SNARE complex at the ribbon synapses of cochlear hair cells: analysis of synaptic vesicle‐ and synaptic membrane‐associated proteins

Abstract

Neurotransmitters are released via exocytosis of synaptic vesicles involving a fusion complex consisting of a set of highly conserved proteins, which form a multiprotein complex resulting in the docking of synaptic vesicles at the site of release. There are three major differences between cochlear hair cell synapses and CNS synapses: (i) hair cells have a specialized structure, the synaptic ribbon, to which synaptic vesicles are attached; (ii) hair cells can maintain high and sustained release of neurotransmitter; and (iii) hair cells lack synaptophysin and synapsin. These differences suggest that an unconventional mechanism of neurotransmitter release may be involved at ribbon synapses. In this study we used different and complementary approaches to determine whether or not ribbon-containing hair cells of the cochlea express any component of the core fusion complex found in conventional synapses. Syntaxin 1, the synaptic membrane synaptosome-associated protein (SNAP)-25 and vesicle-associated membrane protein (VAMP or synaptobrevin) were found to be present in the organ of Corti of both rat and guinea-pig, as shown by reverse transcription polymerase chain reaction and Western blotting. In situ hybridization and immunocytochemistry showed mRNA and protein expression, respectively, in both inner and outer hair cells. Synaptotagmins I and II, generally considered to play major roles in neurotransmitter release at central synapses, were not detected in the organ of Corti.

Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins

“…Attachment Protein] receptors) -SNAP-25, syntaxin-1, and synaptobrevin-1 -are expressed in IHCs 1,3 . In analogy to most neurosecretory preparations investigated so far 6 , it has been assumed that these neuronal SNAREs are also required for vesicle fusion at the IHC ribbon synapse, but this assumption has not yet been tested.…”

Maturation of Ribbon Synapses in Hair Cells Is Driven by Thyroid Hormone

“…Here, we used quantitative RT-PCR to explore whether and how the mRNA levels of synaptic proteins change in the wt organ of Corti from before (P6) to after (P14 -P17) the onset of hearing. The analysis included otoferlin [synaptic vesicle C 2 -domain protein (Roux et al, 2006) (Ravichandran et al, 1996)], synaptobrevin 1 [vesicle SNARE (Safieddine and Wenthold, 1999)], and the Ca V 1.3 L-type Ca 2ϩ channel (Platzer et al, 2000;Brandt et al, 2005). The relative RNA abundance of the individual genes differed to varying degrees among the tissues (differences were largest for RIBEYE and otoferlin mRNAs and smallest for SNAP25; data not shown).…”

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

“…The vesicle fusion step is believed to be mediated by the SNARE proteins synapbtobrevin 2, syntaxin 1A, and SNAP-25 on the vesicle and plasma membranes (Safieddine and Wenthold, 1999). However, SNAREs are not sensitive to calcium, and in reconstituted fusion assays, they mediate fusion on time scales that are orders of magnitude slower than observed in vivo (Weber et al, 1998).…”

“…In neurons, calcium sensitivity and fast kinetics are typically conferred by SNARE-binding proteins, including synaptotagmin I/II (Chapman, 2008). However, mature hair cells have little or no synaptotagmin I/II (Safieddine and Wenthold, 1999), and it has been proposed that the transmembrane protein otoferlin might serve as the calcium sensor that regulates hair cell neurotransmitter release by accelerating SNARE-mediated membrane fusion (Roux et al, 2006).…”