The phosphorylation state of the retinal ribbon synapse t-SNARE syntaxin3B is regulated by neuronal activity.

Abstract

Purpose : Synaptic vesicle exocytosis in conventional synapses requires the formation of SNARE complexes between syntaxin1, SNAP-25 and synaptobrevin/VAMP. Retinal ribbon synapses utilize a specialized isoform of syntaxin, syntaxin3B (STX3B). We have shown previously that STX3B is phosphorylated in vitro by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at threonine 14 (T14), facilitating its interaction with SNAP-25 (Liu et al., 2014). Here, we test the hypothesis that the phosphorylation of STX3B at T14 in vivo is regulated by neuronal activity in a Ca²⁺-dependent manner.

Methods : Phosphorylation of STX3B at T14 (pSTX3B) and total STX3B were quantified by immunofluorescence using antibodies raised against pSTX3 and STX3, respectively. Experiments were performed in 1) light- and dark-adapted mice, 2) mouse retinal eyecups that were light- or dark-adapted in the presence or absence of external Ca²⁺, and 3) acutely isolated mouse rod-bipolar cells (RBCs) in which resting intraterminal Ca²⁺ levels were experimentally manipulated.

Results : The terminals of RBCs in light-adapted mice showed a significant increase in pSTX3B relative to total STX3B when compared to RBC terminals in dark-adapted mice. Conversely, the ratio of pSTX3B to STX3B was significantly increased in rod photoreceptor terminals in dark-adapted mice relative to light-adapted mice. Similarly, when bathed in solution containing physiological Ca²⁺, RBC terminals in light-exposed eyecups showed an increase in the ratio of pSTX3B to STX3B compared to those in dark-exposed eyecups. In dark-exposed eyecups, rod photoreceptor terminals showed an increase in the ratio of pSTX3B relative to STX3B compared to those in light-exposed eyecups. In the absence of external Ca²⁺, the pSTX3B to STX3B ratio remained at basal levels in both cell types irrespective of illumination conditions. In acutely isolated RBCs, STX3B phosphorylation significantly increased in response to the elevation of intraterminal Ca²⁺.

Conclusions : These findings suggest that phosphorylation of STX3B at T14 occurs in vivo in response to stimulus-evoked Ca²⁺ entry. We propose a novel mechanism of retinal ribbon synapse function in which syntaxin3B is phosphorylated during on-going neurotransmitter release, facilitating the formation of new SNARE complexes and thus, the delivery of new vesicles to the fusion-competent state.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.