Karl-Wilhelm Koch

Frequenin—A novel calcium-binding protein that modulates synaptic efficacy in the drosophila nervous system

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

A 26 KD CALCIUM BINDING PROTEIN FROM BOVINE ROD OUTER SEGMENTS AS MODULATOR OF PHOTORECEPTOR GUANYLATE CYCLASE

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 (greater than 0.5 microM) increases when the free calcium concentration is lowered into the nanomolar range (less than 0.1 microM). 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 3‐ to 4‐fold increase of cyclase activity when the free calcium concentration was lowered in the physiological range from 0.5 microM 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.

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

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 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. 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 microM). 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.

Recoverin, a novel calcium-binding protein from vertebrate photoreceptors

Photoreceptor guanylyl cyclase activity is modulated by an endogenous calcium-binding protein called recoverin. A modified isolation procedure for recoverin using gel-filtration chromatography instead of a heat denaturation step is presented. The elution volume of recoverin corresponds to a monomer. Recoverin exhibits a calcium-dependent mobility shift in a native gel electrophoresis. Isoelectric focusing revealed a pI of 5.25. No subspecies of recoverin were detected.

Biochemical mechanism of light adaptation in vertebrate photoreceptors

Vertebrate photoreceptors can adjust their sensitivity to a wide range of light intensities spanning several orders of magnitude, the phenomenon of which is called light adaptation. Electrophysiological and biochemical studies have revealed that calcium can serve as an intracellular transmitter of light adaptation under the control of cGMP metabolism. After illumination, the cytoplasmic calcium concentration of a photoreceptor decreases, which in turn strongly activates photoreceptor guanylate cyclase. This calcium-dependent effect is mediated by a novel calcium-binding protein (recoverin) and leads to the restoration of the depleted cGMP pool after illumination.

Identification of a Domain in Guanylyl Cyclase-activating Protein 1 That Interacts with a Complex of Guanylyl Cyclase and Tubulin in Photoreceptors

The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73–Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73–Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.

Mechanism of photoreception in vertebrate vision

Abstract A change in intracellular Ca 2+ is probably not required for excitation of photoreceptors by light; hence the Ca 2+ hypothesis, which has dominated the field for almost 15 years, needs to be modified. Instead, cGMP controls the light-regulated channels directly by binding cooperatively to a membrane receptor. These results make cGMP the likely candidate for the excitatory messenger. Ca 2+ probably modulates or adapts the responses to cGMP.

Phosphorylation of recoverin, the calcium-sensitive activator of photoreceptor guanylyl cyclase

Recoverin, a new calcium binding protein from bovine rod photoreceptor cells, activates guanylyl cyclase below a free calcium concentration of 200 nM. We show here that recoverin is phosphorylated by an endogeneous kinase and Mg‐ATP at the same decreased calcium concentration. The calcium‐dependent activation of guanylyl cyclase is enhanced in the presence of ATP. We suggest that phosphorylation of recoverin reinforces the stimulation of guanylyl cyclase at decreased calcium concentrations.

Purified retinal nitric oxide synthase enhances ADP-ribosylation of rod outer segment proteins

Nitric oxide synthase is present in different cell layers of vertebrate retina and seems to have neuromodulatory functions in the outer retina. The enzyme, when purified from a bovine retina extract, has an apparent molecular mass of 160 kDa and resembles the neuronal constitutive NOS type I with respect to Ca2+‐calmodulin sensitivity, K m value and inhibition by analogues of l‐arginine. Retinal NOS is present in a preparation of rod outer segments attached to parts of the inner segments, but not in pure outer segments. We describe the enhancement of specific ADP‐ribosylation of outer segment proteins by purified retinal NOS.

Control of photoreceptor proteins by Ca2

A decrease of cytoplasmic Ca(2+)-concentration in vertebrate photoreceptor cells after illumination is necessary for light adaptation. Although the mechanisms of adaptation is not completely understood, several Ca(2+)-dependent cellular processes have been discovered. Some involve calcium-binding proteins like recoverin, guanylyl cyclase-activating protein and calmodulin, and their target proteins rhodopsin kinase, guanylyl cyclase, the cGMP-gated channel, and NO synthase. The activity of several enzymes or channels is directly controlled by Ca2+ and does not involve calcium-binding proteins. These proteins are pyrophosphatase, protein kinase C and the cGMP-gated channel.