Congenital night blindness (CNB) affects retinal rod
photoreceptor cells and is expressed as an inability to
see under dim light conditions. Three different mutations
in rhodopsin have been shown to cause autosomal dominant
CNB in humans; Gly90!Asp, Thr94!Ile, and Ala292!Glu. While
there is general agreement that the disease is caused
by inappropriate stimulation (and consequent desensitization)
of rod cells, there is some controversy regarding the
source of the stimulatory signal. Two models have been
proposed. In Model I, the stimulatory signal is proposed
to come from the constitutively active apoprotein, or
opsin, forms of the mutant rhodopsins. This model was
originally proposed for the A292E and G90D mutants, but
it is now known that all three mutant opsins (including
the more recently identified T92I mutant) are constitutively
active under in vitro assay conditions. In Model
II, the stimulatory signal is postulated to result from
increased thermal isomerization of the 11- cis-
retinal chromophore in the holoprotein, or pigment, forms
of the mutant rhodopsins to generate a metarhodopsin II
intermediate which results in desensitization of the photoreceptor
cell. Model II was originally proposed for the mutant
G90D on the basis of theoretical considerations and was
subsequently promoted on the basis of FTIR studies of
the recombinant mutant.
These two models are similar in that both invoke an active
species that would be present at vanishingly small concentrations
within the rod photoreceptor cell (the dark-adapted thresholds
of G90D patients are elevated by 3 log units, but in the
highly-sensitive rod cell containing 108
– 109 rhodopsin molecules
this level of sensitivity is equivalent to what would
be observed from adaptation to a light stimulus in which
there were only about 10 photoisomerization events per
rod cell). The two models differ, however, in terms of
the chemical makeup of the active species, and they have
exploited this difference to design a simple experimental
test to distinguish these two models using isolated rod
photoreceptor cells from transgenic xenopus laevis
and exogenously added 11- cis-retinal. According,
to both models, they expect rod cells from the CNB mutants
to exhibit significant desensitization of the light-evoked
currents (intensity-response curve shifted to higher light
intensity and more rapid flash-response kinetics). In
Model I, the active signal comes from the apoprotein;
11-cis-retinal would decrease the equilibrium concentration
of opsin, turn off the constitutive activity, and rescue
the wild-type phenotype (intensity- response curve shifted
to lower light intensity and slowed flash-response kinetics).
In Model II, the active signal comes from the holoprotein;
added 11-cis-retinal would have no effect.
Thus, the experimental design is as follows. Rod photoreceptor
cells are isolated from transgenic frogs containing rhodopsin
CNB mutations. Suction micropipette recordings are used
to show that the cells display the desensitized response
to light expected of CNB mutants. The cells are then incubated
with exogenously added 11-cis-retinal and the electrical
recordings repeated to determine if the cells remain desensitized
or if they recover wild-type sensitivity. If Model II
were responsible for the CNB phenotype, thermal isomerization
of the chromophore rhodopsin should be unaffected by added
11-cis-retinal, and we would expect the cells to
remain desensitized after treatment with retinal. In contrast,
if Model I were responsible for the CNB phenotype, the
constitutively active mutant opsin should be converted
to the inactive rhodopsin form following incubation with
retinal, and we would expect the cells to exhibit wild-type
sensitivity following incubation with retinal.
They showed here that the rod cells from all three CNB
mutants are desensitized as isolated but recover wild-type
sensitivity following incubation with 11-cis-retinal.
These data are inconsistent with Model II and eliminate
thermal generation of metarhodopsin II from rhodopsin
as a source of the desensitized CNB phenotype. Furthermore,
while the data do not prove Model I, they are exactly
what would be expected from that model in which constitutively
active mutant opsin causes the desensitization of the
CNB photoreceptor cells.
