the measurements described
above indicate that >96% of the RGS9-1 in chipmunk retina is in
cones, this result indicates that, in contrast to the exclusive
association of RGS9-1 with G5L in bovine and
murine rods, RGS9-1 associates with similar amounts of
G5L and G5S in
chipmunk cones.
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Figure 8.
Coimmunoprecipitation of both long and short
isoforms of G5 with RGS9-1 in chipmunk retina. RGS9-1
and associated proteins were immunoprecipitated with immobilized
RGS9-1-specific antibodies as described in Materials and
Methods. The immunoprecipitates were analyzed by SDS-PAGE and
immunoblotting using antibodies specific for RGS9 (bottom
panel) or G5 (top panel;
antibody recognizes a peptide epitope present in both isoforms). The
left two lanes in each panel are total
retinal homogenates from 50 µg of bovine retina
(B-Ret.) or 10 µg of chipmunk retina
(C-Ret.), and the right two lanes are the
immunoprecipitates (IP; from 100 and 20 µg,
respectively).
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Discussion |
The differences observed in rod and cone photoresponses in the
chipmunk, like differences between rods and cones of other species,
likely arise at several steps in the phototransduction cascade.
Candidates for key biochemical differences giving rise to lower
sensitivity and faster recovery are the photopigments themselves,
lifetimes of activated pigments, activation of the G-proteins by
light-activated pigments, lifetimes of activated G-proteins,
G-protein-effector coupling, cGMP-gated channel properties, and
differences in Ca2+ concentrations and
Ca2+-regulatory mechanisms feeding back on
the other steps and on guanylate cyclase activity. There have been only
a few biochemical studies of isolated cone pigments, but these suggest
that the complex with 11-cis-retinal may be less stable than
that formed by rhodopsin both with respect to thermal isomerization
(Birge and Barlow, 1995), leading to greater dark noise, and with
respect to dissociation of retinal, which could lead both to increased background attributable to weak activation by apo-opsins on
dissociation of 11-cis-retinal in the dark and to rapid
inactivation attributable to rapid dissociation of all-trans
retinal after photoactivation. A recent study of isolated carp cones
(Tachibanaki et al., 2001) reported that the gain of phototransduction
reactions was much lower than in rod outer segments; however, direct
measurements of G-protein activation by human and chicken
green-sensitive cone pigments (Imai et al., 1997; Vissers et al., 1998)
and Xenopus short-wavelength visual pigments (Starace and
Knox, 1997; Babu et al., 2001) suggest that the efficiency of rod
transducin activation by cone pigment equivalents of metarhodopsin II
is only approximately twofold lower than activation by rod
metarhodopsin II. Thermal decay of the metarhodopsin-like species has
been consistently reported to be much faster for cone pigments than for
rhodopsin, and the study of carp cones also revealed much faster
phosphorylation and phosphorylation-induced inactivation. Thus the
catalytic efficiency of photoexcited cone pigments is less likely to
account for the differences in sensitivity than is the dramatically
reduced lifetime of this catalytically active state. Rapid truncation
of the rise in activated transducin is consistent with our observation
of more rapid times to peak in chipmunk cone responses than in rod responses and with the 10-fold lower effective amplification.
Rapid inactivation of photoexcited pigment by itself is not suffici
TOP
ABSTRACT
Introduction
