Abstract
Purpose::
In conventional synapses the interaction between the plasma membrane SNARE proteins syntaxin 1A, and SNAP-25 and the vesicular SNARE synaptobrevin/VAMP is thought to be essential for fast calcium-regulated synaptic vesicle exocytosis. In contrast to conventional synapses, ribbon synapses in the retina lack syntaxin 1A. Instead these synapses contain the related isoform syntaxin 3 (Morgans et al. J. Neurosci. (1996) 16, 6713-6721). Syntaxin 3 is also found in non-neuronal cells, where it has been implicated in transport vesicle trafficking. The syntaxin 3 gene codes for four different splice forms, syntaxin 3A, 3B 3C and 3D. Previous immunostaining studies have not distinguished between the different isoforms. We identified here the isoform of syntaxin 3 in ribbon synapses and studied its biochemical properties.
Methods::
Standard molecular biological and biochemical techniques were described previously (Belizaire et al., J. Comp. Neurol. (2004), 470, 266-281; Lazzell et al. JBC (2004) 279, 52124-52131).
Results::
We analyzed mouse EST databases and found that syntaxin 3B is the major isoform expressed in the eye, whereas non-neuronal tissues express syntaxin 3A. Using RT-PCR we confirmed that syntaxin 3B was expressed at high levels in the retina, very low levels in the brain and undetectable levels in non-neuronal tissue. In contrast to the syntaxin 3B expression profile, syntaxin 3A mRNA was expressed only at very low levels in the retina, but was strongly expressed in non-neuronal tissue. Using in situ hybridization, we demonstrated that syntaxin 3B is specifically expressed in the outer and inner nuclear layers of the retina, whereas syntaxin 3A mRNA was not detectable in the retina. These results demonstrate that ribbon synapses specifically contain the syntaxin 3B isoform.To compare the biochemical properties of the syntaxin isoforms in conventional and ribbon synapses, we studied the binding of different synaptic proteins from brain and retina extracts to immobilized syntaxin 3B or syntaxin 1A. We found that syntaxin 3B had a much lower binding affinity for SNAP-25 than syntaxin 1A.
Conclusions::
We show here that syntaxin 3B is the isofom expressed in ribbon synapses of the retina. The two major syntaxin isoforms (3A and 3B) have an identical N-terminus but differ in the C-terminus. Specifically, they differ in the C-terminal half of the H3 domain and the C-terminal transmembrane domain. These two domains are thought to be involved directly in the vesicle fusion reaction catalyzed by syntaxin. These sequence differences between the two isoforms indicate that syntaxin 3B has a specific role for synaptic vesicle exocytosis in ribbon synapses.
Keywords: synapse • retina