From the
Recoverin is a 23-kDa Ca
A drop in free Ca
Recoverin is expressed only in the retina
and, with the exception of a subset of bipolar cells, is specific to
photoreceptor cells(13) . It is a 23-kDa protein that contains
several EF-hand motifs characteristic of Ca
This paper addresses several
questions that are important in establishing the physiological
relevance of the Ca
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy where Ca denotes free Ca
The
curve fitting shows that parameter a (the amplitude of low
Ca
We have
determined the amount of endogenous recoverin present in our
osmotically intact ROS preparations and found that its molar ratio to
rhodopsin is approximately 1:174 ± 18 (Fig. 2A).
The inhibition of rhodopsin phosphorylation in whole frog ROS as a
function of the total concentration of recoverin at saturating free
Ca
Inhibition of rhodopsin phosphorylation by recoverin can be
observed in vitro above 1 µM free Ca
Our data provide an estimate of the affinity of recoverin for
Ca
On-line formulae not verified for accuracy where Rec is recoverin and K
If, under the high membrane concentrations found
in ROS, myristoylated recoverin is expected to be half-saturated with
Ca
The low
affinity of recoverin for Ca
We thank Art Polans and Krzystof Palczewski for
providing us with unpublished results and for helpful discussions. We
also thank Vadim Arshavsky for commentaries on the initial version of
the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-binding protein found
predominantly in vertebrate photoreceptor cells. Recent
electrophysiological and biochemical studies suggest that recoverin may
regulate the photoresponse by inhibiting rhodopsin phosphorylation. We
find in both cell homogenates and reconstituted systems that the
inhibition of rhodopsin phosphorylation by recoverin occurs over a
significantly higher free Ca
range than previously
reported. Half-maximal inhibition occurs at 1.5-3 µM free Ca
and is cooperative with a Hill
coefficient of
2. Measurements of transducin activation
demonstrate that this inhibition prolongs the lifetime of catalytically
active rhodopsin. Ca
-recoverin directly inhibits
rhodopsin kinase activity, and Ca
-dependent binding
of recoverin to rod outer segment membranes is not required for its
action. Extrapolation of the in vitro data to in vivo conditions based on simple mass action calculations places the
Ca
-recoverin regulation within the physiological free
Ca
range in intact rod outer segment. The data are
consistent with a model in which the fall in free Ca
that accompanies rod excitation exerts negative feedback by
relieving inhibition of rhodopsin phosphorylation.
following illumination
appears to be a key regulatory step during recovery of the
photoresponse and during light adaptation (see Refs. 1 and 2 for
review). It has been shown to regulate a variety of enzymes involved in
the phototransduction cascade (3, 4, 5, 6) that include guanylate
cyclase, the cGMP-gated channel, rhodopsin, and cGMP phosphodiesterase.
Following Kawamura(7) , several studies have suggested that the
Ca
effect on cGMP phosphodiesterase might be mediated
by a Ca
-binding protein, recoverin, through its
inhibition of rhodopsin phosphorylation (8, 9, 10) (recoverin is called S-modulin in the
frog; for simplicity, we refer to it as frog recoverin). Consistent
with this inhibition of rhodopsin phosphorylation, physiological
experiments have shown that recoverin and its homologues can slow
photoresponse recovery(11) . Recent experiments on transgenic
mice lacking recoverin also seem to support this
hypothesis(12) .
-binding
proteins (14, 15) and is heterogenously fatty acid
acylated at its N terminus(16) . This modification allows
recoverin to bind membranes upon binding Ca
,
suggesting that recoverin's biological activity may be related to
its Ca
-dependent membrane
binding(17, 18) .
-recoverin system in
ROS.
(
)1) What is the free Ca
concentration range over which recoverin inhibition of rhodopsin
phosphorylation is relaxed? 2) What is the mechanism of recoverin
inhibition of rhodopsin phosphorylation? 3) Do the in vitro biochemical data support the proposed physiological role of
recoverin in living photoreceptors? The data show that
Ca
-recoverin acts directly on RK to decrease its
catalytic activity and that no components other than rhodopsin, kinase,
and recoverin are required. Extrapolation of the recoverin inhibition
of RK observed in vitro at micromolar free Ca
to conditions in the intact ROS suggests a role for the
Ca
-recoverin system in normal ROS function.
Materials and
Solutions
[-
P]ATP and
[
-
S]GTP were purchased from DuPont NEN;
Percoll and Heparin HiTrap columns were from Pharmacia Biotech Inc.,
potassium isethionate was from Kodak; Chelex 100 resin was from
Bio-Rad; 1 M CaCl
solution was from BDH; BAPTA and
Fluo-3 were from Molecular Probes; and bis-Tris propane was from
Calbiochem. Other chemicals were obtained from Sigma. The standard
buffer used in all experiments contained 105 mM potassium
isethionate, 5 mM sodium isethionate, 10 mM HEPES, 2
mM MgCl
, pH 7.8. It was purified from calcium ion
contamination on a Chelex 100 column prior to addition of MgCl
so that Ca
concentration was below 1
µM.
ROS Preparation
Intact frog ROS were
purified as described(19) , except that CaCl concentration in Percoll was decreased to 0.1 mM. ROS
were resuspended in the standard buffer and homogenized with a
motorized tissue grinder to minimize any diffusional limitations caused
by disk stacks(20) . Bovine ROS were purified under infrared
illumination as described(21) . The same method of bovine ROS
isolation was used for RK purification, but all sucrose solutions were
prepared in 10 mM Tris, 5 mM MgCl
, pH
7.5, and the procedure was performed in room light.
Protein Purification
Bovine and frog
recoverin were purified (22) and stored at -70 °C. The
concentration of recoverin was determined by absorbance at 280 nm using
a molar extinction coefficient of 36,400.(
)RK
was extracted as described (23) and purified based on published
procedures(24, 25) . Briefly, extracted RK was dialyzed
against 10 mM Tris, 0.4% Tween 80, pH 8.0, and loaded on a 1
3.5-cm DEAE-cellulose column at 0.3 ml/min. The column was
washed with 300 ml of buffer A (20 mM Tris, 0.2% Tween 80, pH
8.0) and then with 100 ml of 35 mM NaCl in buffer A, and RK
was eluted with a 35-135 mM NaCl gradient (0.2 ml/min;
total volume, 45 ml). Fractions containing RK were loaded on a 1-ml
Heparin HiTrap column equilibrated with buffer B (10 mM bis-Tris propane, 0.064% Tween 80, 2 mM MgCl
,
pH 7.8). The column was washed with 125 mM KCl in buffer B,
and RK was eluted with 250 mM KCl at 0.06 ml/min.
Calcium Buffering
A set of 4 stock
solutions with different CaCl
concentrations and fixed
BAPTA concentration in standard buffer was prepared (1
=
5 mM BAPTA). Free Ca
in 4-fold diluted
solutions was measured. For the free Ca
range of 10
nM to 5 µM, Fluo-3 dye was used. The K
of Fluo-3 for Ca
, 450
nM, was derived from its fluorescence in solutions of
10-70 nM free Ca
buffered with BAPTA
according to the estimates of the program BAD(26) . Preparation
of accurate BAPTA solutions required gravimetric determination of the
water content (
10%) of the commercially obtained BAPTA. For free
Ca
that was
10 µM, a
Ca
-selective electrode (Microelectrodes, Inc.,
Londonderry, NH) was used following recommendations of the
manufacturer. A control determination showed that frog ROS suspensions
containing up to 20 µM rhodopsin do not change free
Ca
.
Determination of Frog Recoverin/Rhodopsin
Ratio
Samples of intact frog ROS with different rhodopsin
concentrations (19) were mixed with SDS-polyacrylamide gel
electrophoresis loading buffer, heated for 30 min at 60 °C, and run
on a 15% polyacrylamide gel in parallel with purified recoverin
standards. The intensity of recoverin bands was determined by
densitometric analysis with the aid of a Foto/analyst system and
Collage software (Fotodyne, New Berlin, WI) using laplacian edge
detection and local background subtraction. Calculations assumed
molecular masses of 23 and 39 kDa for recoverin and rhodopsin,
respectively.
RK Activity Assay
Frog RK activity was
measured in ROS suspensions of 20 µM rhodopsin unless
otherwise indicated. Bovine RK activity was measured using urea-treated
ROS membranes as a substrate (final concentration, 10 µM rhodopsin). ROS, a CaCl/BAPTA stock solution, and
recoverin if necessary were mixed in a final volume of 15 µl in the
dark, the suspension was illuminated (bleached), and 5 µl of
[
-
P]ATP (0.4-0.8 mM) was
added (control experiments have shown that the order in which reactants
are mixed is not important; the same extent of RK inhibition is
obtained if the reaction is initiated by bleaching of rhodopsin in the
presence of ATP). After 1-2 min, the reaction was stopped by the
addition of 80 µl of 6% trichloroacetic acid or 100 mM EDTA, 100 mM KF, pH 7.5.
P
incorporation was measured using one of the following methods.
For low levels of bleached rhodopsin, the excised rhodopsin bands from
12% SDS-polyacrylamide gel electrophoresis gels were dissolved in 30%
H
O
. For bleaches of 5-100%, samples were
filtered through nitrocellulose filters, washed with 6
1 ml of
100 mM sodium phosphate, pH 7.5, and dissolved in 2 ml of
glacial acetic acid. The dissolved samples were counted by liquid
scintillation.
GTP
The gain of
GTPS Binding Assay
S binding following a calibrated light flash was measured by
filter binding under the same conditions as those used for rhodopsin
phosphorylation with the exception that samples contained 100
µM [
-
S]GTP and cold ATP. The
reaction was allowed to proceed for 5 min and quenched with 100 mM GTP, 100 mM hydroxylamine, pH 7.8.
Curve Fitting and Data Presentation
Curve
fitting used the Marquardt-Levenberg least squares algorithm available
in SigmaPlot software. The data from individual experiments shown in Fig. 1A and 3 were first fit to a double sigmoid
function (see ``Results'') and then normalized by the value
of P (maximal rhodopsin phosphorylation)
obtained. In Fig. 2A, in order to combine data from
individual experiments that showed similar slopes on varying
backgrounds, a first-order regression coefficient b (y = ax + b) for each individual data
set was subtracted from the data, which were then normalized by the
value at 0.1 µg of recoverin. The data from individual experiments
shown in Fig. 2B were first fit to the function,
Figure 1:
Rhodopsin
phosphorylation is inhibited by Ca-recoverin and by
Ca
alone. A, rhodopsin phosphorylation in
fully bleached frog ROS in the absence (
; seven experiments) or
presence of 3 (
; five experiments), 10 (▾; two
experiments), or 30 µM added recoverin (
; two
experiments). The smooth curves represent fits for the data as
explained in the text. The half-maximal free Ca
concentration (K
) and Hill coefficient of
the recoverin effect (n) for the corresponding curves are
shown. B, interpretation of the data and curve fitting in A. Curve 1, rhodopsin phosphorylation is inhibited by
high Ca
and can be described by a sigmoid curve with
parameters K
= 0.75 mM, m = 0.65; curve 2, recoverin inhibits a portion of
rhodopsin phosphorylation activity at lower Ca
(Equation 2 with parameters a = 0.4, K
, K
= 2.5 µM, n = 2); curve
3, simultaneous appearance of the processes illustrated by curves 1 and 2 results the in a biphasic curve that
closely resembles experimental data.
Figure 2:
Estimation of recoverin amount in frog ROS
and inhibition of rhodopsin phosphorylation as a function of recoverin
concentration. A, determination of the rhodopsin/recoverin
ratio in osmotically intact frog ROS. Integrated recoverin band
intensities of recoverin standards and whole ROS samples are shown
(pooled data from 5 separate gels). The data are consistent with a
rhodopsin/recoverin ratio of 174 ± 18. Inset, a
representative gel used for recoverin quantitation (from left to right): 0.03, 0.05, 0.07, 0.09, and 0.11 µg of
recoverin and three ROS samples (15, 20, and 25 µg of rhodopsin).
The upper bands seen in the ROS samples were identified as
recoverin based on Western blotting analysis, and the fact that they
run at exactly the same position as purified bovine recoverin when the
cytosolic ROS fraction is analyzed by electrophoresis. The retardation
in case of whole ROS is caused by gel overloading with respect to
rhodopsin. B, dependence of the inhibition of rhodopsin
phosphorylation on recoverin concentration in 10 mM rhodopsin
whole frog ROS at 25 µM () and 2 mM (
)
free Ca
. The curve is drawn according to the
Michaelis-Menten equation with V
= 71.8
and K = 3.4 µM.
where P is the rhodopsin phosphorylation and R is concentration of recoverin and then normalized by the value of X obtained. Normalized data from different experiments were
combined and curve fits were performed without averaging. All data
points shown are the means ± S.D. of at least two experiments.
Two Ranges of Free Ca
The dependence of rhodopsin phosphorylation
on free CaConcentration Inhibit Rhodopsin
Phosphorylation
for whole frog ROS in the absence and
presence of exogenous recoverin is shown in Fig. 1A. A
biphasic character of the free Ca
titration is
observed. This is best explained by assuming that inhibition of
rhodopsin phosphorylation occurs independently at micromolar and
millimolar free Ca
ranges. The data can be fit to the
relation
concentration and P is the amount of phosphate incorporated into rhodopsin normalized
by maximal rhodopsin phosphorylation P
. As
illustrated in Fig. 1B, this expression describes a
combination of ``low Ca
inhibition'' (a
sigmoid curve with a fractional amplitude a, half-maximal free
Ca
concentration K
, and a Hill
coefficient of n) (Fig. 1B, curve 1)
and ``high Ca
inhibition'' (a sigmoid
function with corresponding parameters K
and m) (Fig. 1B, curve 2) that results in
a biphasic Ca
dependence (Fig. 1B, curve 3) that closely resembles the experimental data.
inhibition) increases with increasing recoverin
concentration, whereas other parameters do not change significantly.
The high Ca
inhibition shows half-saturation in the
range of 0.4-0.7 mM free Ca
and is
unlikely to be of physiological relevance. The reconstitution
experiment shown below (see Fig. 4) demonstrates that it does not
require recoverin and is therefore an intrinsic property of RK. Similar
inhibition of purified RK by near-millimolar Ca
was
previously reported(27) . We thus conclude that the inhibition
of rhodopsin phosphorylation at micromolar free Ca
is
recoverin-specific and refer to it as the ``recoverin
effect.''
Figure 4:
The
recoverin effect can be reconstituted with purified RK alone. A, purified bovine RK (0.05 µM) was combined with
urea-treated bovine ROS (10 µM rhodopsin) in the absence
() or presence (
) of 10 µM recoverin. Rhodopsin
phosphorylation was allowed to proceed for 2 min after bleaching 100%
rhodopsin and adding ATP. The data are consistent with
Ca
-recoverin inhibition with parameters K
= 2.5 µM free
Ca
, n = 1.89, and a =
0.45. Data from one of four similar experiments are shown. B,
SDS-polyacrylamide gel electrophoresis of the protein preparations. Lane 1, molecular mass standards; lane 2,
urea-treated ROS membranes, 5 µg of rhodopsin; lane 3, 3
µg of recoverin; lane 4, 1 µg of
RK.
Qualitatively, the results depicted in Fig. 1are similar to those already
published(8, 10, 28) . The important
distinction, however, is that we find a higher half-saturating free
Ca for the recoverin effect (about 2 µMversus 0.1-0.2 µM). Finding a
half-saturating free Ca
value that is an order of
magnitude higher than that reported by two other laboratories has
compelled us to consider this discrepancy carefully. We cannot
confidently assign a reason for the difference but propose that it is
due to several intrinsic difficulties in calcium buffering with EGTA. A
problem that is frequently overlooked is that the high affinity of EGTA
for Ca
and its strong pH sensitivity result in large
errors in free Ca
if even slight errors in the
concentrations of protons, chelator, or Ca
are
introduced (for detailed discussion, see Refs. 29 and 30). To ensure
accuracy and reproducibility of calcium buffering, we have directly
measured free Ca
in solutions used for the
experiments (see ``Experimental Procedures'') and thus
believe that our approach gives more reliable estimates.
concentrations can then be described by a simple
hyperbola with half-saturation at 3.4 ± 0.3 µM recoverin (Fig. 2B).
Recoverin Prolongs the Lifetime of Catalytically
Active Rhodopsin
To verify that the reported effect of
recoverin on cGMP phosphodiesterase activity (6, 7) is
not a direct stimulation of cGMP phosphodiesterase by
Ca-recoverin and that the recoverin action prolongs
the lifetime of catalytically active rhodopsin, we tested whether the
recoverin effect could be detected on the level of transducin
activation. The longer the lifetime of catalytically active rhodopsin,
the larger the number of transducin molecules it is able to activate,
provided that the availability of transducin is not limiting.
Therefore, the total amount of GTP
S bound to transducin following
a dim flash will be a function of the rate of rhodopsin inactivation. Fig. 3demonstrates that addition of recoverin to ROS in 10
µM free Ca
causes an increased gain of
GTP
S binding that is consistent with an inhibition of rhodopsin
phosphorylation.
Figure 3:
Ca-recoverin prolongs
the lifetime of catalytically active rhodopsin. The gain of GTP
S
binding in frog ROS homogenates after a light flash bleaching 0.00054%
rhodopsin (of a total 20 µM) in the presence of 100
µM GTP
S and ATP without (open bars) and with
5 µM added recoverin (hatched bars) is
shown.
Recoverin Acts Directly on RK
The
simplest hypothesis for the mechanism of recoverin action is direct
inhibition of RK. The Ca dependence of rhodopsin
phosphorylation obtained with purified bovine recoverin, RK, and
urea-treated ROS is shown in Fig. 4A, and the protein
preparations are shown in Fig. 4B. It is clear that all
features of Ca
inhibition of rhodopsin
phosphorylation (high calcium inhibition, the Ca
range for the recoverin effect, and a Hill coefficient close to
2) can be reconstituted with these purified proteins without
involvement of any additional factors.
Recoverin Action Does Not Require Binding to
Rhodopsin or ROS Membranes
Fig. 5summarizes data
from experiments testing several alternative mechanisms of recoverin
inhibition of RK. First, recoverin might prevent access of RK to
bleached rhodopsin. For such a competitive inhibition the extent of the
recoverin effect should be greater at lower substrate concentrations. Fig. 5A demonstrates, to the contrary, that the
recoverin effect does not depend on the amount of bleached rhodopsin
present. Second, Ca-dependent membrane binding of
recoverin might play a role in recoverin function. To find out whether
only membrane-bound recoverin is able to inhibit RK, the extent of the
recoverin effect in the presence of 10 and 100 µM rhodopsin was compared. Because the K
of myristoylated recoverin for ROS membranes is
200
µM rhodopsin(17) , this 10-fold increase of
membrane concentration should correspond to a 10-fold increase of the
amount of membrane-bound recoverin. Only a very small difference was
found (Fig. 5B), probably attributable to less reliable
Ca
buffering in the presence of high concentrations
of proteins and lipids. Finally, there is the possibility that
Ca
-recoverin increases RK's K
for ATP so that the 100-200
µM ATP used in the previous experiments was not saturating
in the presence of recoverin. Under this scenario the recoverin effect
should diminish with increasing ATP concentration. Fig. 5C shows that it is not the case; the same result is obtained with
100 µM and 1 mM ATP.
Figure 5:
The
recoverin effect does not depend on bleached rhodopsin, ROS membranes,
or ATP concentrations. Rhodopsin phosphorylation at 10 µM free Ca (hatched bars) was compared
with rhodopsin phosphorylation at 10 nM free Ca
(open bars) in the presence of recoverin, with the
latter being set at 100%. A, influence of fractional rhodopsin
bleach in frog ROS homogenate at 20 µM rhodopsin with 3
µM recoverin added. B, influence of membrane
concentration was tested using purified bovine proteins and
urea-treated ROS membranes at 10 µM recoverin. C,
influence of ATP concentration was assayed in frog ROS homogenates at
10 µM rhodopsin with 10 µM recoverin
added.
and requires the presence of micromolar concentrations of
recoverin (Fig. 1A). Reconstitution of the recoverin
effect demonstrates that it reflects a direct inhibition of RK by
Ca
-recoverin (Fig. 4, see also Refs. 9 and 10).
The fact that the recoverin effect is independent of the concentration
of RK substrates (Fig. 5, A and C) strongly
suggests that recoverin inhibits the catalytic activity of RK. Two
lines of evidence suggest that membrane binding of recoverin does not
dramatically affect its ability to inhibit RK. First, it has been shown
that nonacylated recombinant recoverin that does not bind to membranes
exhibits the same inhibitory activity as the native protein (28).
(
)Second, the data in Fig. 5B, taken
together with the low affinity of recoverin for membranes(17) ,
indicate that membrane binding is not required for recoverin inhibition
of RK.
. Assuming that
Ca
-recoverin-bound RK does not phosphorylate
rhodopsin, the extent of the recoverin effect is determined by the
percent of RK that is bound to recoverin. This is given by an
equilibrium
and K
are equilibrium dissociation constants for the
two reactions. Because a Hill coefficient value close to 2 was found
experimentally (Fig. 1A and 4A), for simplicity
we presume two binding sites with equal macroscopic binding constants
for Ca
. An analysis of this equilibrium under
conditions of buffered Ca
reveals that 1) When the
total recoverin concentration is 10-fold or more higher than RK, the
recoverin effect is independent of RK concentration and 2) at
saturating free Ca
, the recoverin concentration
required for the half-maximal amplitude of the recoverin effect is
equal to K
. Therefore, only K
remains to be varied in order to find a fit for the experimental
data. A K
of 4.5 µM, along with a K
of 3.4 µM (from Fig. 2B), is found to provide a reasonable fit for the
data (Fig. 6, curve 1). Our indirect estimate of 4.5
µM for K
is in agreement with flow
dialysis measurements of Ca
binding to native
recoverin that showtwo sites with affinities of about 2.7 and 3.8
µM
and to myristoylated recombinant recoverin
that show cooperative Ca
binding with a Hill
coefficient of 1.75 and half-saturation at 17 µM Ca
(31) . This analysis also provides an
indirect but, we think, compelling reason that previously reported free
Ca
ranges for the recoverin effect in vitro are in error: In order to observe a K
of
0.1-0.2 µM free Ca
for the
recoverin effect when recoverin is 5-10
µM(7, 10, 28) , one would need to
set K
at 0.1-0.3 µM, very far
from the values determined experimentally.
Figure 6:
Recoverin inhibition of rhodopsin
phosphorylation may occur at physiological free Ca
concentrations. Theoretical predictions of Ca
dependence of the binding between RK and recoverin. Curve
1, predicted free Ca
dependence for 10.1
µM recoverin; K
= 2.2
µM free Ca
. Rescaled data from Fig.
1A (10 µM recoverin added) are shown (▾). Curve 2, extrapolation to the concentrations of 34 µM myristoylated recoverin that is able to bind to membranes, 6
mM rhodopsin ROS membranes found in intact ROS, and 7
µM RK; K
= 0.27
µM. See details in text.
We have an apparent
problem. The data seem to portray recoverin as a low potency inhibitor
acting at micromolar free Ca levels, whereas the free
Ca
concentration in dark ROS is 200-600 nM and falls to much lower levels on
illumination(32, 33, 34, 35) . The
problem is resolved by extrapolation of the data to in vivo conditions, which brings the recoverin effect into the
physiological free Ca
range. Note first that at 10.1
µM total recoverin the theoretical half-saturating free
Ca
(K
= 2.2
µM; Fig. 6, curve 1) is lower than the K
of 4.5 µM used in the calculations.
The nature of such a shift is easy to understand; the K
of recoverin for Ca
dictates what proportion of recoverin is
Ca
-bound, whereas the percentage of RK bound to
Ca
-recoverin is a function of the absolute
concentration of Ca
-bound recoverin. Under
presumed in vivo conditions of 34 µM recoverin
(based on 6 mM cytoplasmic rhodopsin concentration (36) and the rhodopsin/recoverin ratio of 174 that we
determined), the calculated half-saturating free Ca
for the recoverin effect is 1.4 µM.
(
)A further factor that will affect the free
Ca
range of recoverin action in vivo is the
ability of myristoylated Ca
-recoverin to bind to
membranes. As Zozulya and Stryer (17) point out, at physiologically
high membrane concentrations the Ca
titration curve
of recoverin binding to membranes should have a K
lower than recoverin's affinity for Ca
.
The same holds true for Ca
binding to recoverin.
Binding of Ca
-recoverin to membrane shifts the
equilibrium of the binding between Ca
and recoverin
toward the formation of more Ca
-recoverin complexes.
With K
= 4.5 µM and a
Ca
-recoverin K
for
membranes of 230 µM rhodopsin (from Ref. 16; Fig. 3), a calculated Ca
titration curve of
recoverin under presumed in vivo conditions of 6 mM rhodopsin and 34 µM recoverin has a K
of 0.87 µM free Ca
(not shown).
at 0.87 µM, to estimate the
percentage of RK not bound to Ca
-recoverin in
vivo as a function of free Ca
, one should take
not the true recoverin affinity for Ca
(
4.5
µM), as we did above, but its effective value of 0.87
µM. Fig. 6, curve 2, shows how this results
in a further shift of the predicted range of the recoverin effect
toward lower free Ca
values, yielding a K
of 271 nM free Ca
,
which is within the physiological range. This leads us to an important
conclusion: even though myristoylation of recoverin per se is
not required for its inhibition of RK (Ref. 28 and this study), under in vivo conditions this posttranlational modification might
determine the free Ca
range of the recoverin effect!
As demonstrated by Ames et al.(31) , another important
function of recoverin myristoylation is that it induces positive
cooperativity of Ca
binding by recoverin, making it a
more efficient sensor of Ca
changes.
has an important
physiological implication. Given that the rate of association between
Ca
and Ca
-binding proteins is about
2
10
(37) , the micromolar K
of the Ca
-recoverin
complex means that its dissociation is rapid, and the lifetime of the
complex is on the order of tens of milliseconds. This should allow
photoreceptors to track changes in free Ca
concentration without delay. As we show above, the low affinity
for Ca
does not impair recoverin's ability to
sense free Ca
concentrations significantly lower than K
, provided that the
Ca
-bound form of recoverin selectively binds to
membrane and high enough concentrations of recoverin are present. The
observations presented in this paper, taken together with previous
studies(7, 9, 10, 11, 12) , make
a compelling case for the relevance of recoverin in phototransduction.
The extrapolation presented here is not meant to be definitive but
points to a solution for the apparent discrepancy between in vitro data and the physiological free Ca
range.
Moreover, it suggests the possibility that the efficiency of the
Ca
feedback on the level of rhodopsin phosphorylation
might in turn be modulated by changes in the percentage of N-acylated recoverin and the exact chemical nature of the
modification.
S, guanosine
5`-O-(3-thiotriphosphate); BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid.
range over which it occurs. The maximal inhibition
at 34 µM recoverin, for example, varies from 73 to 90% for
recoverin to RK ratios of 1 and 10, respectively. Because no bands
comparable in intensity to recoverin are found in the region of
65-75 kDa (besides the rhodopsin dimer) and because of the
difference in the molecular weights of the proteins, we presume here
the ratio 5:1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.