Abstract
Purpose: :
Syntaxins mediate synaptic vesicle fusion by interacting with the synaptic proteins SNAP-25 and synaptobrevin/VAMP via the so called SNARE domains. In conventional synapses, this function is subserved by syntaxin 1, which also regulates voltage-gated calcium channels. Retinal ribbon synapses lack syntaxin 1A, but contain the related isoform syntaxin 3 (Morgans et al. J. Neurosci. (1996) 16, 6713-6721). In previous studies, we found that mouse retinal ribbon synapses contain a specific spliceform of the syntaxin 3 gene called syntaxin 3B. As a first step towards assessing the potential roles of syntaxin 3B at a retinal ribbon synapse, we cloned syntaxin 3B from zebrafish (Danio rerio) and goldfish (Carassius auratus).
Methods: :
Zebrafish genome and EST databases were screened using the program tblastn (NCBI) with the mouse syntaxin 3B sequence and an EST clone derived from a retina cDNA library coding for zebrafish syntaxin 3B was identified. A bacterial expression construct coding for a fusion protein between glutathione sulfotransferase (GST) and the SNARE domain of the zebrafish syntaxin 3B was generated. The GST-syntaxin 3 fusion protein was expressed in E. coli, purified and coupled to Alexa 488. This construct was dialyzed into acutely dissociated goldfish Mb1 bipolar cell terminals via a patch pipette placed directly on the terminal and the time course of loading optically monitored.
Results: :
We sequenced the clone and compared the amino acid sequence of the encoded zebrafish syntaxin 3B with the mouse syntaxin 3B. The full length proteins were 75% identical, and the SNARE domains were 90% identical. Thus, syntaxin 3B is strongly conserved between mammals and fish. To verify the presence of syntaxin 3B in the goldfish retina, we designed PCR primers based on the syntaxin 3B zebrafish sequence and performed RT-PCR using mRNA from goldfish retina and brain. As in mouse, syntaxin 3B is expressed at high levels in goldfish retina relative to brain. The sequence of the goldfish syntaxin 3B protein was highly homologous to the zebrafish syntaxin 3B sequence (98 % identity). Loading of the zebrafish GST-syntaxin 3B SNARE domain coupled to Alexa 488 into isolated goldfish Mb bipolar cell terminals reached a steady state equilibrium with the internal solution within about two minutes, similar to that of GST-Alexa 488.
Conclusions: :
Syntaxin 3B is an evolutionary conserved gene product that is strongly expressed in the fish retina. The SNARE domain of syntaxin 3B can be recombinantly expressed and efficiently loaded into synaptic terminals of goldfish bipolar cells.
Keywords: synapse • retina