The Journal of General Physiology
J. Gen. Physiol. © The Rockefeller University Press $8.00
Volume 127 Number 4 April 2006 355–358
Shedding Light On Cones
Barry E. Knox and Eduardo Solessio
Department of Biochemistry and Department of Molecular Biology and Ophthalmology, SUNY Upstate Medical University,
Syracuse, NY 13210
Daytime vision in vertebrates initiates with the absorp-
tion of light by cone photoreceptors (Rodieck, 1998),
which generate signals for color discrimination (Sharpe
et al., 1999). In humans, these cells are concentrated in
a specialized part of the central retina called the fovea.
This region of the eye operates over a wide range of in-
tensities (Aguilar and Stiles, 1954) mediating high tem-
poral (Green, 1970) and spatial visual resolution (Hart,
1987). The importance of the fovea to human vision is
most clearly seen in the devastating disease, age-related
macular degeneration (Bird, 2003). In line with these
observations, cones in lower vertebrates (Normann and
Perlman, 1979; Perry and McNaughton, 1991; Burkhardt,
1994) and primates (Schnapf et al., 1990) exhibit faster
response kinetics and extended adaptation ranges when
compared with rods, although these improved features
are accompanied by a loss in light sensitivity.
Given the importance of cone photoreceptors, it is
problematic that there is not yet a broad understanding
of their unique features. This defi ciency is due to the
lack of an experimental system that provides a physi-
ologically suitable cell preparation that can be manip-
ulated genetically to modulate gene expression. This
elusive goal in phototransduction research has recently
been reached by E. Pugh and colleagues (Daniele et al.,
2005; Nikonov et al., 2005; and on p. 359 of this issue),
who have crossed over the hurdle by establishing a ro-
bust way to record light responses from murine cones.
In this fi rst glimpse of cone responses, these investiga-
tors have uncovered some unique properties of cone
physiology and opened the way for further explorations
using genetic manipulation.
Lessons from Rods
Enormous progress in terms of understanding rod
phototransduction has come from many labs in stud-
ies using the suction electrode recording technique
pioneered by Baylor and colleagues in the late 1970s
(Baylor et al., 1979). In combination with genetically
manipulated mice, a quantitative description of the rod
photoresponse has been established (Lamb and Pugh,
1992; for review see Arshavsky et al., 2002). These stud-
ies established that amplifi cation, a measure of the gain
of the transduction cascade, occurs in three stages and
elucidated the molecular basis for each of these stages.
In the fi rst stage, gain is achieved through the activation
of many transducin (GT) molecules by a light-activated
rhodopsin molecule (R*). In the second stage, many
cGMP molecules are hydrolyzed by activated cGMP phos-
phodiesterase (PDE). Finally, the cGMP-gated channels
have a cooperativity in cGMP binding, leading to an ad-
dition gain step. The rising phase of the response to a
brief fl ash of light is determined by the combined ef-
fects of the three stages and can be described by a para-
bolic equation ( Lamb and Pugh, 1992):
( ). R tAt
R(t) is the normalized response, Φ is the number of
photoisomerizations of rhodopsin, and A is the ampli-
fi cation constant. The amplifi cation constant can be
quantitatively understood in biochemical terms:
where υG is the rate of transducin activation per R*, cGE
is the coupling effi ciency from G* activation to PDE ac-
tivation, βsub is related to the rate of cGMP hydrolysis
per PDE subunit, and ncG is the Hill coeffi cient of cGMP
channel opening. A satisfying aspect of this framework
is that all parameters are linked to measured properties.
For example, βsub identifi es the contribution of a PDE
subunit to the amplifi cation of the photoresponse and
is defi ned using the enzymatic properties of PDE:
where kcat/Km is the apparent second order rate constant
for free PDE and cGMP reactions, Vcyto is the volume of
the cytoplasm, NA is Avogadro’s number, and BPcG is the
cytoplasmic cGMP buffering power. Biochemical and
physiological experiments (for review see Arshavsky
et al., 2002) have shown that changes in the concentra-
tion of effector molecules in the outer segment leads to
alterations in the photoresponse. For example,
large protein translocations into and out of the outer
Correspondence to Barry E. Knox: email@example.com Abbreviation used in this paper: PDE, phosphodiesterase.
356 Shedding Light On Cones
segment can reduce the amplifi cation of the response
(Sokolov et al., 2002).
Response termination is not quite as well under-
stood quantitatively (Hamer et al., 2005). It involves
mechanisms that inactive each of the integrating
stages. First, disruption of GT activation by light-
activated rhodopsin occurs via phosphorylation of
rhodopsin by GRK1 and arrestin binding (Arshavsky,
2002). Second, hydrolysis of GTP bound to activated
GT α subunit is accelerated by the RGS9-Gb5L-R9AP
complex (Chen et al., 2000; Arshavsky et al., 2002).
This is the slowest step in rod recovery from saturat-
ing fl ashes. Finally, cGMP levels are restored to rees-
tablish circulating current via multiple mechanisms
including calcium-dependent activation of guanyl-
ate cyclase via GCAP proteins (Arshavsky et al., 2002;
Korenbrot and Rebrik, 2002; Palczewski et al., 2004).
It has also been recently shown that developmental
changes in calcium feedback through increasing con-
centrations of calcium-binding proteins can change
the functioning of rod vision in amphibians (Solessio
et al., 2004). The impressive progress in a quantitative
understanding of rod phototransduction has framed
the issues of what molecular mechanisms determine
the unique properties of cones.
Cones Preserve Responsiveness in Strong Light
Cone and rod photoreceptors contain similar types of
proteins for phototransduction, though they often are
encoded in distinct genes. Physiologically, however,
their response properties are quite different. Although
the rising phase appears to be quite similar in both rods
and cones, the responses terminate much more quickly
in cones and overshoots the dark current level (Baylor,
1987). This may be related to quantitative differences
in the level of expression of proteins involved in inac-
tivating GT (Cowan et al., 1998), activating guanylate
cyclase (Palczewski et al., 2004), or calcium homeostasis
(Koren brot and Rebrik, 2002). More signifi cantly, cones
do not saturate in response to background illumination
but remain responsive over more than seven orders of
magnitude (Perlman and Normann, 1998). Rods satu-
rate (i.e., become unresponsive to incremental fl ashes)
at much lower photobleaching levels.
How do cones maintain their sensitivity? There appears
to be two different realms in cone physiology: a high-
intensity range where pigment depletion dominates
the response sensitivity and a low-intensity range where
mechanisms involving other adaptive methods must be
active (Burkhardt, 1994; Perlman and Normann, 1998).
To further understand the adaptive properties of cones,
we need to understand how cones terminate their re-
sponses so quickly and their behavior in bright lights.
Until now, however, it has not so far been possible to
investigate these questions in mice cones, where genetic
manipulations are enormously powerful.
Recording from Mouse Cones
Recording from mouse cones has proved challenging
because they are only a small fraction of the total photo-
receptor population in this rod-dominated retina
(Carter-Dawson and LaVail, 1979). A breakthrough was
reported by Pugh and colleagues last year, who took ad-
vantage of the Nrl knockout mouse (Mears et al., 2001)
in which photoreceptor cell fate was drastically altered
(Daniele et al., 2005; Nikonov et al., 2005).
Pugh and colleagues showed quite convincingly that
photoreceptors in the Nrl−/− mouse closely resemble
cone photoreceptors, using both morphological and
molecular techniques (Daniele et al., 2005). They also
studied the functional properties of Nrl−/− cone cells
at the single cell level. Because of their abundance, it
was possible to improve techniques for long and stable
recordings. It turned out that the suction pipette
approach, which had been successful for mouse rods,
was not tolerated well by the more fragile cone outer
segments. By drawing the inner segments of the cones
into the recording pipette, Nikonov et al. (2005) deter-
mined that the responses had faster kinetics and re-
duced sensitivity compared with wild-type mouse rods.
In a twist from many other species, mouse cones coex-
press both S- and M-cone opsin. Thus, the authors char-
acterized the dim fl ash responses from cells obtained
from mice lacking both Nrl and Grk1 function. These
cells exhibit differences in the recoveries to stimuli that
activate the M- and S-pigment; only the M-pigment–
driven responses are slowed down in the double knockout.
This is a surprising and very signifi cant result as it reveals
an unexpected complexity in the light responses
of cones. One possible explanation is that there is an ad-
ditional inactivation mechanism (e.g., another kinase)
that is specifi c for S-opsin. Another possibility is that
S- and M-opsin–activated states have different stabilities
(Vought et al., 1999). To have sensitivity in the ultraviolet
range (λmax ?360 nm), the retinylidene Schiff base link-
age apparently must be unprotonated (Babu et al., 2001;
Kusnetzow et al., 2004). Thus, the different inactivation
mechanism for S-opsin may be a tradeoff for stability of
the activated form in order to achieve spectral tuning.
In the article published in this issue, Nikonov et al.
applied their novel recording approach to study cones
in a wild-type retina. One complication not present in
the Nrl−/− retina is that the cells are not isolated
from other photoreceptors (e.g., rods), so background
illumination is required to isolate the cone responses.
In a further refi nement of the experimental design,
the authors studied cone responses in the GNAT1−/−
mouse, in which rod transduction has been specifi cally
disabled. The combination of WT and KO mice shows
convincingly that reliable cone responses can be re-
corded from many cells, permitting a thorough quan-
titative analysis of the photoresponses under dim light
and stronger background illumination. The fi ndings
Knox and Solessio357
from this initial characterization will form the basis for
future mechanistic explorations of the shape and size
of the photoresponse. For now, the amplifi cation con-
stants were two- to threefold lower for cones than rods,
indicating either a reduced effi ciency of transducin
activation (υG) or differences in cone PDE properties
(kcat/KM). The dominant time constant for recovery
from a bright fl ash is much faster in mouse cones than
rods; and most interestingly, the circulating currents
recover substantially in both S- and M-type cones fol-
lowing a fl ash of light that bleaches a substantial frac-
tion (>50%) of the pigment. This immunity is in stark
contrast to rods, which do not recover signifi cantly.
In salamanders, it has been proposed that the apopro-
tein (the bleached pigment) has an activity that may
act somewhat like light (Cornwall and Fain, 1994;
Cornwall et al., 1995) and thus play an adaptive role in
desensitizing the photoresponse. Perhaps this is not an
important mechanism in mouse cone responses to
bright backgrounds, which could point to key differ-
ences in terms of setting the dynamic range for photo-
transduction. As stated above, one of the key features
of cones is that they adapt to a wide range of light
intensities, which is the most central property required
by these cells to function in bright light. We can look
forward to more mechanistic information in this power-
ful animal model now that the technical challenges
have been met.
The authors are supported by the National Institutes of Health
grants EY-11256 and EY-12975 (B.E. Knox), Research to Prevent
Blindness (unrestricted grant to SUNY UMU Department of
Ophthalmology) and Lions of CNY.
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