Calcium-binding proteins: intracellular sensors from the calmodulin superfamily.

In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.     

TRP, TRPL and Cacophony Channels Mediate Ca(2+) Influx and Exocytosis in Photoreceptors Axons in Drosophila

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, we studied the distribution and physiological properties of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

Speed, adaptation, and stability of the response to light in cone photoreceptors: the functional role of Ca-dependent modulation of ligand sensitivity in cGMP-gated ion channels

The response of cone photoreceptors to light is stable and reproducible because of the exceptional regulation of the cascade of enzymatic reactions that link visual pigment (VP) excitation to the gating of cyclic GMP (cGMP)-gated ion channels (cyclic nucleotide-gated [CNG]) in the outer segment plasma membrane. Regulation is achieved in part through negative feedback control of some of these reactions by cytoplasmic free Ca(2+). As part of the control process, Ca(2+) regulates the phosphorylation of excited VP, the activity of guanylate cyclase, and the ligand sensitivity of the CNG ion channels. We measured photocurrents elicited by stimuli in the form of flashes, steps, and flashes superimposed on steps in voltage-clamped single bass cones isolated from striped bass retina. We also developed a computational model that comprises all the known molecular events of cone phototransduction, including all Ca-dependent controls. Constrained by available experimental data in bass cones and cone transduction biochemistry, we achieved an excellent match between experimental photocurrents and those simulated by the model. We used the model to explore the physiological role of CNG ion channel modulation. Control of CNG channel activity by both cGMP and Ca(2+) causes the time course of the light-dependent currents to be faster than if only cGMP controlled their activity. Channel modulation also plays a critical role in the regulation of the light sensitivity and light adaptation of the cone photoresponse. In the absence of ion channel modulation, cone photocurrents would be unstable, oscillating during and at the offset of light stimuli.     

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.     

Metal-ion affinity and specificity in EF-hand proteins: coordination geometry and domain plasticity in parvalbumin.

BACKGROUND: The EF-hand family is a large set of Ca(2+)-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization and vision. RESULTS: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca(2+)-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca(2+)-binding affinity 100-fold and increases the Mg(2+)-binding affinity 10-fold. Both the Ca(2+)- and Mg(2+)-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. CONCLUSIONS: The E101D mutation does not affect the Mg(2+) coordination geometry of the binding loop, but it does pull the F helix 1.1 A towards the loop. The E101D-Ca(2+) structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca(2+), presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca(2+)-binding loop delimit the allowable geometries for the coordinating sphere.     

Mutations in the rod outer segment membrane guanylate cyclase in a cone-rod dystrophy cause defects in calcium signaling.

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a member of the subfamily of Ca(2+)-regulated membrane guanylate cyclases; and it is pivotal for vertebrate phototransduction. Two opposing regulatory modes control the activity of ROS-GC1. At nanomolar concentrations of Ca(2+), ROS-GC1 is activated by Ca(2+)-binding proteins named guanylate cyclase activating proteins (GCAPs). However, at micromolar concentrations of Ca(2+), ROS-GC1 is stimulated by S100beta [also named calcium-dependent (CD) GCAP]. This mode is not linked with phototransduction; instead, it is predicted to be involved in retinal synaptic activity. Two point mutations, E786D and R787C, in ROS-GC1 have been connected with cone-rod dystrophy (CORD6), with only one type of point mutation occurring in each family. The present study shows that the E786D mutation has no effect on the basal catalytic activity of ROS-GC1 and on its activation by GCAP1 and S100beta; however, the mutated cyclase becomes more activated by GCAP2. The R787C mutation has three consequences: (1) it causes major damage to the basal cyclase activity, (2) it makes the cyclase 5-fold more sensitive to activation by GCAP1; and 3) converts the cyclase into a form that is less sensitive to activation by GCAP2 and S100beta. Thus, the two CORD6-linked mutations in ROS-GC1, which occur at adjacent positions, result in vastly different biochemical phenotypes, and they are connected with very specific molecular defects in the Ca(2+) switching components of the cyclase. These defects, in turn, are proposed to have a profound effect on both the machinery of phototransduction and the retinal synapse. The study for the first time defines the biochemistry of CORD6 pathology in precise molecular terms.     

Ca(2+)-binding proteins in the retina: from discovery to etiology of human disease(1)

Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.     

Comparative analysis of the isoform expression pattern of Ca(2+)-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequency stimulated muscle fibers

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.     

Conformational changes in guanylyl cyclase-activating protein 1 (GCAP1) and its tryptophan mutants as a function of calcium concentration.

Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca2+-binding proteins belonging to the calmodulin superfamily. Ca2+-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. In this study, we characterized GCAP1 mutants in which three endogenous nonessential Trp residues were replaced by Phe residues, eliminating intrinsic fluorescence. Subsequently, hydrophobic amino acids adjacent to each of the three functional Ca2+-binding loops were replaced by reporter Trp residues. Using fluorescence spectroscopy and biochemical assays, we found that binding of Ca2+ to GCAP1 causes a major conformational change especially in the region around the EF3-hand motif. This transition of GCAP1 from an activator to an inhibitor of GC requires an activation energy Ea = 9.3 kcal/mol. When Tyr99 adjacent to the EF3-hand motif was replaced by Cys, a mutation linked to autosomal dominant cone dystrophy in humans, Cys99 is unable to stabilize the inactive GCAP1-Ca2+ complex. Stopped-flow kinetic measurements indicated that GCAP1 rapidly loses its bound Ca2+ (k-1 = 72 s-1 at 37 degrees C) and was estimated to associate with Ca2+ at a rate (k1 > 2 x 10(8) M-1 s-1) close to the diffusion limit. Thus, GCAP1 displays thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response.     

Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina

Calcium feedback in vertebrate photoreceptors regulates synthesis of cGMP, a second messenger in phototransduction. The decrease in the free intracellular Ca(2+) concentrations caused by illumination stimulates two isoforms of retinal membrane guanylyl cyclase (RetGC) via Ca(2+)-sensor proteins and thus contributes to photoreceptor recovery and light adaptation. Unlike other members of the membrane guanylyl cyclase family, retinal guanylyl cyclases do not have identified extracellular peptide ligands. Recoverin-like proteins, GCAP-1 and GCAP-2, interact with the intracellular portion of the cyclases and stimulate its activity through dimerization of the cyclase subunits. Several mutations that affect the function of photoreceptor guanylyl cyclase and the activator protein have been linked to various forms of congenital human retinal diseases, such as Leber congenital amaurosis, cone and cone-rod dystrophy.     

Responses of Ca2+-binding proteins to localized,transient changes in intracellular [Ca2+

In smooth muscle cells, various transient, localized [Ca(2+)] changes have been observed that are thought to regulate cell function without necessarily inducing contraction. Although a great deal of effort has been put into detecting these transients and elucidating the mechanisms involved in their generation, the extent to which these transient Ca(2+) signals interact with intracellular Ca(2+)-binding molecules remains relatively unknown. To understand how the spatial and temporal characteristics of an intracellular Ca(2+) signal influence its interaction with Ca(2+)-binding proteins, mathematical models of Ca(2+) diffusion and regulation in smooth muscle cells were used to study Ca(2+) binding to prototypical proteins with one or two Ca(2+)-binding sites. Simulations with the models: (1) demonstrate the extent to which the rate constants for Ca(2+)-binding to proteins and the spatial and temporal characteristics of different Ca(2+) transients influence the magnitude and time course of the responses of these proteins to the transients; (2) predict significant differences in the responses of proteins with one or two Ca(2+)-binding sites to individual Ca(2+) transients and to trains of transients; (3) demonstrate how the kinetic characteristics determine the fidelity with which the responses of Ca(2+)-sensitive molecules reflect the magnitude and time course of transient Ca(2+) signals. Overall, this work demonstrates the clear need for complete information about the kinetics of Ca(2+) binding for determining how well Ca(2+)-binding molecules respond to different types of Ca(2+) signals. These results have important implications when considering the possible modulation of Ca(2+)- and Ca(2+)/calmodulin-dependent proteins by localized intracellular Ca(2+) transients in smooth muscle cells and, more generally, in other cell types.     

The functions of Ca(2+) in bacteria: a role for EF-hand proteins?

In bacteria, Ca(2+) is implicated in a wide variety of cellular processes, including the cell cycle and cell division. Dedicated influx and efflux systems tightly control the low cytoplasmic Ca(2+) levels in prokaryotes. Additionally, the growing number of proteins containing various Ca(2+)-binding motifs supports the importance of Ca(2+), which controls various protein functions by affecting protein stability, enzymatic activity or signal transduction. The existence of calmodulin-like proteins (containing EF-hand motifs) in bacteria is a long-standing hypothesis. Analysis of the prokaryotic protein sequences available in the databases has revealed the presence of several calmodulin-like proteins containing two or more authentic EF-hand motifs, suggesting that calmodulin-like proteins could be involved in Ca(2+) regulation in bacteria.     

Metal-driven operation of the human large-conductance voltage- and Ca2+-dependent potassium channel (BK) gating ring apparatus

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.     

Impairment of the rod outer segment membrane guanylate cyclase dimerization in a cone-rod dystrophy results in defective calcium signaling

Rod outer segment membrane guanylate cyclase1 (ROS-GC1) is the original member of the membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca(2+) signals in the sensory neurons. In the vertebrate retinal neurons, ROS-GC1 is pivotal for the operations of phototransduction and, most likely, of the synaptic activity. The phototransduction- and the synapse-linked domains are separate, and they are located in the intracellular region of ROS-GC1. These domains sense Ca(2+) signals via Ca(2+)-binding proteins. These proteins are ROS-GC activating proteins, GCAPs. GCAPs control ROS-GC1 activity through two opposing regulatory modes. In one mode, at nanomolar concentrations of Ca(2+), the GCAPs activate the cyclase and as the Ca(2+) concentrations rise, the cyclase is progressively inhibited. This mode operates in phototransduction via two GCAPs: 1 and 2. The second mode occurs at micromolar concentrations of Ca(2+) via S100beta. Here, the rise of Ca(2+) concentrations progressively stimulates the enzyme. This mode is linked with the retinal synaptic activity. In both modes, the final step in Ca(2+) signal transduction involves ROS-GC dimerization, which causes the cyclase activation. The identity of the dimerization domain is not known. A heterozygous, triple mutation -E786D, R787C, T788M- in ROS-GC1 has been connected with autosomal cone-rod dystrophy in a British family. The present study shows the biochemical consequences of this mutation on the phototransduction- and the synapse-linked components of the cyclase. (1) It severely damages the intrinsic cyclase activity. (2) It significantly raises the GCAP1- and GCAP2-dependent maximal velocity of the cyclase, but this compensation, however, is not sufficient to override the basal cyclase activity. (3) It converts the cyclase into a form that only marginally responds to S100beta. The mutant produces insufficient amounts of the cyclic GMP needed to drive the machinery of phototransduction and of the retinal synapse at an optimum level. The underlying cause of the breakdown of both types of machinery is that, in contrast to the native ROS-GC1, the mutant cyclase is unable to change from its monomeric to the dimeric form, the form required for the functional integrity of the enzyme. The study defines the CORD in molecular terms, at a most basic level identifies a region that is critical in its dimer formation, and, thus, discloses a single unifying mechanistic theme underlying the complex pathology of the disease.     

Characterization of p26olf, a novel calcium-binding protein in the frog olfactory epithelium

We have previously shown that p26olf is a novel S100-like Ca(2+)-binding protein in the frog olfactory epithelium. In this paper, we characterized the Ca(2+) binding property of p26olf, examined the precise localization in the frog olfactory epithelium, and searched for the possible target proteins of p26olf. By flow dialysis experiments using (45)Ca, p26olf was suggested to bind approximately 4 Ca(2+). Circular dichroism measurement showed that binding of Ca(2+) to p26olf induces an increase in the apparent content of both alpha-helix and beta-sheet with an apparent K(d) value of 2.4 micrometer. Electron microscopic observation disclosed p26olf immunoreactivity in the cilia, dendritic knob, and dendrite of olfactory receptor cells. Blot overlay analysis and affinity purification of p26olf-binding proteins showed that p26olf binds to a frog beta-adrenergic receptor kinase-like protein in a Ca(2+)-dependent manner. These results suggested that p26olf has some roles in the olfactory transduction or adaptation.     

Structure-function relationships in sorcin, a member of the penta EF-hand family. Interaction of sorcin fragments with the ryanodine receptor and an Escherichia coli model system

Sorcin, a 21.6 kDa cytosolic EF-hand protein which undergoes a Ca(2+)-induced translocation from cytoplasm to membranes, has been assigned to the newly defined penta EF-hand family. A molecular model of the C-terminal Ca(2+)-binding domain has been generated using as a template the X-ray coordinates of the corresponding domain in the calpain light subunit, the family prototype [Lin, G., et al. (1997) Nat. Struct. Biol. 4, 539-546]. The model indicates that in sorcin the three-dimensional structure is conserved and in particular that of EF1, the novel EF-hand motif characteristic of the family. On this basis, two stable fragments have been obtained and characterized. Just like the native protein, the sorcin Ca(2+)-binding domain (residues 33-198) is largely dimeric, interacts with the ryanodine receptor at physiological calcium concentrations, and undergoes a reversible, Ca(2+)-dependent translocation from cytosol to target proteins on Escherichia coli membranes. In contrast, the 90-198 fragment (residues 90-198), which lacks EF1 and EF2, does not bind Ca(2+) with high affinity and is unable to translocate. Binding of calcium to the EF1-EF2 pair is therefore required for the activation of sorcin which uses the C-terminal calcium-binding domain for interaction with the ryanodine receptor, a physiological target in muscle cells.     

Ca2+ differently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.