Karl-wilhelm Koch - Academia.edu (original) (raw)

Papers by Karl-wilhelm Koch

The EMBO Journal, Apr 1, 1991

The resynthesis of cGMP in vertebrate photoreceptors by guanylate cyclase is one of the key event... more The resynthesis of cGMP in vertebrate photoreceptors by guanylate cyclase is one of the key events leading to the reopening of cGMP-gated channels after photoexcitation. Guanylate cyclase activity in vertebrate rod outer segments is dependent on the free calcium concentration. The basal activity of the enzyme observed at high concentrations of free calcium (>0.5 /M) increases when the free calcium concentration is lowered into the nanomolar range (< 0.1 /tM). This effect of calcium is known to be mediated by a soluble calcium-sensitive protein in a highly cooperative way. We here show that this soluble protein, i.e. the modulator of photoreceptor guanylate cyclase, is a 26 kd protein. Reconstitution of the purified 26 kd protein with washed rod outer segment membranes containing guanylate cyclase revealed a 3to 4-fold increase of cyclase activity when the free calcium concentration was lowered in the physiological range from 0.5 ,tM to 4 nM. Guanylate cyclase in whole rod outer segments was stimulated 10-fold in the same calcium range. The activation process in the reconstituted system was similar to the one in the native rod outer segment preparation, it showed a high cooperativity with a Hill coefficient n between 1.4 and 3.5. The half-maximal activation occurred between 110 and 220 nM free calcium. The molar ratio of the modulator to rhodopsin is 1:76 32. The protein is a calcium binding protein as detected with 45Ca autoradiography. Partial amino acid sequence analysis revealed a 60% homology to visinin from chicken cones.

Human Molecular Genetics, Sep 10, 2015

Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for ... more Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca 2+ with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca 2+. The latter mutant also shows a significantly high affinity for Mg 2+ , which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca 2+ , so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca 2+-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highestaffinity Ca 2+ binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca 2+. We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca 2+ and cyclic GMP homeostasis and result in retinal degeneration.

International Journal of Molecular Sciences, Mar 17, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Frontiers in Molecular Neuroscience, Sep 28, 2018

Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise ... more Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca 2+ sensor (NCS) proteins. Among them, the Ca 2+ sensor recoverin undergoes a pronounced Ca 2+-dependent conformational change, a prototypical so-called Ca 2+myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different recoverin isoforms (zRec1a, zRec1b, zRec2a, and zRec2b) that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca 2+-sensitive working modes for zebrafish recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca 2+-sensing properties of the different recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca 2+ resemble that one observed with mammalian recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular recoverin. Ca 2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca 2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of recoverin isoforms to changing intracellular Ca 2+ during illumination.

PLOS ONE, Aug 5, 2013

The zebrafish guanylate cyclase type 3 (zGC3) is specifically expressed in cone cells. A specifc ... more The zebrafish guanylate cyclase type 3 (zGC3) is specifically expressed in cone cells. A specifc antibody directed against zGC3 revealed expression at the protein level at 3.5 dpf in outer and inner retinal layers, which increased in intensity between 3.5 and 7 dpf. This expression pattern differed from sections of the adult retina showing strong immunostaining in outer segments of double cones and short single cones, less intense immunoreactivity in long single cones, but no staining in the inner retina. Although transcription and protein expression levels of zGC3 are similar to that of the cyclase regulator guanylate cyclase-activating protein 3 (zGCAP3), we surprisingly found that zGCAP3 is present in a 28-fold molar excess over zGC3 in zebrafish retinae. Further, zGCAP3 was an efficient regulator of guanylate cyclases activity in native zebrafish retinal membrane preparations. Therefore, we investigated the physiological function of zGCAP3 by two different behavioral assays. Using the morpholino antisense technique, we knocked down expression of zGCAP3 and recorded the optokinetic and optomotor responses of morphants, control morphants, and wild type fish at 5-6 dpf. No significant differences in behavioral responses among wild type, morphants and control morphants were found, indicating that a loss of zGCAP3 has no consequences in primary visual processing in the larval retina despite its prominent expression pattern. Its physiological function is therefore compensated by other zGCAP isoforms.

Scientific Reports, Jun 10, 2015

Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that sha... more Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca 2+-binding properties, Ca 2+-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca 2+-sensing, namely how similar Ca 2+-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca 2+-binding sites with Mg 2+. All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca 2+ with K D values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg 2+ either occupied the low affinity Ca 2+-binding site or it shifted the affinities for Ca 2+-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca 2+ than by Mg 2+ , although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca 2+-sensors. Calcium sensor proteins mediate signaling processes that respond to changing concentrations of Ca 2+-ions 1,2. The binding of Ca 2+ to intracellular calcium sensor proteins can trigger conformational transitions, which constitute a crucial step to regulate further downstream signaling proteins. One family of Ca 2+-binding proteins named neuronal calcium sensor (NCS) proteins are predominantly expressed in neuronal tissue and are involved in diverse intracellular processes 2,3. All NCS proteins harbor four EF-hand Ca 2+-binding motifs, of which in most cases three (sometimes only two) motifs can bind micromolar to submicromolar Ca 2+. One group of the NCS proteins is expressed in sensory cells and among them the guanylate cyclase-activating proteins (GCAPs) perform an important function in controlling the membrane bound guanylate cyclases (GCs) in retinal rod and cone cells 4-6. In their Ca 2+-free, Mg 2+-bound form GCAPs activate GCs, but they switch to an inhibitory mode, when all Ca 2+-binding sites are filled with Ca 2+ 7,8. Changing levels of cytoplasmic Ca 2+ in rod and cone outer segments are linked to changing levels of the intracellular messenger cGMP. After light activation of the photoreceptor cell the intracellular cGMP level is depleted, leading to a shutdown of cyclic nucleotide gated (CNG) channels in the outer segment of the cell. This stops the influx of Ca 2+ , which is however still extruded by the continuous operation of a Na + / Ca 2+ , K + exchanger leading to a net decrease of cytoplasmic Ca 2+. This decrease is sensed by GCAPs which in turn increase the GC activity,

Journal of Biological Chemistry, Mar 1, 2019

The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase... more The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca 2؉ concentrations decline, and decelerates the cyclase in the dark, when Ca 2؉ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca 2؉-dependent activatorto-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca 2؉ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca 2؉ concentrations characteristic of darkadapted photoreceptors. Ca 2؉-dependent increase in Trp 94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca 2؉ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca 2؉ , and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca 2؉ sensitivity of the cyclase within the physiological range of intracellular Ca 2؉ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly 86 , leading to abnormally high affinity for the target enzyme and reduced Ca 2؉ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca 2؉ influx in photoreceptors in the dark. Guanylyl cyclase-activating proteins (GCAPs), 4 N-myristoylated calcium/magnesium-binding proteins of the EF-hand superfamily, are comprised of two pairs of EF-hand domains connected via a "hinge" region (reviewed in Refs. 1 and 2). Among several isoforms of GCAPs expressed in the vertebrate retinas (3-6) two, GCAP1 and GCAP2, regulate visual signaling in all species by properly shaping the sensitivity and kinetics of rod and cone responses (7-10). Vertebrate rods and cones respond to light stimuli by closing cGMP-gated channels in their outer segments via phototransduction cascade-mediated hydrolysis of cGMP (reviewed in Refs. 11 and 12). Following the excitation, cGMP production by retinal membrane guanylyl cyclase (RetGC) (13-15) first becomes accelerated, to speed up the recovery and light adaptation of photoreceptors, and then decelerated again as photoreceptors recover from the excitation back to their dark-adapted state (7, 16). Negative Ca 2ϩ feedback regulates the activity of RetGC via its Ca 2ϩ sensor proteins, GCAPs, such that in the light, when cGMP channels are closed and the influx of Ca 2ϩ through the channels stops, GCAPs release Ca 2ϩ and convert into a Mg 2ϩ-liganded state that stimulates RetGC. Once the photoreceptors return to their dark-adapted state, when cGMP channels reopen and the influx of Ca 2ϩ resumes, GCAPs undergo the reverse, activatorto-inhibitor, transition, by replacing Mg 2ϩ in their EF-hands with Ca 2ϩ , and decelerate RetGC (reviewed in Refs. 2 and 12). Failure of RetGC to accelerate or decelerate cGMP production within the normal range of the intracellular free Ca 2ϩ alters light sensitivity and kinetics of rod and cone response to light (7-9, 16-18) and has been linked to various forms of retinal blindness in humans, such as Leber congenital amaurosis, dominant cone or cone-rod degenerations (reviewed in Ref. 19-22), and a recessive night blindness (23). Multiple mutations linked to these blinding disorders have been found in the genes coding for RetGC1 isozyme (GUCY2D) (19-27) and GCAP1 (GUCA1A) (28-40). GUCA1A mutations linked to the domi

The EMBO Journal, Apr 15, 1998

contributed equally to this work Calmodulin (CaM) controls the activity of the rod cGMP-gated ion... more contributed equally to this work Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of αand β-subunits, display a high CaM sensitivity (EC 50 ഛ5 nM) similar to the native channel. Using surface plasmon resonance spectroscopy, we identified two unconventional CaM-binding sites (CaM1 and CaM2), one in each of the N-and the C-terminal regions of the β-subunit. Ca 2⍣ cooperatively stimulates binding of CaM to these sites exactly within the range of [Ca 2⍣ ] occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects. We discuss different models to explain the high-affinity binding of CaM.

Frontiers in Molecular Neuroscience, 2012

The membrane guanylate cyclase family has been branched into three subfamilies: natriuretic pepti... more The membrane guanylate cyclase family has been branched into three subfamilies: natriuretic peptide hormone surface receptors, Ca 2+-modulated neuronal ROS-GC, and Ca 2+-modulated odorant surface receptor ONE-GC. The first subfamily is solely modulated by the extracellularly generated hormonal signals; the second, by the intracellularly generated sensory and sensory-linked signals; and the third, by combination of these two. The present study defines a new paradigm and a new mechanism of Ca 2+ signaling. (1) It demonstrates for the first time that ANF-RGC, the prototype member of the surface receptor subfamily, is stimulated by free [Ca 2+ ] i. The stimulation occurs via myristoylated form of neurocalcin δ, and both the guanylate cyclase and the calcium sensor neurocalcin δ are present in the glomerulosa region of the adrenal gland. (2) The EF-2, EF-3 and EF-4 hands of GCAP1 sense the progressive increment of [Ca 2+ ] i and with a K 1/2 of 100 nM turn ROS-GC1 "OFF." In total reversal, the same EF hands upon sensing the progressive increment of [Ca 2+ ] i with K 1/2 turn ONE-GC "ON." The findings suggest a universal Ca 2+-modulated signal transduction theme of the membrane guanylate cyclase family; demonstrate that signaling of ANF-RGC occurs by the peptide hormones and also by [Ca 2+ ] i signals; that for the Ca 2+ signal transduction, ANF-RGC functions as a two-component transduction system consisting of the Ca 2+ sensor neurocalcin δ and the transducer ANF-RGC; and that the neurocalcin δ in this case expands beyond its NCS family. Furthermore, the study shows a novel mechanism of the [Ca 2+ ] i sensor GCAP1 where it acts as an antithetical NCS for the signaling mechanisms of ROS-GC1 and ONE-GC.

Molecular BioSystems, Mar 20, 2014

Vertebrate visual phototransduction is perhaps the most well-studied G-protein signaling pathway.... more Vertebrate visual phototransduction is perhaps the most well-studied G-protein signaling pathway. A wealth of available biochemical and electrophysiological data has resulted in a rich history of mathematical modeling of the system. However, while the most comprehensive models have relied upon amphibian biochemical and electrophysiological data, modern research typically employs mammalian species, particularly mice, which exhibit significantly faster signaling dynamics. In this work, we present an adaptation of a previously published, comprehensive model of amphibian phototransduction that can produce quantitatively accurate simulations of the murine photoresponse. We demonstrate the ability of the model to predict responses to a wide range of stimuli and under a variety of mutant conditions. Finally, we employ the model to highlight a likely unknown mechanism related to the interaction between rhodopsin and rhodopsin kinase.

The EMBO Journal, Jul 1, 1994

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indic... more Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 gM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterine. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 gM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.

ACS Chemical Neuroscience, Mar 21, 2013

Calcium-signaling in cells requires a fine-tuned system of calcium-transport proteins involving i... more Calcium-signaling in cells requires a fine-tuned system of calcium-transport proteins involving ion channels, exchangers, and ion-pumps but also calcium-sensor proteins and their targets. Thus, control of physiological responses very often depends on incremental changes of the cytoplasmic calcium concentration, which are sensed by calcium-binding proteins and are further transmitted to specific target proteins. This Review will focus on calcium-signaling in vertebrate photoreceptor cells, where recent physiological and biochemical data indicate that a subset of neuronal calcium sensor proteins named guanylate cyclase-activating proteins (GCAPs) operate in a calcium-relay system, namely, to make gradual responses to small changes in calcium. We will further integrate this mechanism in an existing computational model of phototransduction showing that it is consistent and compatible with the dynamics that are characteristic for the precise operation of the phototransduction pathways.

International Journal of Molecular Sciences, Apr 5, 2022

PLOS ONE, Aug 2, 2011

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAP... more Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca 2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca 2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca 2+ around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca 2+ /Mg 2+-dependent conformational changes and in the Ca 2+-dependent monomer-dimer equilibrium. Direct Ca 2+-binding revealed that all zGCAPs bound at least three Ca 2+. The corresponding apparent affinity constants reflect binding of Ca 2+ with high (#100 nM), medium (0.1-5 mM) and/or low ($5 mM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca 2+-sensor system in zebrafish rod and cone cells supporting a Ca 2+-relay model of differential zGCAP operation in these cells.

Genes, Mar 2, 2021

Frontiers in Molecular Neuroscience, Jan 17, 2023

Cone photoreceptor cells of night-migratory songbirds seem to process the primary steps of two di... more Cone photoreceptor cells of night-migratory songbirds seem to process the primary steps of two different senses, vision and magnetoreception. The molecular basis of phototransduction is a prototypical G protein-coupled receptor pathway starting with the photoexcitation of rhodopsin or cone opsin thereby activating a heterotrimeric G protein named transducin. This interaction is well understood in vertebrate rod cells, but parameter describing protein-protein interactions of cone specific proteins are rare and not available for migratory birds. European robin is a model organism for studying the orientation of birds in the earth magnetic field. Recent findings showed a link between the putative magnetoreceptor cryptochrome 4a and the cone specific G-protein of European robin. In the present work, we investigated the interaction of European robin cone specific G protein and cytoplasmic regions of long wavelength opsin. We identified the second loop in opsin connecting transmembrane regions three and four as a critical binding interface. Surface plasmon resonance studies using a synthetic peptide representing the second cytoplasmic loop and purified G protein α-subunit showed a high affinity interaction with a K D value of 21 nM. Truncation of the G protein α-subunit at the C-terminus by six amino acids slightly decreased the affinity. Our results suggest that binding of the G protein to cryptochrome can compete with the interaction of G protein and non-photoexcited long wavelength opsin. Thus, the parallel presence of two different sensory pathways in bird cone photoreceptors is reasonable under dark-adapted conditions or during illumination with short wavelengths.

International Journal of Molecular Sciences, 2020

Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the reti... more Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-π interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well ...

Scientific Reports, Mar 31, 2017

Quantification of protein binding to membrane proteins is challenging and a limited set of method... more Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a freesolution label-free method with high sensitivity, to quantify the interaction of neuronal Ca 2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase-activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent K D-values of 663 ± 121 nM and 231 ± 63 nM for Ca 2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca 2+ levels. Photoreceptor cells efficiently respond to changing light conditions on a millisecond time scale by a well-balanced interplay of two second-messenger species, cGMP and calcium 1-3. Light excitation of the G protein-coupled receptor rhodopsin triggers a downstream signaling cascade leading to hydrolysis of cGMP, subsequently leading to a decrease of cytoplasmic Ca 2+-concentration [Ca 2+ ]. A network of Ca 2+-sensor proteins in rod and cone photoreceptor cells can detect subtle changes in intracellular [Ca 2+ ]. These Ca 2+-sensors precisely regulate the enzymatic activity of their targets 4-7 providing an efficient Ca 2+-mediated feedback loop to restore second messenger levels prior to illumination. Among them the guanylate cyclase-activating proteins (e.g. GCAP1 and GCAP2 in mammalians) control the synthesis of cGMP by sensory guanylate cyclases (GCs) in a Ca 2+-dependent manner and in a step-by-step Ca 2+-relay mechanism 4,6. GCAPs contain three functional and one non-functional EF-Hand type of Ca 2+-binding motifs and are Ca 2+-sensor proteins belonging to the family of neuronal Ca 2+-sensor (NCS) proteins 4-7. Mammalians express two or three, while teleost fish express six to eight different GCAP isoforms in their rod ancd cone cells 8 , which all regulate enzymatic activity of sensory GCs at different Ca 2+ levels 9. Synthesis of cGMP under control of cytoplasmic Ca 2+ contributes to the restoration of the dark adapted state and mediates light adaptation of photoreceptors 1-3. Mammalian rod and cone cells express two forms of a sensory GC, assigned as GC-E and GC-F (alternatively named ROS-GC1 or 2, retGC1 or 2). Both GCs form homo-dimers in disc membranes of rod and cone outer segments, but recent research was mainly focused on GC-E, probably for the following reasons: (i) the fraction of total GC-E exceeds that of GC-F by 25-fold in bovine 10 and 4-fold in mouse outer segments 11 ; (ii) various

Biophysical Chemistry, Oct 1, 2019

• Zebrafish cones were recorded in whole-cell employing pressure-polished pipettes. • GCAP overex... more • Zebrafish cones were recorded in whole-cell employing pressure-polished pipettes. • GCAP overexpression or knock-down was performed by injecting GCAP or its antibody. • Guanylate cyclase was already saturated with endogenous GCAP. • The antibody slowed down photoresponse recovery and reduced lightsensitive current. • Possibly, the antibody inhibited both the Ca 2+-regulated and the basal GC activity.

Cell Calcium, Mar 1, 2013