VAMP4 Maintains a Ca²⁺-Sensitive Pool of Spontaneously Recycling Synaptic Vesicles

Abstract

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca 21 signaling pathways. Here, we probed the mechanisms that maintain Ca 21 sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca 21 -dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augments Ca 21 -sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release.

Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes

“…Furthermore, the previously observed reduction in asynchronous release ( 14 ) could be an indirect consequence of the increased Pr at VAMP4-depleted synapses, since synchronous and asynchronous releases are inversely correlated, due to sharing the same pool of releasable SVs ( 51 , 52 ). We do not observe any change in mEPSC frequency in VAMP4 KO slices, in contrast to previous observations ( 50 ). At present, we cannot provide a mechanistic explanation for these differences; however, it may reflect the experimental systems in which they were performed (shRNA knockdown in primary culture versus genomic deletion in intact neuronal circuits).…”

“…VAMP4 plays a role in two other calcium-dependent aspects of neurotransmission, asynchronous release ( 14 , 50 ) and the frequency of calcium-dependent mEPSCs ( 50 ). Asynchronous release performs important roles in certain forms of plasticity at specialized synapses but is not likely to be involved in the fast neuronal processing in the CNS, which instead is mediated by synchronous release ( 15 , 16 ).…”

SNARE Regulatory Proteins in Synaptic Vesicle Fusion and Recycling

“…Alternatively, n-Syb may be specialized for evoked SV release, with spontaneous fusion supported by c-Syb and other Drosophila v-SNAREs (Littleton, 2000). Although no other v-SNARE beyond c-Syb has been shown to function in SV fusion in Drosophila, multiple v-SNAREs support spontaneous and asynchronous SV release at mammalian synapses (Ramirez et al, 2012;Lin et al, 2020). Similar to Drosophila, C. elegans null mutations in the Syb2 homolog (snb-1) are embryonic lethal, but retain uncoordinated movements that indicate a low level of residual SV release (Nonet et al, 1998).…”

The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites

“…We used a KD strategy with shRNAs to suppress the expression of VAMP4, as done before in neurons (Lin et al, 2020;Nicholson-Fish et al, 2015;Raingo et al, 2012). We selected two different shRNAs: KD1, which targets the 3 0 UTR of VAMP4 mRNA, and KD2, which targets the coding sequence (see STAR Methods).…”