Clint Makino,
Ph.D.
Associate Professor of Ophthalmology (Neuroscience)
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We are investigating the earliest events
of vision wherein light is converted into an electrical signal
by the retinal photoreceptors.
What sets the speed of the photoresponse?
Rods contain an enormous number of rhodopsin
molecules densely packed in membranous disks, in order to
achieve high sensitivity. But dense packing could impair phototransduction
by restricting the lateral mobility of rhodopsin as well as
that of other proteins in the membrane. To test the hypothesis
that the speed of the photoresponse is limited by the rates
with which molecules collide with each other on the membrane,
we recorded photoresponses of single rods containing fewer
rhodopsins in their membranes. This condition was achieved
by hemizygous knockout of the rod opsin gene in transgenic
mice (R+/-). Photoresponses were accelerated by the reduction
in rhodopsin packing density, supporting the hypothesis (Fig.
1). The key step in the activation of the photoresponse was
identified as the collision between rhodopsin and transducin.
Response recovery appears to be limited by the collision rate
between rhodopsin and rhodopsin kinase and/or that between
between transducin and RGS9. These results predict that under
scotopic conditions, humans heterozygous for a null mutation
in rhodopsin will have higher than normal temporal resolution.
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Figure 1. Top panels:
membrane surfaces of wild type and transgenic mouse rod
membranes. Rhodopsins and transducins are depicted by
red and blue circles, respectively. The green area shows
the surface area "sampled" by a photoexcited rhodopsin
(green star) every few milliseconds as it diffuses. The
area expands and the number of transducins contacted increases
as the packing density of rhodopsins decreases. Bottom
panels: flash responses of wild type (left) and R+/- (right)
rods recorded with suction electrode. Responses of R+/-
rods rise and recover more rapidly than those of the wild
type rods. |
What is the time course of light adaptation?
Upon exposure to dim steady light, rods
summate the effects of single photon absorptions. With brighter
intensities, the response sags over time as the rod desensitizes
or light adapts (Fig. 2). Very bright light saturates the
rod, but eventually, there may be a partial recovery. The
magnitude of adaptation increases with response amplitude.
We have observed two temporal phases of desensitization. The
fast phase, which is largely complete 5 s after light onset,
is calcium dependent and has been well described by others.
The slow phase develops ten times more slowly, but the full
magnitude of its effect is nearly the same as that of the
fast phase. The slow phase is also calcium dependent and while
the underlying mechanisms are not yet known, regulation over
the catalytic lifetime or activity of rhodopsin appears to
be involved.
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Figure 2. Averaged responses
of a frog rod to steps of light. With the exception of
the dimmest and brightest steps, the responses exhibited
a fast and a slow sag. The fast sag was not observed in
the response to the brightest step, but the slow sag was
present. Bright,saturating flashes were given 61 s after
step onset to show that the current recovered during the
sag was suppressible by light. |
What determines the spectral sensitivity
of a photoreceptor?
The spectral sensitivity of a photoreceptor
is largely determined by the visual pigment that it contains.
Although photoreceptors were generally thought to express
only one type of pigment, there are a number of examples where
a cone has been shown to contain two different types of pigments.
However, we discovered that the UV-sensitive cone in tiger
salamander contains three different types of pigments,
all of which are functional (Fig. 3). In addition to a UV-absorbing
pigment, the pigments of blue- and red-sensitive cones are
also present. Selectivity of pigment expression is not completely
lacking in the UV-sensitive cone because the green-sensitive
rod pigment is absent. Interestingly, the blue-sensitive cone
pigment is also expressed in a blue-sensitive rod (Fig. 3),
the first example of a rod and a cone utilizing the same visual
pigment.
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Figure 3. Averaged spectral
sensitivities of salamander UV-sensitive cones (violet
circles), blue-sensitive rods (blue diamonds) and cones
(blue circles) and red-sensitive cones (red circles).
Continuous lines show fits of the spectra with a template.
The fit to the UV-sensitive cone spectrum was made assuming
that the long wavelength limb included components that
were the same as those giving rise to the spectra of the
other two cone types. |
Selected Publications:
Calvert, P. D., Govardovskii, V. I., Arshavsky,
V. Y. and Makino, C. L. (2002). Two temporal phases of light
adaptation in retinal rods. Journal of General Physiology
119: 129-145.
Calvert, P. D., Govardovskii, V. I., Krasnoperova,
N., Anderson, R. E., Lem, J. and Makino, C. L. (2001). Membrane
protein diffusion sets the speed of rod phototransduction.
Nature 411: 90-94.
Ma, J.-x., Znoiko, S., Otherson, K. L.,
Ryan, J. C., Das, J., Isayama, T., Kono, M., Oprian, D. D.,
Corson, D. W., Cornwall, M. C., Cameron, D. A., Harosi, F.
I., Makino, C. L. and Crouch, R. K. (2001). A visual pigment
expressed in both rod and cone photoreceptors. Neuron
32: 451-461.
Roof, D. J. and Makino, C. L. (2000). The
structure and function of retinal photoreceptors, in Principles
and Practice of Ophthalmology 2nd edition,
eds. F. A. Jakobiec and D. Alberts. W.B. Saunders Co., Philadelphia,
pp. 1624-1673.
Lem, J., Krasnoperova, N. V., Calvert, P.
D., Kosaras, B., Cameron, D. A., Nicolo, M., Makino, C. L.
and Sidman, R. L. (1999). Morphological, physiological and
biochemical changes in rhodopsin knockout mice. Proceedings
of the National Academy of Sciences of the USA 96: 736-741.
Makino, C. L. and Dodd, R. L. (1996). Multiple
visual pigments in a photoreceptor of the salamander retina.
Journal of General Physiology 108: 27-34.
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