No abstract

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in non-neuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxin 3A, 3B, 3C and 3D. We demonstrate here using analysis of EST databases, RT-PCR, in situ hybridization and Northern blot analysis that cells in the mouse retina only express syntaxin 3B. In contrast non-neuronal tissues, such as kidney express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP-25 compared to that of syntaxin1A. Using an in vitro fusion assay we could demonstrate that vesicles containing syntaxin 3B and SNAP-25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) PCR and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

Nitric oxide (NO) is a potent cell‐signaling molecule that plays important roles in diverse range biological processes within various tissues. Almost in every cell of our body, NO is generated by certain enzymes called nitric oxide synthases (NOS). NO is synthesized constitutively in nervous systems and endothelial cells, whereas in some other cells NO production is induced. Excessive NO production is linked to carcinogenesis, septic shock, asthma, stroke, etc. On the other hand, insufficient NO is involved with hypertension, impotence, arteriosclerosis, etc. So, there are dual roles for NO molecule at different tissue level. Since three isoforms of NOS responsible for NO production, an isoform‐specific inhibitor(s) needs to be designed that would not interfere with another NOS. In this regard, we have employed BD‐Clontech made Yeast Two‐Hybrid System to identify the putative proteins that would specifically bind iNOS protein for regulation. An N‐terminal iNOS‐bait protein has been utilized to identify a mammalian cDNA library. Employing all sorts of negative and positive controls necessary to interpret Yeast Two‐hybrid data, we could identify a number protein that shows possible protein‐protein interaction with iNOS. The probable role of these proteins in the regulation or modulation of iNOS function will be discussed.

No abstract