A novel Ca2+-feedback mechanism extends the operating range of mammalian rods to brighter light

Sensory cells adjust their sensitivity to incoming signals, such as odor or light, in response to changes in background stimulation, thereby extending the range over which they operate. For instance, rod photoreceptors are extremely sensitive in darkness, so that they are able to detect individual photons, but remain responsive to visual stimuli under conditions of bright ambient light, which would be expected to saturate their response given the high gain of the rod transduction cascade in darkness. These photoreceptors regulate their sensitivity to light rapidly and reversibly in response to changes in ambient illumination, thereby avoiding saturation. Calcium ions (Ca²⁺) play a major role in mediating the rapid, subsecond adaptation to light, and the Ca²⁺-binding proteins GCAP1 and GCAP2 (or guanylyl cyclase–activating proteins [GCAPs]) have been identified as important mediators of the photoreceptor response to changes in intracellular Ca²⁺. However, mouse rods lacking both GCAP1 and GCAP2 (GCAP^−/−) still show substantial light adaptation. Here, we determined the Ca²⁺ dependency of this residual light adaptation and, by combining pharmacological, genetic, and electrophysiological tools, showed that an unknown Ca²⁺-dependent mechanism contributes to light adaptation in GCAP^−/− mouse rods. We found that mimicking the light-induced decrease in intracellular [Ca²⁺] accelerated recovery of the response to visual stimuli and caused a fourfold decrease of sensitivity in GCAP^−/− rods. About half of this Ca²⁺-dependent regulation of sensitivity could be attributed to the recoverin-mediated pathway, whereas half of it was caused by the unknown mechanism. Furthermore, our data demonstrate that the feedback mechanisms regulating the sensitivity of mammalian rods on the second and subsecond time scales are all Ca²⁺ dependent and that, unlike salamander rods, Ca²⁺-independent background-induced acceleration of flash response kinetics is rather weak in mouse rods.     

Calcium-dependent control of temporal processing in an auditory interneuron: a computational analysis

Sensitivity to acoustic amplitude modulation in crickets differs between species and depends on carrier frequency (e.g., calling song vs. bat-ultrasound bands). Using computational tools, we explore how Ca²⁺-dependent mechanisms underlying selective attention can contribute to such differences in amplitude modulation sensitivity. For omega neuron 1 (ON1), selective attention is mediated by Ca²⁺-dependent feedback: [Ca²⁺]_internal increases with excitation, activating a Ca²⁺-dependent after-hyperpolarizing current. We propose that Ca²⁺ removal rate and the size of the after-hyperpolarizing current can determine ON1’s temporal modulation transfer function (TMTF). This is tested using a conductance-based simulation calibrated to responses in vivo. The model shows that parameter values that simulate responses to single pulses are sufficient in simulating responses to modulated stimuli: no special modulation-sensitive mechanisms are necessary, as high and low-pass portions of the TMTF are due to Ca²⁺-dependent spike frequency adaptation and post-synaptic potential depression, respectively. Furthermore, variance in the two biophysical parameters is sufficient to produce TMTFs of varying bandwidth, shifting amplitude modulation sensitivity like that in different species and in response to different carrier frequencies. Thus, the hypothesis that the size of after-hyperpolarizing current and the rate of Ca²⁺ removal can affect amplitude modulation sensitivity is computationally validated.     

Plasma membrane calcium ATPase isoform 3 expression in single cells isolated from rat liver

Photon absorption by photoreceptors activates hydrolysis of cGMP, which shuts down cGMP-gated channels and decreases free Ca²⁺ concentrations in outer segment. Suppression of Ca²⁺ influx through the cGMP channel by light activates retinal guanylyl cyclase through guanylyl cyclase activating proteins (GCAPs) and thus expedites photoreceptors recovery from excitation and restores their light sensitivity. GCAP1 and GCAP2, two ubiquitous among vertebrate species isoforms of GCAPs that activate retGC during rod response to light, are myristoylated Ca²⁺/Mg²⁺-binding proteins of the EF-hand superfamily. They consist of one non-metal binding EF-hand-like domain and three other EF-hands, each capable of binding Ca²⁺ and Mg²⁺. In the metal binding EF-hands of GCAP1, different point mutations can selectively block binding of Ca²⁺ or both Ca²⁺ and Mg²⁺ altogether. Activation of retGC at low Ca²⁺ (light adaptation) or its inhibition at high Ca²⁺ (dark adaptation) follows a cycle of Ca²⁺/Mg²⁺ exchange in GCAPs, rather than release of Ca²⁺ and its binding by apo-GCAPs. The Mg²⁺ binding in two of the EF-hands controls docking of GCAP1 with retGC1 in the conditions of light adaptation and is essential for activation of retGC. Mg²⁺ binding in a C-terminal EF-hand contributes to neither retGC1 docking with the cyclase nor its subsequent activation in the light, but is specifically required for switching the cyclase off in the conditions of dark adaptation by binding Ca²⁺. The Mg²⁺/Ca²⁺ exchange in GCAP1 and 2 operates within different range of intracellular Ca²⁺ concentrations and provides a two-step activation of the cyclase during rod recovery.     

New insights into the fatty acid-binding protein (FABP) family in the small intestine

Cyclic GMP is essential for the ability of rods and cones to respond to the light stimuli. Light triggers hydrolysis of cGMP and stops the influx of sodium and calcium through the cGMP-gated ion channels. The consequence of this event is 2-fold: first, the decrease in the inward sodium current plays the major role in an abrupt hyperpolarization of the cellular membrane; secondly, the decrease in the Ca²⁺ influx diminishes the free intracellular Ca²⁺ concentration. While the former constitutes the essence of the phototransduction pathway in rods and cones, the latter gives rise to a potent feedback mechanism that accelerates photoreceptor recovery and adaptation to background light. One of the most important events by which Ca²⁺ feedback controls recovery and light adaptation is synthesis of cGMP by guanylyl cyclase. Two isozymes of membrane photoreceptor guanylyl cyclase (retGC) have been identified in rods and cones that are regulated by Ca²⁺-binding proteins, GCAPs. At low intracellular concentrations of Ca²⁺ typical for light-adapted rods and cones GCAPs activate RetGC, but concentrations above 500 nM typical for dark-adapted photoreceptors turn them into inhibitors of retGC. A variety of mutations found in GCAP and retGC genes have been linked to several forms of human congenital retinal diseases, such as dominant cone degeneration, cone-rod dystrophy and Leber congenital amaurosis.     

Rhodopsin kinase and recoverin modulate phosphodiesterase during mouse photoreceptor light adaptation

Light stimulates rhodopsin in a retinal rod to activate the G protein transducin, which binds to phosphodiesterase (PDE), relieving PDE inhibition and decreasing guanosine 3′,5′-cyclic monophosphate (cGMP) concentration. The decrease in cGMP closes outer segment channels, producing the rod electrical response. Prolonged exposure to light decreases sensitivity and accelerates response kinetics in a process known as light adaptation, mediated at least in part by a decrease in outer segment Ca²⁺. Recent evidence indicates that one of the mechanisms of adaptation in mammalian rods is down-regulation of PDE. To investigate the effect of light and a possible role of rhodopsin kinase (G protein–coupled receptor kinase 1 [GRK1]) and the GRK1-regulating protein recoverin on PDE modulation, we used transgenic mice with decreased expression of GTPase-accelerating proteins (GAPs) and, consequently, a less rapid decay of the light response. This slowed decay made the effects of genetic manipulation of GRK1 and recoverin easier to observe and interpret. We monitored the decay of the light response and of light-activated PDE by measuring the exponential response decay time (τ_REC) and the limiting time constant (τ_D), the latter of which directly reflects light-activated PDE decay under the conditions of our experiments. We found that, in GAP-underexpressing rods, steady background light decreased both τ_REC and τ_D, and the decrease in τ_D was nearly linear with the decrease in amplitude of the outer segment current. Background light had little effect on τ_REC or τ_D if the gene for recoverin was deleted. Moreover, in GAP-underexpressing rods, increased GRK1 expression or deletion of recoverin produced large and highly significant accelerations of τ_REC and τ_D. The simplest explanation of our results is that Ca²⁺-dependent regulation of GRK1 by recoverin modulates the decay of light-activated PDE, and that this modulation is responsible for acceleration of response decay and the increase in temporal resolution of rods in background light.     

Effects of Ca2+ and calmodulin on adenylyl cyclase activity in sheep olfactory epithelium

Sheep olfactory epithelium contains an adenylyl cyclase which is stimulated by many but not all odorants. Here we report that this enzyme is activated by calmodulin in a dose-dependent manner, and that calcium ions are required for this response. Odorant stimulation of adenylyl cyclase is unaffected by the complex Ca²⁺/calmodulin, as suggested by the results obtained both in Ca²⁺/calmodulin-depleted membranes and under calmodulin antagonist treatment; this confirms the prediction that the Ca²⁺ binding protein and odorants stimulate the olfactory adenylyl cyclase through parallel mechanisms. The persistent activation of the regulatory component of adenylyl cyclase by GppNHp does not alter the response of the enzyme to either odorant or Ca²⁺/calmodulin. In sheep olfactory epithelium a cAMP-phosphodiesterase activity is also present, which is highly inhibited by IBMX and aminophylline, searcely by RO 20-1724, and unaffected by Ca²⁺/calmodulin. The modulatory role exerted by calcium on cAMP system in sheep olfactory signal transduction is discussed.     

NOS Inhibition Enhances Myogenic Tone by Increasing Rho-Kinase Mediated Ca2+ Sensitivity in the Male but Not the Female Gerbil Spiral Modiolar Artery

Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-N^G-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca²⁺ concentration and/or the Ca²⁺ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca²⁺, and Ca²⁺ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K⁺ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca²⁺ concentration in smooth muscle cells but increased the Ca²⁺ sensitivity. This increase in the Ca²⁺ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca²⁺ sensitivity and abolished the LNNA-induced increase in Ca²⁺ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca²⁺ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca²⁺ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.     

Duck Pancreatic Acinar Cell as a Unique Model for Independent Cholinergic Stimulation–Secretion Coupling

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 μM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose–response curve. ACh dose dependently induced increases in cytosolic Ca²⁺ concentration ([Ca²⁺] _c ), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 μM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca²⁺] _c increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.     

The presence of membranes or micelles induces structural changes of the myristoylated guanylate-cyclase activating protein-2

Guanylate cyclase-activating proteins (GCAPs) are neuronal Ca²⁺ sensors that play a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase. GCAPs are N-terminally myristoylated, and the role of the myristoyl moiety is not yet fully understood. While protein lipid chains typically represent membrane anchors, the crystal structure of GCAP-1 showed that the myristoyl chain of the protein is completely buried within a hydrophobic pocket of the protein, which stabilizes the protein structure. Therefore, we address the question of the localization of the myristoyl group of GCAP-2 in the absence and in the presence of lipid membranes as well as DPC detergents (as a membrane substitute amenable to solution state NMR). We investigate membrane binding of both myristoylated and nonmyristoylated GCAP-2 and study the structure and dynamics of the myristoyl moiety of GCAP-2 in the presence of POPC membranes. Further, we address structural alterations within the myristoylated N-terminus of GCAP-2 in the presence of membrane mimetics. Our results suggest that upon membrane binding the myristoyl group is released from the protein interior and inserts into the lipid bilayer.     

Adaptive potentiation in rod photoreceptors after light exposure

Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After exposure to periods of saturating illumination, rods show a 10–35% increase in circulating dark current, an adaptive potentiation (AP) to light exposure. This potentiation grows as exposure to light is extended up to 3 min and decreases with longer exposures. Cells return to their initial dark-adapted sensitivity with a time constant of recovery of ∼7 s. Halving the extracellular Mg concentration prolongs the adaptation, increasing the time constant of recovery to 13.3 s, but does not affect the magnitude of potentiation. In rods lacking guanylate cyclase activating proteins 1 and 2 (GCAP^−/−), AP is more than doubled compared with WT rods, and halving the extracellular Mg concentration does not affect the recovery time constant. Rods from a mouse expressing cyclic nucleotide–gated channels incapable of binding calmodulin also showed a marked increase in the amplitude of AP. Application of an insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitor (Tyrphostin AG1024) blocked AP, whereas application of an insulin receptor kinase inhibitor (HNMPA(AM)₃) failed to do so. A broad-acting tyrosine phosphatase inhibitor (orthovanadate) also blocked AP. Our findings identify a unique form of adaptation in photoreceptors, so that they show transient hypersensitivity to light, and are consistent with a model in which light history, acting via the IGF-1R, can increase the sensitivity of rod photoreceptors, whereas the photocurrent overshoot is regulated by Ca-calmodulin and Ca²⁺/Mg²⁺-sensitive GCAPs.     

Intracellular mechanisms of adaptation and self-regulation in self-organizing networks: The role of chemical transducers

This paper describes mechanisms of intracellular and intercellular adaptation that are due to spatial or temporal factors. The spatial mechanisms support self-regulating pattern formation that is capable of directing self-organization in a large class of systems, including examples of directed intercellular growth, transmitter production, and intracellular conductance changes. A balance between intracellular flows and counterflows causes adaptation. This balance can be shifted by environmental inputs. The decrease in Ca²⁺-modulated outward K⁺ conductance in certain molluscan nerve cells is a likely example. Examples wherein Ca²⁺ acts as a second messenger that shunts receptor sensitivity can also be discussed from this perspective. The systems differ in basic ways from recent diffusion models. Chemical transducers driven by membrane-bound intracellular signals can establish long-range intercellular interactions that compensate for variable intercellular distances and are invariant under developmental size changes; diffusional signals do not. The intracellular adaptational mechanisms are formally analogous to intercellular mechanisms that include cellular properties which are omitted in recent reaction-diffusion models of pattern formation. The cellular models use these properties to compute size-invariant properties despite wide variations in their intercellular signals. Mechanisms of temporal adaptation can be derived from the simplest laws of chemical transduction by using a correspondence principle. These mechanisms lead to such properties of intercellular signals as transient overshoot, antagonistic rebound, and an inverted U in sensitivity as intracellular signals or adaptation levels shift. Such effects are implicated in studies of behavioral, reinforcement, motor control, and cognitive coding. Supported in part by the National Science Foundation (NSF MCS 77-02958).     

Regulation of Ca<Superscript>2+</Superscript> influx in proliferating and differentiating murine myoblasts with participation of L-type Ca<Superscript>2+</Superscript> channels

The basic mechanisms of regulation of Ca²⁺ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca²⁺ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca²⁺ through these channels is regulated by the adrenergic system. The influx of Ca²⁺ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca²⁺ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca²⁺ influx is substantially lower than in proliferating cells, and maximal influx of Ca²⁺ may be reached only upon exhaustion of reticular stocks.     

PCNA Up-Regulation in Cerebral Vessels with Angiotensin II-Hypertension: Abnormal Regulation of Ca2+ Channel and Nitrate Tolerance Associated with Alternative Splicing of cGKI

We studied the responses of the basilar arteries from control rats and from rats infused with angiotensin II (Ang; 240 μg/kg/h × 4 weeks), which were hypertensive (137 ± 13 vs 205 ± 10 mm Hg). Ang-hypertensive rats (AHR) showed significant up-regulation of the expression of proliferative cell nuclear antigen (PCNA) (PCNA index, 0.65) in vascular smooth muscle cell (VSMC) layers. Both hypertension and PCNA up-regulation were absent in animals co-treated with the hydrophilic dihydropyridine Ca²⁺ channel blocker amlodipine (100 mg/liter in the drinking water). Quantitative patch-clamp analysis of freshly isolated VSMC showed a significant increase in L-type Ca²⁺ channel currents in AHR that was attributed to an increase in the open channel probability, with no change in other biophysical properties, pharmacological characteristics, or in the channel expression. Compared with controls, regulation of Ca²⁺ channels in AHR was abnormal, with nitrate tolerance manifested as a reduction in down-regulation of the Ca²⁺ channel activity in response to the NO donor Na nitroprusside. A diminished sensitivity to 8-Br-cGMP was also observed, consistent with a mechanism downstream of soluble guanylyl cyclase. The nitrate tolerance was found to be attributable to alternative splicing of cGKI, with a decrease in the cGKIalpha expression and an increase in the cGKIbeta expression, with the latter known to be less sensitive to activation by cGMP and related analogs. We conclude that the increase in the proliferative response of VSMC in AHR is associated with an abnormal response to NO by VSMC due to alternative splicing of cGKI, resulting in an increase in the Ca²⁺ channel activation and up-regulation of the pro-proliferative Ca²⁺-sensitive gene for PCNA.     

Ca2+sensitivity and caffeine-induced changes in skinned cardiac muscle fibers of the carp,Cyprinus carpio

 Ca²⁺ sensitivity and caffeine-induced sensitivity changes in skinned carp heart fibers were compared with those of guinea pig and rat heart. The Ca²⁺ concentration-response curves of saponin-treated left atrial skinned fibers obtained from guinea pig and rat were almost identical. Doses of 5 and 20 mmol ⋅ l^-1 caffeine shifted this curve to the left. However, when a relatively high concentration (50 mmol ⋅ l^-1) of caffeine was used, the left-ward shift was reduced. Caffeine reduced the peak of the Ca²⁺ concentration-response curve. The Ca²⁺ concentration-response curve of carp atrial skinned fiber is almost identical to that of guinea pig and rat. However, a further increase in Ca²⁺ sensitivity was observed even when 50 mmol ⋅ l^-1 caffeine was added. Similarly, a decrease in the response curve peak was also observed. Ca²⁺ sensitivity in ventricular skinned fibers obtained from carp was almost the same as that observed for the atrial, but the increase in Ca²⁺ sensitivity due to caffeine was larger. In addition, a further increase was also observed when 50 mmol ⋅ l^-1 caffeine was added. These results indicate that the Ca²⁺ sensitivity of contractile proteins in atrial muscles from carp heart is the same as that of guinea pig and rat. It is, however, assumed that there are some differences in properties in the contractile proteins. It is also assumed that there are some differences between the atrial and ventricular muscles of carp heart. Accepted: 17 May 1996     

Backbone 1H, 13C, and 15N resonance assignments of guanylyl cyclase activating protein-1, GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca²⁺-dependent activation of retinal guanylyl cyclase that regulates the visual light response. GCAP1 is genetically linked to retinal degenerative diseases. We report backbone NMR chemical shift assignments of Ca²⁺-saturated GCAP1 (BMRB no. 18026).     

Enhanced sensitivity to stimulation of sodium transport and cyclic AMP by antidiuretic hormone after Ca2+ depletion of isolated frog skin epithelium

Summary The role of Ca²⁺ in the stimulation by antidiuretic hormone (ADH) of active sodium transport across the isolated epithelium of frog skin was investigated. This has been done by bathing the blood side with Ca²⁺-free solution containing 0.1mm EGTA. This Ca²⁺ depletion halved the resistance but had no significant effect on the short-circuit current (SCC). The sensitivity of both cAMP- and SCC-stimulation to ADH was increased 40-fold by Ca²⁺ depletion. Sensitivity to stimulation by theophylline was only changed a little, while stimulation by exogenous cAMP was completely unaltered. The increase in sensitivity to ADH was dependent on the duration of preincubation in Ca²⁺-free solution, which indicates that a slowly exchanging Ca²⁺ pool is involved in the determination of sensitivity to ADH. We suggest this pool is of cellular origin and the increased sensitivity is due to the decrease of a Ca²⁺ inhibition of the ADH-stimulated adenylate cyclase. But a direct effect of Ca²⁺ on binding of ADH to the receptor cannot be excluded. Our results are not compatible with the hypothesis that entry of extracellular Ca²⁺ is an obligatory step in the natriferic action of ADH, although it may be so in the hydroosmotic action of ADH. We also found the maximal response to ADH to be higher after Ca²⁺ depletion. This is in agreement with the hypothesis of intracellular Ca²⁺ as a modulator of the sodium permeability of the outward-facing membrane.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Regulation of Calmodulin- and Dopamine-Stimulated Adenylate Cyclase Activities by Light in Bovine Retina

Abstract: Neural retina from most species contains 3,4-dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca²⁺-binding protein, cal-modulin, and that calmodulin can increase dopamine-sen-sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca²⁺ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua-nosine-5′-(β,γ-imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn²⁺-, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down-regulation of dopamine receptor activity. The down-regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.     

Functional EF-Hands in Neuronal Calcium Sensor GCAP2 Determine Its Phosphorylation State and Subcellular Distribution In Vivo,and Are Essential for Photoreceptor Cell Integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca²⁺-free and Ca²⁺-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca²⁺ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca²⁺. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF⁻GCAP2), we show that GCAP2 locked in its Ca²⁺-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca²⁺-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF⁻GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca²⁺-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca²⁺ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in “equivalent-light” scenarios.     

Calcium-induced modulation of synaptic transmission

Calcium (Ca²⁺) is a second messenger regulating a wide variety of intracellular processes. Using GABA-and glycinergic synapses as examples, this review analyzes two functions of this unique ion: postsynaptic Ca²⁺-dependent modulation of receptor-operated channels and Ca²⁺-induced retrograde regulation of neurotransmitter release from the presynaptic terminals. Phosphorylation, rapid Ca²⁺-induced modulation via intermediate Ca²⁺-binding proteins, and changes in the number of functional receptors represent the main pathways of short-and long-term plasticity of postsynaptic receptor-operated channel machinery. Retrograde signaling is an example of synaptic modulation triggered by stimulation of postsynaptic cells and mediated via regulation of presynaptic neurotransmitter release. This mechanism provides postsynaptic neurons with efficient tools to control the presynaptic afferents in an activity-dependent mode. Elevation of intracellular Ca²⁺ in a postsynaptic neuron triggers the synthesis of endocannabinoids (derivatives of arachidonic acid). Their retrograde diffusion through the synaptic cleft and consequent activation of presynaptic G-protein coupled to CB1 receptors inhibits the release of neurotransmitter. These mechanisms of double modulation, which include control over the function of postsynaptic ion channels and retrograde suppression of the release machinery, play an important role in Ca²⁺-dependent control of the main excitatory and inhibitory synaptic pathways in the mammalian nervous system.