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Electrical coupling has been reported to occur only between homotypic retinal ganglion cells, in line with the concept of parallel processing in the early visual system. Here, however, we show reciprocal correlated firing between heterotypic ganglion cells in multielectrode array recordings during light stimulation in retinas of adult guinea pigs of either sex. Heterotypic coupling was further confirmed via tracer spread after intracellular injections of single cells with neurobiotin. Both electrically coupled cell types were sustained ON center ganglion cells but showed distinct light response properties and receptive field sizes. We identified one of the involved cell types as sustained ON α-ganglion cells. The presence of electrical coupling between heterotypic ganglion cells introduces a network motif in which the signals of distinct ganglion cell types are partially mixed at the output stage of the retina.SIGNIFICANCE STATEMENTThe visual information is split into parallel pathways, before it is sent to the brain via the output neurons of the retina, the ganglion cells. Ganglion cells can form electrical synapses between dendrites of neighboring cells in support of lateral information exchange. To date, ganglion-to-ganglion cell coupling is thought to occur only between cells of the same type. Here, however, we show that electrical coupling between different types of ganglion cells exists in the mammalian retina. We provide functional and anatomical evidence that two different types of ganglion cells share information via electrical coupling. This new network motif extends the impact of the heavily studied coding benefits of homotypic coupling to heterotypic coupling across parallel neuronal pathways.

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse.SIGNIFICANCE STATEMENTHorizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light responses of transient OFF-α retinal ganglion cells in a newly generated mouse line. In this mouse line, horizontal cell signals were no longer modulated by light. With light response recordings, we show that horizontal cells increase the dynamic range of retinal ganglion cells for contrast and temporal changes and contribute to the center/surround organization of their receptive fields.

By use of the quartz crystal microbalance technique, the interaction of the Raf-Ras binding domain (RafRBD) and the cysteine-rich domain Raf-C1 with lipids was quantified by using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf-C1 linked to the maltose binding protein and a His tag, termed MBP-Raf-C1, was used. Dissociation constants and rate constants of the association and dissociation were obtained for various 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) lipid mixtures. Independently of the phosphatidylserine (PS) content, the dissociation constants were in the order of 5x10(-7) M, while the on-rate constants were in the range of 2x10(3) (M s)(-1) and the off-rate constants in the range of 1x10(-3) s(-1). The maximum frequency shift increased significantly with increasing amounts of DMPS; this indicates that this negatively charged lipid is the primary binding site for MBP-Raf-C1. Exchange of DMPS for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) did not alter the thermodynamics and kinetics of protein binding, which implies that the protein interaction is mainly electrostatically driven. Scanning force microscopy (SFM) was employed to render protein adsorption visible and to confirm the assumption of a protein monolayer on the lipid layer. SFM images clearly revealed that the protein binds preferentially, but not solely, to negatively charged phosphatidylserine headgroups. We hypothesize that PS-enriched domains are initial binding sites with high affinity for Raf-C1, but that lateral interactions may account for protein domain growth.

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI3-kinase) in cultured hepatocytes. Ras activation was dependent on PI3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI3-kinase/Ras/Erk pathway in the choleretic effect of TUDC.

Cadmium is a carcinogen whose genotoxicity is only weak. Besides its tumor-initiating capacity, cadmium may be tumor-promoting, since it interferes with several steps of cellular signal transduction. We have investigated effects of cadmium(II) on protein kinase C (PKC), which is a key enzyme in the control of cellular growth and differentiation. Tumor-promoting phorbol esters cause an activation and translocation of PKC from the cytosol to the plasma membrane and to the nucleus of mammalian cells. In mouse 3T3/10 T 1/2 fibroblasts, cadmium(II) potentiated the effect of phorbol ester on nuclear binding and activation of PKC. Furthermore, in a reconstituted system consisting of rat liver nuclei and rat brain PKC, cadmium stimulated the binding of the enzyme to a 105-kDa protein. We propose a model in which cadmium(II) substitutes for zinc(II) in the regulatory domain of PKC, thus rendering the putative protein-protein binding site exposed. Further work is required to elucidate the potential role of the nuclear PKC binding protein(s) in the control of cell proliferation.

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ras is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras. AF6 complex in the millisecond range. As probed by 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.

GTP-binding proteins of the Ras subfamily act as molecular switches which regulate cellular proliferation and differentiation. They are switched “ON” by binding of GTP and “OFF” by the hydrolysis of GTP to GDP. Ras acts as an oncogene when mutated to a constitutively activated form which reflects its crucial role in the control of cell proliferation and differentiation. The interaction between the GTPase switch Ras and the downstream effector protein kinase c-Raf-1 represents a paradigm for the control of mitogenic signalling. Ras, when activated, binds to and activates its downstream effector, Raf, which in turn translates the GTP-dependent “ON” signal into protein phosphorylation. This signal is propagated within a setup of kinases (MEK, ERK) which is commonly referred to as the “MAP-kinase module”, finally leading to the induction of gene expression (reviewed in Block & Wittinghofer, 1995).

Within the sequence of protein kinase C (PKC), a Ca2+ and phospholipid dependent enzyme (reviewed in Nishizuka 1989), no conserved Ca2+ binding site could be identified and the enzyme does not bind Ca2+ as an isolated enzyme at physiological Ca2+ concentrations (Bazzi & Nelsestuen 1990). The primary structure of PKC reveals within the phorbol ester binding domain two “zinc finger like” consensus sequences as well as a number of highly conserved histidine and cysteine residues (Parker et al. 1986). Ono et al. (1989) demonstrated the necessity of putative Zn2+ ligands within the CX2CX13CX2C sequence for phorbol ester binding. Yet the functional consequences of metal binding to these structures or to other conserved potential ligands are poorly understood. Studies investigating the effects of divalent metal ions on PKC led to contradictory results, especially reporting activatory or inhibitory effects of zinc on the enzyme (Csermely et al. 1988, Speizer et al. 1989). Hence we developed a purification protocol which is suitable to characterize metal binding dependent properties of protein kinase C. Immobilized metal ion chromotography is suitable for preparative purification and subserves an analytical tool to evaluate surface accessible metal ion binding sites (Hemdan et al. 1989). The elucidation of metal binding properties of PKC may enhance our understanding of yet another mechanism regulating enzyme activation.

5-Aminolevulinate dehydratase from bovine liver requires Zn(II) for its activity and is inhibited by micromolecular concentrations of Pb(II). To elucidate the structure of the active site and its interactions between the active site and the metal binding site we labeled the active site for fluorescence studies and ESR spectroscopy. o-Phthalaldehyde reacted with active site lysyl and cysteinyl residues to form a fluorescent isoindole derivative. The fluorescence energy was independent of the deprivation of Zn(II) and of its substitution by the inhibitory Pb(II). For ESR-studies five iodoacetamide and four isothiocyanate pyrrolidine-N-oxyl derivatives with various spacer lengths were used to label the active site cysteinyl and lysyl residues, respectively. The ESR spectra of the modified enzyme preparations exhibited a significant immobilization of all labels, even with the longest spacers employed. Obviously the reactive cysteine is buried more than 12 A, and the active site lysine more than 11 A in a cleft of the enzyme structure. Zn(II) deprivation from the iodoacetamide spin-labeled enzyme caused a marked reversible increase in label mobility, whereas the Pb(II) substituted enzyme exhibited a smaller mobilization of the label. These results are interpreted by a model of the active site where the reactive cysteinyl and the lysyl side groups are close enough to be crosslinked by o-phthalaldehyde within a distance of 3 A. A structural role is assigned to Zn(II) in the enzyme, since Zn(II) deprivation does not alter the fluorescence of the isoindole derivative and increases the mobility of the cysteine-bound spin labels in the active site cleft.

The zinc coordination in 5-aminolevulinate dehydratase was investigated by extended x-ray absorption fine structure (EXAFS) associated with the zinc K-edge. The enzyme binds 8 mol of zinc/mol of octameric protein, but only four zinc ions seem sufficient for full activity. The authors have undertaken a study on four forms of the enzyme: (a) the eight-zinc native enzyme; (b) the enzyme with only the four zinc sites necessary for full activation occupied; (c) the enzyme with the vacant sites of (b) occupied by four lead ions; (d) the product complex between (b) and porphobilinogen. They have shown that two structurally distinct types of zinc sites are available in the enzyme. The site necessary for activity has an average zinc environment best described by two/three histidines and one/zero oxygen from a group such as tyrosine or a solvent molecule at 2.06 {plus minus} 0.02 {angstrom}, one tyrosine or aspartate at 1.91 {plus minus} 0.03 {angstrom}, and one cysteine sulfur at 2.32 {plus minus} 0.03 {angstrom} with a total coordination of five ligands. The unoccupied site in (b) is dominated by a single contribution of four cysteinyl sulfur atoms at 2.28 {plus minus} 0.02 {angstrom}. Spectra from samples (c) and (d) show onlymore » small changes from that of (b), reflecting a slight rearrangement of the ligands around the zinc atom.« less