©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Rhodopsin Kinase by Recoverin
FURTHER EVIDENCE FOR A NEGATIVE FEEDBACK SYSTEM IN PHOTOTRANSDUCTION (*)

Vadim A. Klenchin (1)(§), Peter D. Calvert (1) (2), M. Deric Bownds (1) (2) (3)

From the (1)Laboratory of Molecular Biology, the (2)Neuroscience Training Program, and the (3)Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recoverin is a 23-kDa Ca-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.


INTRODUCTION

A drop in free Ca 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) .

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-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) .

This paper addresses several questions that are important in establishing the physiological relevance of the Ca-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.


EXPERIMENTAL PROCEDURES

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% HO. 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.

GTPS Binding Assay

The gain of GTPS 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,

On-line formulae not verified for accuracy


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.


RESULTS

Two Ranges of Free CaConcentration Inhibit Rhodopsin Phosphorylation

The dependence of rhodopsin phosphorylation on free Ca 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

On-line formulae not verified for accuracy

where Ca denotes free Ca 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.

The curve fitting shows that parameter a (the amplitude of low Ca 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.

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 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 GTPS 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 GTPS binding that is consistent with an inhibition of rhodopsin phosphorylation.


Figure 3: Ca-recoverin prolongs the lifetime of catalytically active rhodopsin. The gain of GTPS binding in frog ROS homogenates after a light flash bleaching 0.00054% rhodopsin (of a total 20 µM) in the presence of 100 µM GTPS 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.




DISCUSSION

Inhibition of rhodopsin phosphorylation by recoverin can be observed in vitro above 1 µM free Ca 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.

Our data provide an estimate of the affinity of recoverin for Ca. 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

On-line formulae not verified for accuracy

where Rec is recoverin and K 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).

If, under the high membrane concentrations found in ROS, myristoylated recoverin is expected to be half-saturated with Ca 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.

The low affinity of recoverin for Ca 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant EY 00463. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: 327 Molecular Biology, 1525 Linden Dr., Madison, WI 53706. Tel.: 608-262-4380; Fax: 608-262-4570.

The abbreviations used are: ROS, rod outer segment(s); RK, rhodopsin kinase; GTPS, guanosine 5`-O-(3-thiotriphosphate); BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid.

A. Polans, personal communication.

P. D. Calvert, V. A. Klenchin, and M. D. Bownds, manuscript in preparation.

For this calculation to be strictly performed, one also needs to know the in vivo RK concentration. However, its variation would only slightly affect the amplitude of the inhibition and would not affect the free Ca 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.


ACKNOWLEDGEMENTS

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.


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