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J Physiol 594.19 (2016) pp 5415–5426 5415
The Journal of Physiology
Neuroscience
TOPICAL REVIEW
Why are rods more sensitive than cones?
Norianne T. Ingram1, Alapakkam P. Sampath2and Gordon L. Fain1,2
1Department of Integrative Biology and Physiology, University of California, Los Angeles, CA 90095–7239, USA
2Department of Ophthalmology and Jules Stein Eye Institute, University of California, Los Angeles, CA 90095–7000, USA
Cone Rod
Photopigment:
Cone Rh Rod Rh
G protein (transducin):
Phosphodiesterase:
PDE6C PDE6A&B
PDE6G
cGMP-gated channels:
A3+B3 A1+B1
Lower GAP expression
Slower turnover of cGMP
α2
Lower cyclase expression
β3γ8α1β1γ1
PDE6H
Abstract One hundred and fifty years ago Max Schultze first proposed the duplex theory of
vision, that vertebrate eyes have two types of photoreceptor cells with differing sensitivity: rods
for dim light and cones for bright light and colour detection. We now know that this division
is fundamental not only to the photoreceptors themselves but to the whole of retinal and visual
processing. But why are rods more sensitive, and how did the duplex retina first evolve? Cells
resembling cones are very old, first appearing among cnidarians; the emergence of rods was
a key step in the evolution of the vertebrate eye. Many transduction proteins have different
isoforms in rods and cones, and others are expressed at different levels. Moreover rods and cones
Gordon L. Fain is Distinguished Professor of Integrative Biology/Physiology and of Ophthalmology at the
University of California Los Angeles (UCLA) and a member of the Jules Stein Eye Institute. His laboratory
works on the physiology of vertebrate photoreceptors. NorianneIngram is a graduate student in the Molecular,
Cellular, and Integrative Physiology Program at UCLA and is presently doing her thesis on conephotoreceptors
and retinal signal processing. Alapakkam P. Sampath is Professor of Ophthalmology at UCLA and a member
of the Jules Stein Eye Institute. His laboratory works on vertebrate photoreceptor physiology and signal
integration in vertebrate retina.
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5416 N. T. Ingram and others J Physiol 594.19
have a different anatomy, with only rods containing membranous discs enclosed by the plasma
membrane. These differences must be responsible for the difference in absolute sensitivity, but
which are essential? Recent research particularly expressing cone proteins in rods or changing the
level of expression seem to show that many of the molecular differences in the activation and decay
of the response may have each made a small contribution as evolution proceeded stepwise with
incremental increases in sensitivity. Rod outer-segment discs were not essential and developed
after single-photon detection. These experiments collectively provide a new understanding of
the two kinds of photoreceptors and help to explain how gene duplication and the formation
of rod-specific proteins produced the duplex retina, which has remained remarkably constant in
physiology from amphibians to man.
(Received 1 April 2016; accepted after revision 16 May 2016; first published online 24 May 2016)
Corresponding author G. L. Fain: 2129 Terasaki Life Sciences, 610 Charles E Young East, University of California, Los
Angeles, Los Angeles, CA 90095–7239, USA. Email: gfain@ucla.edu
Abstract figure legend Rods evolved from cones or their progenitors through the emergence of distinct isoforms
and altered expression levels of proteins required for transducing light into an electrical signal. These changes must
collectively explain why rods are more sensitive.
Abbreviations A, activation constant; cGMP, cyclic guanosine monophosphate; CNG, cyclic-nucleotide gated;
GAP, GTPase-accelerating protein; GC, guanylyl cyclase; GCAP, guanylyl cyclase-activating protein; GRK, G-protein
receptor kinase; hν, light (photon); mG, mouse 508 nm cone rhodopsin; mS, mouse 360 nm cone rhodopsin; PDE6,
phosphodiesterase 6; PDE6∗, light-activated phosphodiesterase 6; Rh∗, light-activated rhodopsin or metaII; Tα,α
subunit of photoreceptor G protein; WT, wild-type.
Introduction
In 2016 we celebrate the 150th anniversary of the
groundbreaking article of Max Schultze (1866), who first
proposed that rod and cone photoreceptors have different
functions. Schultze noticed that retinas of nocturnal
animals tend to have a larger proportion of cells with
rod-shaped outer segments (Fig. 1A), and that diurnal
animals have greater numbers of cells with outer segments
tapering like cones (Fig. 1B). He then proposed the duplex
theory of vision: that rods mediate perception in dim light
and cones are specialized for bright light and colour vision.
We now know that his division of visual detection into
two systems is fundamental not only to the properties
of photoreceptors but also to the connections these cells
make with other neurons and to the whole of retinal and
visual processing (Masland, 2012).
Since the publication of Schultze’s paper, we have
wondered why rod vision is more sensitive. The first intra-
cellular recordings showed that most of the sensitivity
difference is inherent in the photoreceptors themselves:
single rods are more sensitive than single cones (Fain
& Dowling, 1973). Soon afterward, biochemists and
molecular biologists discovered that the two photo-
receptors have many of the same kinds of proteins and
detect light in a similar way. Cones are much older
than rods: from the sequences of a very large number
of vertebrate photopigments, we can infer that gene
duplication produced all of the different kinds of cone
pigments before the evolution of rod pigments (Nickle
& Robinson, 2007; Shichida & Matsuyama, 2009). Along
with the pigment came the many other molecular and
anatomical differences between the two kinds of cells,
with the result that rods are able to integrate incoming
light over a longer period and operate at the theoretical
limit of single-photon detection, whereas cones are less
sensitive but exhibit adaptive properties that allow them
to detect luminance changes and motion when the photon
flux is less limiting. These differences in physiology must
ultimately derive from differences in the mechanism of
transduction in the two kinds of photoreceptors.
Recent experiments are beginning to clarify these
differences. Some of the most interesting observations
have been made from the combined efforts of molecular
biologists and physiologists inserting cone genes into
mouse rods. These experiments along with more
traditional observations by biochemists and single-cell
physiologists are gradually clarifying the roles of different
proteins in rod sensitivity. Our initial expectation had
been that one particular alteration might dramatically
change the properties of the photoreceptor. Instead we
have discovered what we should have suspected all along,
that evolution proceeded by making small changesin many
transduction proteins, incrementally increasing sensitivity
toproducetherodsandconesthatemergedaslongas500
million years ago. Although the present state of research
leaves many questions unanswered, we can now begin to
see how rods became more sensitive.
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J Physiol 594.19 Why are rods more sensitive than cones? 5417
Mechanism of transduction: rod/cone protein
isoforms
Both rods and cones detect light according to the same
basic scheme (Fig. 2). They use similar photopigments,
which were once given distinct names but are now usually
called rod or cone opsin or rhodopsin. The absorption
of light produces a change in the pigment conformation
to an intermediate called metaII or Rh∗,whichtriggers
a G-protein cascade (for an overview, see for example
Fain, 2014). The heterotrimeric G proteins of rods and
cones (called transducins) are different: rods express α1,
β1andγ1, whereas cones express α2, β3andγ8 (Sakmar
& Khorana, 1988; Kubo et al. 1991; Ong et al. 1995; Deng
et al. 2009). The G protein binds to Rh∗,andexchangeof
GTP for GDP on the transducin αsubunit (Tα)produces
the active form Tα•GTP.
Tα•GTP binds to the photoreceptor effector enzyme,
which is phosphodiesterase 6 (PDE6). This protein has
four subunits, two catalytic and two inhibitory. The
catalytic subunits are slightly different from one another
in rods and are called PDE6αand PDE6β(or PDE6A
and PDE6B), whereas the two in cones are the same and
called PDE6α(or PDE6C). Each PDE tetramer also has
two inhibitory subunits, one for each catalytic subunit,
which have somewhat different sequences in the two types
of photoreceptors and are called rod or cone PDE6γ,or
PDEG (in rods) and PDEH (in cones). Activated PDE6
hydrolyses cGMP, which acts as the second messenger
of the cascade by binding to cGMP-gated channels. The
channels are tetramers again with different protein sub-
units called CNGA1 and CNGB1 in rods and CNGA3 and
CNGB3 in cones (see Kaupp & Seifert, 2002; Zhong et al.
2002; Shuart et al. 2011).
Based on this general scheme, the activation of a single
rhodopsin molecule is amplified across these stages to lead
ultimately to the destruction of as many as one million
cGMP molecules per Rh∗in rods (Yee & Liebman, 1978).
ThisreductionincGMPconcentrationacrossvertebrate
species is sufficient to reduce the cGMP-gated current by
more than its intrinsic noise in darkness (Baylor et al.
1979, 1984; Nakatani et al. 1991). The natural question to
ask then is, can the lower sensitivity of cones be the result
purely of reduced amplification within these steps? Let us
suppose that rod and cone responses were to inactivate
at the same rate. A reduced rate of activation would then
cause cone responses to reach smaller peak amplitudes and
might account entirely for the difference in sensitivity.
But do rod and cone responses inactivate at the same
rate? Not even close! In every vertebrate species from
lamprey (Morshedian & Fain, 2015; Asteriti et al, 2015)
to mouse (see Fig. 3), the rate is much faster in cones, and
this difference must also contribute to the reduced cone
sensitivity.
The rate of inactivation is determined by the rates at
which Rh∗, transducin and PDE return to their basal
AB
Bat Hedgehog Rat Northern pike European perch Pigeon
Figure 1. Rods and cones in nocturnal and diurnal animals
Drawings from Schultze’s original paper (1866) of photoreceptors from nocturnal animals (A) and diurnal animals
(B), magnification approximately 350–400 times. Schultze claimed that the bat retina lacked even a trace of cones,
but in rat he noticed occasional gaps (L¨
ucken) which he speculated could possibly correspond to cones, as we
now know to be true. Fish and pigeon on the other hand have many easily observable cones in addition to rods.
Schultze commented that these observations ‘would seem to indicate that rods are more advantageous than cones
for quantitative light perception’, but that ‘cones would seem to be the nerve end-organs for colour perception’.
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conformations and the cGMP concentration goes back to
its dark level. Rh∗is silenced like other G-protein receptors
by phosphorylation and binding of arrestin. Rods and
cones can have two different G-protein receptor kinases,
GRK1 in rods and GRK1 and/or GRK7 in cones, but
rodents, including mice, have only GRK1 in both kinds
of photoreceptors. Rods in mice have arrestin-1 and cones
both arrestin-1 and arrestin-4, though arrestin-1 is by far
the predominant species in both kinds of photoreceptors
(Nikonov et al. 2008).
Activated transducin and phosphodiesterase are
extinguished as in other G-protein cascades by hydro-
lysis of Tα•GTP to Tα•GDP with the assistance of PDEγ
and three GTPase-accelerating proteins (GAPs): RGS9-1,
Gβ5 and the R9AP-1 binding protein (see Arshavsky &
Wensel, 2013). These proteins are required to speed PDE
deactivation into the functional range of tens to hundreds
of milliseconds, compared to the secondsor tens of seconds
required in their absence (Hollinger & Hepler, 2002).
Although these GAP complex proteins are the same in
rods and cones, expression is significantly higher in cones
(Cowan et al. 1998; Zhang et al. 2003), a point we return
to later.
The cGMP concentration is restored by guanylyl cyclase
(GC), which in photoreceptors is a member of the
membrane guanylyl cyclase family (Potter, 2011). There
are two cyclases in mammalian photoreceptors called
retGC1 (or GC-E) and retGC2 (or GC-F); in mouse, rods
have mostly retGC1 with some retGC2, whereas cones have
only retGC1 (Wen et al. 2014). This difference is unlikely
to be physiologically significant because when the gene for
retGC2 is deleted there is little effect on rod sensitivity
or response waveform (Baehr et al. 2007). The rate of
cyclase activity is controlled by small molecular weight
Ca2+-binding proteins called guanylyl cyclase-activating
proteins or GCAPs. There are again two in mouse, GCAP1
and GCAP2, with somewhat different sensitivities for
divalent ion binding (Dizhoor et al. 2010); rods express
both GCAPs but cones mostly express GCAP1 (Dizhoor
et al. 1995; Xu et al. 2013; Boye et al. 2015).
The differences in transduction proteins for rods and
cones are summarized in Table 1. Rods and cones also
display differences in anatomy: the photopigment in rods
is contained almost entirely within the membrane of intra-
cellular discs, whereas cone outer segments are formed
from infoldings of the plasma membrane. We have long
wondered whether this difference in anatomy might hold
the key to the difference in sensitivity, but we now know
the answer. Nature did the experiment for us: the rods and
cones of lamprey have an identical morphology, which
is like that of cones (see for example Dickson & Graves,
1979), but lamprey rods are nearly as sensitive as mouse
rods and about 70 times more sensitive than lamprey cones
(Morshedian & Fain, 2015; Asteriti et al. 2015). The discs of
GMP
cGMP Na+
Na+
K+
Ca2+
Ca2+
Na+/Ca2+
channel
Na+/Ca2+–K+
exchanger
Disc
Cyclase
Rh*
Rh
PDE
Transducin
αβ
GDP
GTP
Plasma
membrane
GTP
cGMP
GTP
Pi
GDP
GDP
GDP
αβγ
GAP
complex
γ
γ
hν
Figure 2. Phototransduction in vertebrate photoreceptors
Redrawn and printed with permission from Fain et al. (2010).
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J Physiol 594.19 Why are rods more sensitive than cones? 5419
rods do not seem to be essential for high sensitivity vision
(see also Ma et al. 2001) but may instead have evolved to
allow more efficient renewal of outer segment membrane
(Morshedian & Fain, 2015).
Activation of transduction
Although rods are universally more sensitive than cones,
the value of the sensitivity difference varies among
vertebrates, ranging from 25-fold in mudpuppy (Fain
& Dowling, 1973) to 1000-fold between red-sensitive
cones and rods in carp (Tachibanaki et al. 2001). In our
examination of the cause of this sensitivity difference, we
will take as our example the mouse, because many of the
most recent experiments have utilized transgenic mice.
In Fig. 3Aand B, we show mean responses of mouse
rods and cones recorded with suction electrodes. Rod
responses decay much more slowly than cone responses
(note ten-fold difference in the scale of the abscissa)
and are typically about twice as large; after normalizing
response amplitudes to their maximum values, rods are
a little more than 100 times more sensitive than cones
(Fig. 3C), as previously reported (see for example Nikonov
et al. 2006). Part of this difference is the result of the
larger volume of the rod outer segment, which increases
the probability of absorption of a photon by pigment
molecules. We can, however, correct for these differences
by calculating the percentage decrease in photocurrent
per photon absorbed. Calculations of this kind give about
0.2–0.25% per Rh∗for cones (Nikonov et al. 2006; Sakurai
et al. 2011; Cao et al. 2014) and 5% for rods (Sampath et al.
2005; see Reingruber et al. 2015). The resulting factor of
between 20 and 30 is the difference in sensitivity produced
by the transduction cascade.
One reason rods are more sensitive is that early events
in the transduction cascade have greater gain and close
channels more rapidly, as alluded to previously. As a
consequence, rod responses rise more quickly per photon
absorbed; with everything else being equal, rod responses
would reach a commensurately larger peak amplitude for
the same intensity of stimulus. Following the theoretical
treatment of Pugh and Lamb (1993, 2000), we can use
the rising phases to calculate values of an amplification
constant A(see Fig. 4Aand legend), equal to the product
of (1) the rate of formation of light-activated PDE6∗
by the photopigment, (2) the rate of decline of cGMP
16
A
B
C
1. 0
0.8
0.6
0.4
0.2
0.0
10010110 210310 4105106
Photocurrent (pA)
Normalized Current
12
8
4
0
8
4
0
0 2 4 0.0 0.2 0.4
Time (s)
Rods
Rods
Photons μm–2
Cones
Cones
Time (s)
Figure 3. Responses of mouse rods and cones
A, mean responses of 11 WT mouse rods to 20 ms flashes of 500 nm illumination from 0.5 to 2000 photons
µm−2.B, mean responses of 18 mouse M (508 nm) cones to 20 ms flashes of 500 nm illumination from 200
to 500,000 photons µm−2. Responses in Aand Bwere filtered with an 8-pole Bessel filter with a low-pass filter
setting of 75 Hz. C, mean peak amplitudes (with SEM) of responses of mouse rods (•) and mouse cones (◦)to
20 ms flashes of 500 nm illumination, normalized to maximum response and plotted as a function of flash intensity.
Curves give best-fitting Michaelis–Menten equation with flash intensities at half-maximal amplitude of 25.3 (for
rods) and 2960 (for cones) photons µm−2. All recordings were made from C57BL/6 mice from Jackson Laboratory
(Bar Harbor, ME, USA), dark adapted for at least 4 h and usually overnight. All experiments were performed on
mice of either sex in accordance with the rules and regulations of the NIH guidelines for research animals, as
approved by the institutional animal care and use committee (IACUC) of the University of California, Los Angeles.
Animals were kept in cyclic 12 h/12 h on/off lighting in approved cages and supplied with ample food and water.
Animals in all experiments were killed before tissue extraction by approved procedures, usually CO2inhalation or
decerebration. Recordings were made at 37°C in Ames solution. Light intensities are given as photons effective at
the lambda max of the rod or cone pigment calculated by convolving the spectrum of the stimulating beam with
the rod or cone photopigment absorption curves.
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5420 N. T. Ingram and others J Physiol 594.19
Table 1. Photoreceptor transduction protein isoforms in mouse rods and cones
Rod Cone
Photopigment Rod opsin (or rhodopsin) Cone opsin (or rhodopsin)
G protein (transducin) α1, β1andγ1α2, β3andγ8
Phosphodiesterase 6 PDE6A and PDE6B PDE6C
Rod PDE6γ(PDE6G) Cone PDE6γ(PDE6H)
cGMP-gated channels CNGA1 and CNGB1 CNGA3 and CNGB3
Rhodopsin kinase GRK1 GRK1
Arrestin Arrestin-1 Arrestin-1 and arrestin-4
GAPs RGS9-1, Gβ5 and R9AP-1 RGS9-1, Gβ5 and R9AP-1
Guanylyl cyclase retGC1 and retGC2 retGC1
GCAPs GCAP1 and GCAP2 GCAP1
Na+/Ca2+–K+exchanger NCKX1 NCKX2 and NCKX4
concentration per PDE6∗molecule, and (3) the Hill
coefficient of binding of cGMP to the channels. The value
of Ais somewhat dependent on the frequency response of
the recording (Chen et al. 2010b) but is at least 2–3 times
larger in rods than in cones (see Pugh & Lamb, 1993;
Nikonov et al. 2006; Cao et al. 2014). This difference must
be produced by the collective properties of the proteins
responsible for activation. Since the Hill coefficient of
rod and cone cGMP-gated channels is nearly the same
(Picones & Korenbrot, 1992; see Kaupp & Seifert, 2002), we
can focus our attention on the photopigments, G proteins
and PDE6s, which, as we have seen, all have different iso-
forms in rods and cones.
One way to test the role of these proteins is by exogenous
expression of cone proteins in rods or rod proteins in
cones. Gene incorporation is easier for rods because there
is only one rod photopigment in mouse with a reliable and
widelyusedpromoter,androdsaremoreconvenientfor
physiology; so most experiments have put cone genes into
rods. There is one complication: the value of Adepends
upon the rate of change of the cGMP concentration
which is inversely proportional to cytoplasmic volume,
because the larger the volume, the smaller the change in
concentration per activated enzyme. Since mouse rods are
about 2.5 times larger in volume than mouse cones, A
wouldbe2.5timessmallerinrodseveniftheproperties
of all of the proteins were the same. To account for the
greater value of Aactually recorded from rods, activation
would need to proceed at a rate at least 5–10 times faster
(Nikonov et al. 2006). That is, if we could express the cone
variants of all the activation proteins in a rod, activation
should be at least something like 5–10 times slower. No
one has yet expressed all of the proteins together, but many
attempts have been made to express them one by one.
We begin with the photopigments. Sakurai and
colleagues (2007) inserted the mouse 508 nm cone
pigment gene (mG) in place of mouse rhodopsin. They
found that mG/mG rods were about a factor of 3–4 less
sensitive than wild-type (WT) rods and gave smaller values
for the activation constant A,butmG/mG rods expressed
considerably less pigment and transducin, had smaller
outer segments, and showed signs of degeneration. Clearer
perhaps were experiments expressing the mG pigment on
a background of mutant E112Q rod rhodopsin (Sakurai
et al. 2007), whose peak absorbance is shifted into the blue
so that rod and cone pigments in mG/RhEQ rods can be
stimulated selectively. The cone mG pigment produced a
response per Rh∗only about a third as large as the rod
E112Q rhodopsin.
In a similar study, Shi and colleagues (2007) expressed
the mouse short wavelength-sensitive (360 nm) pigment
(mS) in mouse rods and recorded from homozygous
mS/mS rodslackingrodrhodopsinaswellasfrom
heterozygous photoreceptors expressing both the mS
pigment and rod rhodopsin. Although the single-photon
response of mS/mS rods was smaller than in WT rods,
confirming the study of Sakurai et al. (2007), recordings
from heterozygotes expressing both the mS cone and WT
rod pigments and selectively stimulated with short- and
long-wavelength light showed no differences in sensitivity
or response waveform. The two pigments seemed to
produce nearly identical responses when expressed in the
same rod.
Fu and colleagues (2008) then expressed the human
long-wavelength pigment in mouse rods. Responses to
the rod and cone pigments were indistinguishable in
sensitivity and waveform. The cone pigment produced
greater dark noise as also in the experiments of Sakurai
et al. (2007; but see Shi et al. 2007), perhaps as a result
of the lower stability of cone pigments generally (Rieke &
Baylor, 2000; Sampath & Baylor, 2002; Kefalov et al. 2003;
Kefalov et al. 2005; but see Angueyra & Rieke, 2013). This
increase in noise was, however, not large enough to affect
photoreceptor sensitivity. In conclusion, cone pigments
expressed in rods either have no effect on sensitivity or
reduce it by as much as a factor of 2–3.
The first experiments expressing transducin used a viral
vector approach to inject the rod or cone Tαgene into
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J Physiol 594.19 Why are rods more sensitive than cones? 5421
a mouse line that lacked both rod and cone transducins
(Deng et al. 2009). Only a small fraction of the rods had
any light response, probably reflecting the variability
in expression level. From the few cells that could be
recorded, there was no marked difference between cells
expressing rod Tαand those expressing cone Tα.Since,
however, the sensitivity of the rod is heavily dependent on
1. 0
0.8
0.6
0.4
0.2
0.0
0.00 0.05 0.10 0.15
0.00
0.0 0.2 0.4 0.6 0.8 1.0
Time (s)
GNAT2C
GNAT2C
WT
WT
A
B
C
Normalized Photocurrent
0.05 0.10 0.15
0.8
0.6
0.4
0.2
0.0
0.8
1. 0
0.6
0.4
0.2
0.0
Figure 4. Differences in rate of activation and decay of WT
and GNAT2C rods
A, black traces are mean initial time courses of responses of 16 WT
rods to 10 ms flashes at intensities of 8.6, 21 and 79 photons µm−2,
after filtering with an 8-pole Bessel filter with a low-pass filter setting
of 70 Hz. Responses have been normalized to the peak amplitude of
the response. Red traces are fits to the data of the function
r
rmax =1−exp[−1
2IA(t−teff)2]
where r/rmax is the normalized flash response, Iis the flash intensity
in photoisomerizations, Ais the amplification constant, tis time, and
teff is the effective delay time of transduction (Pugh & Lamb, 1993),
with the same mean values of Aof 20.5 s−2and teff of 18 ms at all
three intensities. B, black traces are mean initial time courses of
responses recorded and normalized as in Abut of 14 GNAT2C rods
to 10 ms flashes at intensities of 21, 79 and 227 photons µm−2.
Blue traces are fits to the data with an Aof 10.2 s−2and teff of
19.3 ms. Single red curve gives prediction for brightest intensity with
WT rod value of A(20.5 s−2). The value of Ais about two times
smaller in GNAT2C rods. C, mean small-amplitude responses of 21
WT rods and 9 GNAT2C rods to flashes of intensities
17 photons µm−2(WT) and 79 photons µm−2(GNAT2C).
Responses have been normalized rod by rod to the peak amplitude
of the response to compare waveforms of response decay.
Responses have been fitted with single exponentials of 258 ms (red
trace, WT) and 122 ms (blue trace, GNAT2C). Responses of GNAT2C
rods decay significantly more rapidly. (Panels A–C reprinted with
permission from Chen et al. 2010b).
transducin expression level (Sokolov et al. 2002), which
was not (and could not) be measured for individual cells
with this technique, the results were inconclusive.
Chen et al. (2010b) used a more traditional transgenic
approach to express cone transducin in Gnat1−/−mice
lacking rod transducin. They were fortunate to isolate a
GNAT2C line in which the level of cone transducin was
nearly the same as the WT rod transducin level. Sensitivity
in GNAT2C rods was reduced by a factor of about 3, and the
amplification constant Awas about a factor of 2 smaller.
This effect on amplification can be seen in Fig. 4Aand
B, which shows that the initial phase of the WT response
rises more rapidly than that of GNAT2C rods.
Mao and colleagues (2013) then did a similar
experiment also using a transgenic approach but with a
different result. Rods in their mice expressed less cone Tα
than GNAT2C rods and were less sensitive than WT rods,
but the decrease in sensitivity seemed to depend only upon
the expression level of the transducin and not upon the
properties of cone Tα. They concluded that the species
of transducin has no effect on the sensitivity difference
between rods and cones. Thus incorporation of cone Tα
in rods either has no effect on sensitivity or decreases it by
as much as a factor of 3. No attempts have been made to
express cone β3orγ8inplaceofrodβ1orγ1.
Two groups have attempted to express cone PDE6C in
rods. Deng et al. (2013) injected viral vectors containing
the PDE6C gene into the eyes of rd10 mice, a line that
is deficient in rod PDE6 but does not lack it entirely.
Rods with cone PDE6C were surprisingly about twice as
sensitive as those with the rod PDE6 proteins and showed
a slower time course of decay. This anomalous result
may have been produced by an unphysiological level of
expression of PDE6, which again could not be measured.
A clearer result was obtained by Majumder and colleagues
(2015), who used a transgenic approach and were able to
compare rod and cone PDE6 at the same expression level.
Rods with cone PDE6C had a higher PDE6 basal activity
and a single-photon response between 1.5 and 2 times
smaller than WT rods, with a more rapid time course of
decay (Fig. 5A). No attempt has been made to substitute
cone PDE6γfor rod PDE6γ. This experiment could be
revealing in view of Muradov et al. (2007), who showed
that lamprey rods and cones have the same catalytic PDE6
subunits but different γsubunits. In conclusion, sub-
stitutionofconePDE6forrodPDE6eitherhasnoeffect
or decreases sensitivity by about a factor of two.
In summary, activation in mouse cones is at least 2-
to 3-fold slower than activation in mouse rods. Taking
outer segment volumes into account, we would predict
that expression of cone pigment, cone transducin and
cone PDE into a rod should together decrease the rate
of activation by at least a factor of 5 with a commensurate
decrease in sensitivity. Experiments expressing cone iso-
forms have, however, given conflicting results, with some
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5422 N. T. Ingram and others J Physiol 594.19
showing a 2- to 3-fold difference and some none at
all. There are three possibilities: either papers showing
significant differences are at least partially correct, or cone
isoforms have to be expressed together (for example cone
pigment with cone transducin), or other proteins (such
1. 0
0.5
0.0
0.0 0.5 1.0
Rod PDE6
Cone PDE6
Photocurrent (pA)
Time (s)
0.2
0.1
0.0
0.0 1.0 2.0
Time (s)
Normal GAP
6-fold GAP
Normalized photocurrent
A
B
Figure 5. Single-photon responses of mouse rods with altered
transduction proteins
A, derived average single-photon responses from control rods (black;
rod PDE6) and cone-PDE6C-expressing rods (red; cone PDE6)
(redrawn and reprinted with permission from Majumder et al. 2015).
B, superimposed single-photon responses of WT mouse rods and of
R9AP95 rods with six times the normal expression of GAP proteins
(Chen et al. 2010a). Responses were plotted as a fraction of the
peak current of the rod, effectively giving the fraction of channels
closed per photon. Recordings were made from animals on a
GCAPs−/−background to remove the effects of cyclase modulation
on response amplitude and waveform (Gross et al. 2012). All
experiments were performed on pigmented mice of either sex in
accordance with the rules and regulations of the NIH guidelines for
research animals, as approved by the institutional animal care and
use committees (IACUCs) of the Virginia Commonwealth University
and the University of California, Los Angeles. Animals were kept in
cyclic 12 h/12 h on/off lighting in approved cages and supplied with
ample food and water. Animals in all experiments were killed before
tissue extraction by approved procedures, usually CO2inhalation or
decerebration. Rods were perfused at 37°C with Dulbecco’s
modified Eagle’s medium (Sigma Chemical, St Louis, MO, USA),
supplemented with 15 mMNaHCO3,2mMsodium succinate,
0.5 mMsodium glutamate, 2 mMsodium gluconate, and 5 mMNaCl,
bubbled with 95% O2–5% CO2(pH 7.4). Unless otherwise
indicated, data were filtered at 35 Hz (8 pole, Bessel) and sampled at
100 Hz. (M. L. Woodruff, C. K. Chen & G. L. Fain, unpublished data).
as PDEγor G-protein βand γ) also have a role. One
conclusion, however, seems clear: the contribution of any
one isoform is individually small, such that no one protein
by itself is responsible for the entire difference in activation
or sensitivity between the two kinds of photoreceptors.
Inactivation
If the response per Rh∗is 20–30 times smaller in mouse
cones than in mouse rods and activation accounts for
only part of this difference, the remainder must emerge
from mechanisms of inactivation. The records in Fig. 3
show that rods decay much more slowly than cones and
integrate incoming photons over a longer time period.
This difference in decay could in theory be produced by
any of the reactions terminating the response.
We begin with extinction of Rh∗. Rods and cones in
mouse both phosphorylate photopigment with the same
GRK1 kinase with no marked difference in antibody
labelling and presumably expression (Lyubarsky et al.
2000; Weiss et al. 2001). Moreover both rods and cones
use arrestin-1 with the small amount of arrestin-4 in cones
unlikely to affect the rate of Rh∗decay (Nikonov et al.
2008). The mean lifetime of Rh∗in rods is probably as
short as 40–45 ms (see Burns & Pugh, 2011), which is
already so short that it is difficult to understand how even
two or three serines or threonines could be phosphorylated
and arrestin bind in so little time (Gurevich et al. 2011).
If phosphorylation is faster in cones as Tachibanaki and
colleagues have argued (2005), it is probably not much
faster at least in mouse, whose rods and cones both express
GRK1 at a similar level. More likely suspects for the slower
rate of rod inactivation may be differences in the rates of
decay of light-activated PDE6∗and restoration of cGMP
concentration by the cyclase.
Decay of PDE6∗is produced by hydrolysis of Tα•GTP
to Tα•GDP and rebinding of the PDE6γinhibitory
subunits to the PDE catalytic subunits. The rate of
hydrolysis of Tα•GTP may be affected by the particular
isoforms of transducin and PDE6: both cone transducin
andconePDE6Cexpressedinrodshavebeenreported
to produce responses that decay more rapidly than WT
rod responses (see Figs 4Cand 5A). The rate of hydrolysis
may also be affected by the GAP proteins which, as we
have said, are the same in rods and cones but are more
abundantly expressed in cones at perhaps a 10-fold higher
concentration (Cowan et al. 1998; Zhang et al. 2003). This
difference in expression could have a significant effect on
sensitivity. In Fig. 5B, we compare single-photon responses
from rods with the normal GAP level and mutant R9AP95
rods in which the GAP proteins are 6-fold over-expressed
(Chen et al. 2010a). This experiment was done on a
GCAPs−/−background to obviate any effect of cyclase
feedback on response waveform or amplitude (Gross et al.
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J Physiol 594.19 Why are rods more sensitive than cones? 5423
2012). Rods with over-expressed GAPs are about a factor
of 2–3 less sensitive and decay more rapidly.
There are two ways guanylyl cyclase could produce a
difference in the rate of cGMP synthesis between rods
and cones and alter sensitivity. Both rods and cones in
mouse use the same retGC1 cyclase, but the expression
level is likely to be higher in cones. Staining with a retGC1
antibody is brighter in cones than in rods (Dizhoor et al.
1994), and unpublished measurements on retinas lacking
the neural retina leucine zipper (Nrl) transcription factor
(Mears et al. 2001), where all photoreceptors are cone-like,
indicate that mouse cones may have something like 2–3
times more retGC1 than rods (A. Dizhoor, personal
communication). Cone PDE6 expression may also be
greater than rod but probably by no more than a factor of
1.5 (Zhang et al. 2003; Lobanova et al. 2010); however, cone
PDE6 has a higher basal activity (Majumder et al. 2015).
Together the cyclase and PDE6 would produce a higher
rate of cGMP turnover in darkness, which in salamander
has actually been measured and is about 3-fold greater in
cones than in rods (Cornwall & Fain, 1994; Cornwall et al.
1995). This increase in turnover rate would produce both
an increase in the rate of response decay and a decrease in
sensitivity (Rieke & Baylor, 1996; Nikonov et al. 2000; Fain
et al. 2001). Measurements in salamander indicate that if
turnover in a rod were increased by a factor of 3, sensitivity
would be reduced by about a factor of about 2 (Cornwall
& Fain, 1994; Nikonov et al. 2000).
The rate of cGMP synthesis is also controlled by
GCAP proteins, which in turn are regulated by the
outer-segment Ca2+concentration. Although the GCAPs
themselves are similar in rods and cones, the change
in Ca2+concentration is considerably faster in cones,
at least in salamander (Sampath et al. 1999). Rods and
cones express different isoforms of the Na+/Ca2+–K+
exchanger (Lytton, 2007; Vinberg et al. 2015) and may
have different concentrations or isoforms of Ca2+buffers.
This accelerated decline in Ca2+would produce a more
rapid modulation of the GCAPs and faster activation of
the cyclase, which could in theory decrease cone sensitivity.
This notion has, however, been tested by deleting the genes
for the GCAP proteins, which increases sensitivity by about
the same factor of 3 in both rods (M. L. Woodruff & G. L.
Fain, unpublished observations; Gross et al. 2012) and
cones (Sakurai et al. 2011). These observations indicate
that GCAP-mediated feedback makes little contribution
to the sensitivity difference (however, see Wen et al.
2014). A similar conclusion emerges from comparison of
salamander rod and cone responses under conditions that
suppress changes in outer-segment Ca2+(Matthews et al.
1988, 1990; Nakatani & Yau, 1988, 1989).
In conclusion, the rate of inactivation of transduction
is slower in rods than in cones, with the major effects
apparently produced by the species of transducin and
PDE6, the expression level of cyclase, PDE6 basal activity,
and the expression level of the GAP proteins. Each of
these differences seems, however, to make a relatively
small contribution, and once again no single change
predominates.
We have based our conclusions on results from mouse,
but it is possible and even likely that additional adaptations
are present in other species that contribute to the difference
in rod and cone inactivation. Fish are of particular inter-
est, because the difference in rod and cone sensitivity can
be much larger than in mouse (Tachibanaki et al. 2001).
Kawamura’s laboratory has shown that fish cones have
a very high rate of pigment phosphorylation by GRK7
(Tachibanaki et al. 2005), an enzyme highly expressed in
fish but not present in mouse (Liu et al. 2005). Moreover
carp also show a much higher rate of cGMP synthesis
in cones than in rods, and therefore a higher cGMP
turnover rate (Takemoto et al. 2009). These changes would
collectively cause photoresponses from carp cones to be
smaller and faster (Tachibanaki et al. 2005;Liu et al.
2005; Takemoto et al. 2009). In addition, Rebrik and
Korenbrot have identified a Ca2+-binding protein present
in fish cones but not fish rods that reduces the affinity
of cyclic nucleotide-gated ion (CNG) channels for cGMP
in high [Ca2+]i, a protein they first called CNG-modulin
(Rebrik et al. 2012), but later identified as echinoderm
microtubule-associated protein-like 1 (EML-1, Korenbrot
et al. 2013). Knockdown of this protein in zebrafish
produced a 5-fold increase in cone sensitivity (Korenbrot
et al. 2013), presumably by slowing the rate at which CNG
channels open following illumination. This protein has
not as yet been identified in mammalian cones.
Why are rods more sensitive?
The key step in the formation of the duplex retina of
vertebrates was the evolution of more sensitive rods
to accompany cones, so that the entire range of light
intensities could be encoded by the photoreceptors.
Molecular and biochemical studies tell us that rods and
cones have many of the same transduction proteins but
use different isoforms probably arising by gene duplication
(see Table 1); in some cases they use the same isoform but
at a different level of expression. No one change accounts
for the difference in absolute sensitivity between rods and
cones. Instead, each of the differences we have described
seems to have produced a small increase in the rate of
activation or prolongation of response decay, conferring
an incremental advantage to the organism.
Accumulated changes in a large number of proteins
eventually produced a sensitivity great enough in the
rod to allow it to operate in dim light, with cones
remaining for enhanced temporal resolution when photon
flux is no longer limiting. These changes also have
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5424 N. T. Ingram and others J Physiol 594.19
implications for the dynamic properties of rods and cones,
namely their ability to adapt to increasing light intensity.
While we have not discussed these mechanisms in this
review, the fundamental tradeoff between sensitivity and
dynamic range between rods and cones will also depend
upon differences in their transduction mechanisms. The
properties of the two receptor types form the basis of our
duplex visual system, whose fundamental nature was first
proposed by Schultze 150 years ago.
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Additional information
Competing interests
None declared.
Funding
This work was supported by individual grants from the National
Eye Institute of the NIH to G.L.F. (EY001844) and to A.P.S.
(EY017606), as well as a core grant to the Jules Stein Eye Institute
(EY000331).
Acknowledgements
We are grateful to past and present members of our laboratories
for many useful discussions and for participating in some of the
experiments we have described, and to Margery J. Fain for help
with the figures.
C
2016 The Authors. The Journal of Physiology C
2016 The Physiological Society
