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Originally published In Press as doi:10.1074/jbc.C400065200 on March 1, 2004

J. Biol. Chem., Vol. 279, Issue 17, 16903-16906, April 23, 2004
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Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors

IMPLICATIONS FOR PHOTORECEPTOR GUANYLYL CYCLASE (RetGC) REGULATION IN MAMMALIAN PHOTORECEPTORS*

Igor V. Peshenko and Alexander M. Dizhoor{ddagger}

From the Hafter Research Laboratory, Pennsylvania College of Optometry, Elkins Park, Pennsylvania 19027

Received for publication, February 8, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca2+-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca2+ and inhibit RetGC in a Ca2+-sensitive manner. The reported data for the RetGC inhibition by Ca2+/GCAPs in vitro are in disagreement with the free Ca2+ levels found in mammalian photoreceptors (Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843–854). We have found that binding of Mg2+ dramatically affects both Ca2+-dependent conformational changes in GCAP-1 and Ca2+ sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg2+ concentrations ([Mg]f) from 5.0 mM to 0.5 mM decreases the free Ca2+ concentration required for half-maximal inhibition of RetGC ([Ca]1/2) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 µM to 0.16 and 0.03 µM, respectively. A similar effect of Mg2+ on Ca2+ sensitivity of RetGC by endogenous GCAPs was observed in mouse retina. Analysis of the [Ca]1/2 changes as a function of [Mg]f in mouse retina shows that the [Ca]1/2 becomes consistent with the range of 23–250 nM free Ca2+ found in mouse photoreceptors only if the [Mg]f in the photoreceptors is near 1 mM. Our data demonstrate that GCAPs are Ca2+/Mg2+ sensor proteins. While Ca2+ binding is essential for cyclase activation and inhibition, Mg2+ binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca2+.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
GCAPs1 are Ca2+-binding proteins of the EF-hand superfamily that act as Ca2+ sensors during excitation and recovery in vertebrate photoreceptors (1–4). In dark-adapted photoreceptor a small percent of cGMP-gated Na+/K+,Ca2+ channels remain open. Light triggers hydrolysis of cGMP, thus causing cGMP-gated channels to close and resulting in hyperpolarization of the cell plasma membrane. In addition to hyperpolarization, closure of cGMP-gated Na+/K+,Ca2+ channels decreases the intracellular Ca2+ level by stopping Ca2+ influx through the channels. The change in intracellular Ca2+ accelerates recovery phase of phototransduction by stimulating cGMP synthesis by retinal guanylyl cyclase (RetGC), a process that is mediated by GCAPs (5–9).

Purified GCAPs activate RetGC in the absence of or at low Ca2+ concentrations, but they become RetGC inhibitors when saturated with Ca2+ (1, 4). However, the reported range for the RetGC regulation in vitro by Ca2+/GCAPs (1, 3, 4, 10–14) only partially overlaps with the free intracellular Ca2+ levels found in mammalian photoreceptors (approximately 250 nM in dark and as low as 23 nM in light; Ref. 15). At the same time, the free Ca2+ concentration required for half-maximal RetGC inhibition ([Ca]1/2), determined in vitro using recombinant GCAPs, varies between 220 nM and 1 µM for GCAP-1 and between 200 nM and 300 nM for GCAP-2. These values appear relatively high for mammalian rods, because at such high [Ca]1/2 the dynamic range for cyclase regulation in response to light would be significantly reduced with regard to the actual free intracellular Ca2+ range. They are also much higher than the [Ca]1/2 reported for RetGC regulation estimated in electrophysiological recordings (<100 nM) (16–18). In this study we have found that CGAPs are not just Ca2+-binding proteins but rather Ca2+/Mg2+-binding proteins, and the Mg2+ binding is essential for adjusting the Ca2+ sensitivity of GCAPs to the actual physiological range of free Ca2+ concentrations.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Recombinant GCAP-1 and GCAP-2—GCAP-1 and GCAP-2 coding regions were expressed as previously described from a pET11d vector (Calbiochem/Novagen) in a BLR (DE3) Escherichia coli strain carrying a pBB131 plasmid that encoded N-myristoyltransferase (10, 11, 19). The cells were grown in standard LB medium containing 40 µg/ml kanamycin and 100 µg/ml ampicillin. Free myristic acid was added from a concentrated ethanol solution to the suspension of bacterial cells to a final concentration of 100 µg/ml 30 min prior to the induction with 1mM isopropyl-{beta}-D-thiogalactopyranoside. Three hours after the induction the bacterial pellet was harvested, and the recombinant GCAPs were purified by chromatography as previously described in detail (19).

Tryptophan Fluorescence Measurements—Fluorescence emission at 332 nm (excitation at 290 nm, slit 10 nm) was recorded at 23 °C using 2 µM GCAP-1 in 0.6 ml of 100 mM MOPS/KOH, pH 7.2, 40 mM KCl, 1 mM EGTA and various concentrations of MgCl2. Small aliquots of concentrated CaCl2 solution were added to obtain the desired free Ca2+. The free Ca2+ and Mg2+ concentrations in the solution were calculated according to the method of Brooks and Storey (20) and utilizing the algorithm of Marks and Maxfield (21). All data shown were from several independent experiments producing similar results.

RetGC Assay—All experiments were conducted under infrared light. The assay mixture (25 µl) contained 30 mM MOPS/KOH, pH 7.2, 60 mM KCl, 5 mM NaCl, 1 mM dithiothreitol, 2 mM Ca/EGTA buffer, different concentrations of MgCl to vary the free Mg2+2 between 0.5 and 6 mM, zaprinast and dipyridamole (25 µM each), leupeptin and aprotinine (10 µg/ml each), 0.3 mM ATP, 4 mM cGMP, 1 mM GTP, 1 µCi of [{alpha}-32P]GTP, 0.1 µCi of [8-3H]cGMP, and 1 µl of washed bovine outer segment membranes (3.7 mg/ml rhodopsin) or mouse retina homogenate (0.2 retina per assay). Dark-adapted mouse retinas were homogenized in buffer containing 60 mM MOPS/KOH, pH 7.2, 120 mM KCl, 10 mM NaCl, 2 mM dithiothreitol, zaprinast, dipyridamole (50 µM each), leupeptin, aprotinine (20 µg/ml each) and used immediately. The reaction mixture was incubated for 12 min at 30 °C, then the reaction was stopped by heating for 2.5 min at 95 °C, and the aliquots were analyzed by TLC using fluorescent plastic-backed polyethylenimine cellulose plates (Merck) as described previously (3). Ca2+/EGTA buffers were prepared as described previously (22), and the final free Ca2+ and Mg2+ concentrations in reaction mixture were calculated using the algorithm of Marks and Maxfield (21). Ca2+/EGTA buffers were calibrated using Ca2+ fluorescence dyes Fluo-3 and X-rhod FF (Kd of 325 nM and 17 µM, respectively) (Molecular Probes, Eugene, OR).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Bovine GCAP-1 exhibits Ca2+-sensitive changes in the intrinsic tryptophan fluorescence as a result of cation binding to its EF-hands (23–25). The fluorescence of myristoylated bovine GCAP-1 is at its maximum in the absence of Ca2+ and Mg2+, and the changes in fluorescence as a function of free Ca2+ ([Ca]f) are biphasic (Fig. 1A). In the absence of Mg2+, increasing [Ca]f initially decreases the intensity of the fluorescence (phase I) until it reaches its minimum at 100–200 nM [Ca]f, followed by a small increase in the fluorescence intensity (phase II) until it reaches a plateau at ~1 µM [Ca]f (Fig. 1A). All GCAPs contain four helix-turn-helix EF-hand structures, of which only three can bind Ca2+ (10, 14, 25, 26). The affinity of the individual EF-hands to Ca2+ and the order in which the Ca2+ ions bind EF-hands in GCAP-1 are not immediately apparent; however, the biphasic profile of the Ca2+-dependent changes in fluorescence of GCAP-1 implies that by using this method, the interaction of at least two EF-hands with Ca2+ can be observed. Apparently, conformational changes in GCAP-1 corresponding to phase I reflect higher affinity binding of Ca2+ (it starts at [Ca]f as low as 10 nM), while much higher [Ca]f is necessary to attain the phase II. Surprisingly, we have found that not only Ca2+ but also Mg2+ inhibits fluorescence of GCAP-1. Unlike Ca2+, the Mg2+-dependent change in fluorescence appears monophasic, and the concentration of free Mg2+ ([Mg]f) required for the half-maximal decrease in GCAP-1 tryptophan fluorescence is near 0.25 mM (Fig. 1B). More importantly, Mg2+ also dramatically alters Ca2+-dependent changes in GCAP-1 fluorescence by affecting both phase I and II in a different manner (Fig. 1A). First, Mg2+ strongly diminishes the fluorescence of the Ca2+-free GCAP-1, such that the fluorescence corresponding to the phase I becomes eliminated within the millimolar range of [Mg]f, and the amplitude of the Ca2+-dependent fluorescence change corresponding to the phase II becomes more prominent in the presence of Mg2+ (Fig. 1A). Second, Mg2+ affects the Ca2+-sensitivity of the phase II in such a manner that higher [Ca]f is required to reach the halfmaximal amplitude corresponding to the phase II (Fig. 1A). Taken together, these observations indicate that GCAP-1 directly binds Mg2+, and the Mg2+ binding strongly influences both conformation of GCAP-1 and its affinity for Ca2+. Ca2+/Mg2+-dependent changes in the tryptophan fluorescence of myristoylated GCAP-2 were much smaller than in GCAP-1, probably because of higher tryptophan content in GCAP-2.



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  		FIG. 1.
Ca2+- and Mg2+-dependent changes in tryptophan fluorescence of GCAP-1. A, the Ca2+-dependent changes in the fluorescence intensity of 2 µM GCAP-1 were recorded as described under "Experimental Procedures" at various [Mg]f: no Mg2+ ({circ}), 1.24 mM ({blacktriangleup}), 2.9 mM (), 6.7 mM ({diamond}), 9.0 mM ({blacktriangledown}), and 14 mM ({square}). B, changes in the tryptophan fluorescence intensity of 2 µM GCAP-1 as a function of [Mg]f. The assay mixture contained 1 mM EGTA and no Ca2+. Small aliquots of concentrated MgCl2 solution were added to obtain the desirable [Mg]f.

 
In addition to the conformational changes in GCAP-1, we found that Mg2+ binding dramatically affected Ca2+ sensitivity of RetGC regulation in bovine rod outer segment membranes reconstituted with recombinant myristoylated GCAP-1 or GCAP-2 (Fig. 2, A and B). Lowering [Mg]f from 5.0 to 0.5 mM decreases the free Ca2+ concentrations required for half-maximal inhibition of RetGC ([Ca]1/2) by GCAP-1 and GCAP-2 from 1.3 and 0.2 µM to 0.16 and 0.03 µM, respectively. While the [Ca]1/2 for both purified GCAPs increases proportionally with the increase in [Mg]f (Fig. 2C), the basal RetGC activity in washed membranes that lack GCAPs remains insensitive to Ca2+ at all tested concentrations of Mg2+. Mg2+ needs to be present as a part of Mg-GTP substrate complex in the catalytic center of RetGC and is essential for its activity (27); therefore, the decrease in RetGC activity at [Mg]f below 1 mM (Fig. 2D) could be explained by a decrease in concentration of Mg-GTP substrate complex (inset on Fig. 2D). However, even at [Mg]f as low as 0.5 mM RetGC retains more than 70% of its catalytic activity. Despite the small decrease in absolute RetGC activity at low Mg2+, the amplitude of its stimulation by GCAPs remains virtually the same (Fig. 2, A and B) and varying [Mg]f at above 1 mM affects neither basal nor GCAP-stimulated RetGC activity (Fig. 2D). Hence, Mg2+ changes Ca2+ sensitivity of RetGC by affecting Ca2+ binding properties of GCAPs rather than catalytic activity of the cyclase itself.



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  		FIG. 2.
Ca2+ sensitivity of RetGC regulation in bovine rod outer segment membranes as a function of free Mg2+. Washed outer segment membranes were reconstituted with 10 µM GCAP-1 (A) or GCAP-2 (B) at [Mg]f of 0.5 mM ({square}), 1.0 mM ({blacktriangleup}), 2.0 mM (), 5 mM ({blacktriangledown}) versus basal RetGC activity in the absence of GCAPs at 0.5 mM ({blacksquare}), 1.0 mM ({diamondsuit}), 2.0 mM ({circ}), 5 mM ({diamond}) [Mg]f. The data were fitted by the equation, V = (Vmax – Vmin) /(1 + ([Ca]f/[Ca]1/2))n + Vmin; V is the activity of RetGC-1, Vmax and Vmin are the maximal and minimal activity of RetGC, respectively, n is the cooperativity coefficient. C, the values of [Ca]1/2 for RetGC activation by 10 µM GCAP-1 ({square}) or GCAP-2 ({circ}) determined at various [Mg]f. D, RetGC activity in washed outer segment membranes, basal () or stimulated by 10 µM of Ca2+-free GCAP-1 ({circ}) or GCAP-2 ({square}) was measured at various [Mg]. Inset, concentration of the Mg-GTP substrate complex in the reaction mixture at various [Mg]f calculated using algorithm of Brooks and Storey (20).

 
We have found a similar effect of Mg2+ on Ca2+ sensitivity of RetGC regulation by the endogenous GCAPs in mouse retina (Fig. 3). The overall Ca2+ sensitivity of RetGC regulation in mouse retina homogenate differs from that of individual bovine recombinant GCAPs reconstituted with washed bovine ROS membranes (Fig. 2, A and B). For example at 2 mM [Mg]f the [Ca]1/2 is 113 nM for mouse retina homogenate and 530 and 87 nM for bovine ROS reconstituted with GCAP-1 and GCAP-2, respectively. Apparently, the integral Ca2+ sensitivity of RetGC in retinal homogenates, which contain both GCAP-1 and GCAP-2 (13), reflects the combined effect of both GCAPs. Importantly, the effect of Mg2+ on Ca2+ sensitivity of RetGC regulation by the endogenous GCAPs in whole mouse retina homogenates remained and was similar to that observed in the case of recombinant GCAPs. The dependence of [Ca]1/2 on [Mg]f is also linear within a wide range of [Mg]f (Fig. 3B). While Mg2+ dramatically changes the Ca2+ sensitivity of RetGC regulation, it does not affect its cooperativity, which remains close to 2.0 at all free Mg2+ concentrations tested in our experiments (Fig. 3C).



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  		FIG. 3.
Ca2+ sensitivity of RetGC regulation in mouse retina homogenate as a function of free Mg2+. A, the freshly prepared mouse retina homogenate was assayed for RetGC activity at various [Ca]f and [Mg]f as described under "Experimental Procedures" and in the legend to Fig. 2. The [Mg]f in different series was: 0.5 mM ({square}), 0.8 mM ({blacktriangledown}), 2.0 mM ({circ}), 5.0 mM ({blacktriangleup}), 6.0 mM ({diamond}). The shaded area corresponds to the 23–250 nM free Ca2+ range in mouse photoreceptors between light and dark. B, [Ca]1/2 for RetGC as a function of [Mg]f. C, a Hill plot of the data from A.Mg2+ does not affect cooperativity of RetGC regulation by Ca2+ in mouse retina homogenate.

 
In this study we have found that the key factor affecting Ca2+ sensitivity of GCAPs is their binding of Mg2+; therefore, GCAPs should be more correctly defined as Ca2+/Mg2+ sensor proteins. Mg2+ does not affect the extent to which RetGC becomes activated by GCAPs at low [Ca]f or inhibited at high [Ca]f, and it does not change the cooperativity of RetGC regulation by Ca2+, either. However, it critically affects the Ca2+ sensitivity of RetGC regulation. The shaded area in Fig. 3A corresponds to the 23–250 nM free Ca2+ range in mouse photoreceptors within which [Ca]f changes between light and dark (15). Only at free Mg2+ concentrations between 0.5 and 2 mM the Ca2+ sensitivity of RetGC regulation fits with the actual free Ca2+ found in mouse photoreceptors. The possible explanation why most of the [Ca]1/2 values for GCAPs reported in the literature do not fit well with the 23–250 nM free Ca2+ range is that those values were typically obtained using Mg2+ concentrations that were much higher than physiological. The majority of published RetGC assay mixtures contained nearly 10 mM total Mg2+, which corresponds to 6–8 mM [Mg]f depending on the particular composition of the reaction (1, 3, 4, 10–14). According to our data, for GCAP Ca2+ sensitivity to better correspond to the physiological range of the intracellular Ca2+, the intracellular [Mg]f in the photoreceptors has to be close to 1 mM. The actual concentration of the free Mg2+ in mouse photoreceptor has not been determined; however, 0.81–0.86 mM [Mg]f was recently measured in salamander photoreceptors (28), which agrees well with our data. The effect of Mg2+ on Ca2+ sensitivity of RetGC regulation can also explain why [Ca]1/2 observed in whole-cell recordings (16, 17) was much lower than in most of biochemical assays in vitro that used recombinant GCAPs, because [Mg]f used in those electrophysiological experiments typically corresponded to 0.5 mM.

To conclude, our data demonstrate that GCAPs are Ca2+/Mg2+ sensor proteins. Intracellular Ca2+ is changed in response to light. Therefore, the primary function of GCAPs is to react to the changes in free Ca2+ concentrations caused by light and thus to activate or inhibit RetGC. In contrast, [Mg]f remains virtually unchanged by light (28). However Mg2+ binding by GCAPs contributes to the cyclase regulation by setting the proper dynamic range for the RetGC regulation corresponding to the physiological change in free Ca2+. It remains to be determined which EF-hand(s) in GCAP-1 and GCAP-2 is responsible for adding Mg2+ sensitivity to the cyclase regulation.

GCAPs are members of a separate family of neuron-specific recoverin-like proteins within the EF-hand superfamily. Unlike calmodulin, all proteins of the recoverin family have N-terminal myristoylation and four EF-hand like structures, of which only two or three can bind Ca2+ (29, 30). It was recently observed that recoverin binds Mg2+ at high concentrations; however, the physiological importance of the Mg2+ binding by recoverin has not been revealed (31). Our finding of GCAPs being Ca2+/Mg2+ sensors raises the possibility that Ca2+/Mg2+ binding is a mechanism that adjusts Ca2+ sensitivity of other recoverin-like proteins to the physiological range of the intracellular Ca2+.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant EY11522 and by the Pennsylvania Lions Sight Conservation and Eye Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} A Martin and Florence Hafter Professor of Pharmacology. To whom correspondence should be addressed: Pennsylvania College of Optometry, 8360 Old York Rd., Elkins Park, PA 19027. E-mail: adizhoor{at}pco.edu.

1 The abbreviations used are: GCAP, guanylyl cyclase-activating protein; RetGC, photoreceptor membrane guanylyl cyclase; [Ca]1/2, free Ca2+ concentration required for half-maximal inhibition of RetGC; [Ca]f, free Ca2+ concentrations; MOPS, 4-morpholinopropanesulfonic acid; [Mg]f, free Mg2+ concentrations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Yiannis Koutalos and Dr. Felix Barker for valuable discussion.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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C. L. Makino, X.-H. Wen, N. Michaud, I. V. Peshenko, B. Pawlyk, R. S. Brush, M. Soloviev, X. Liu, M. L. Woodruff, P. D. Calvert, et al.
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Structural Analysis of Mg2+ and Ca2+ Binding to CaBP1, a Neuron-specific Regulator of Calcium Channels
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M. Osawa, A. Dace, K. I. Tong, A. Valiveti, M. Ikura, and J. B. Ames
Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM
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I. V. Peshenko, E. V. Olshevskaya, and A. M. Dizhoor
Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis
J. Biol. Chem., November 26, 2004; 279(48): 50342 - 50349.
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E. V. Olshevskaya, P. D. Calvert, M. L. Woodruff, I. V. Peshenko, A. B. Savchenko, C. L. Makino, Y.-S. Ho, G. L. Fain, and A. M. Dizhoor
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