Connexin30.2:<i>In vitro</i> interaction with connexin36 in hela cells and expression in AII amacrine cells and intrinsically photosensitive ganglion cells in the mouse retina

Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36 – both expressed in AII amacrine cells – are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners

Tamoxifen and raloxifene modulate gap junction coupling during early phases of retinoic acid-dependent neuronal differentiation of NTera2/D1 cells

Gap junctions (GJ) represent a cellular communication system known to influence neuronal differentiation and survival. To assess a putative role of this system for neural effects of tamoxifen (TAM) and raloxifene (RAL), we used the human teratocarcinoma cell line NTera2/D1, retinoic acid (RA)-dependent neuronal differentiation of which is regulated by gap junctions formed of connexin43 (Cx43). As demonstrated by Western blot analysis, concentrations above 1 µmol/l for TAM, and 0.1 µmol/l for RAL lead to a temporary time- and concentration-dependent increase in Cx43 immunoreactivity, which reached a peak for TAM after 1 day and for RAL after 2 days. Immunocytochemical stainings revealed the increase in Cx43 immunoreactivity to result from an accumulation in intracellular compartments such as the Golgi apparatus or lysosomes. In addition, TAM and RAL were able to prevent the RA-dependent decrease of Cx43 immunoreactivity in NTera2/D1 cells, normally observed during neuronal differentiation. This suggested a suppression of neuronal differentiation to result from these substances. According to this, treatment of NTera2/D1 cells with 10 µmol/l TAM or RAL during weeks 1 and 2 of a 6 weeks RA-driven differentiation schedule impaired, whereas treatment during weeks 5 and 6 did not impair, neuronal differentiation of these cells. Modulation of GJ coupling between NTera2/D1 cells by TAM and RAL seems therefore to perturb early neuronal differentiation, whereas differentiated neurons in the mature brain seem to be not affected. These effects could be of importance for actions of TAM and RAL on early embryonic steps of nervous system formation

Significance of Input Correlations in Striatal Function

The striatum is the main input station of the basal ganglia and is strongly associated with motor and cognitive functions. Anatomical evidence suggests that individual striatal neurons are unlikely to share their inputs from the cortex. Using a biologically realistic large-scale network model of striatum and cortico-striatal projections, we provide a functional interpretation of the special anatomical structure of these projections. Specifically, we show that weak pairwise correlation within the pool of inputs to individual striatal neurons enhances the saliency of signal representation in the striatum. By contrast, correlations among the input pools of different striatal neurons render the signal representation less distinct from background activity. We suggest that for the network architecture of the striatum, there is a preferred cortico-striatal input configuration for optimal signal representation. It is further enhanced by the low-rate asynchronous background activity in striatum, supported by the balance between feedforward and feedback inhibitions in the striatal network. Thus, an appropriate combination of rates and correlations in the striatal input sets the stage for action selection presumably implemented in the basal ganglia

Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states

Connexin genes in the mouse and human genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes

Structural and functional diversity of connexin genes in the mouse and human genome

Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders

Three-Dimensional Reconstruction of a Calyx of Held and Its Postsynaptic Principal Neuron in the Medial Nucleus of the Trapezoid Body

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted ˜35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised ˜5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 µm2, and the nearest neighbors were separated, on average, by 0.59 µm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of ˜60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were anatomically docked. Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) ˜35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP