Synaptic co-release and VNUT, a not so “Vesicular” Nucleotide Transporter?

Synopsis

The sympathetic nervous system mediates the “fight or flight” side of our autonomic responses to daily life, and the nerve terminals in sympathetic nerves are the archetypal model of nerves that co-release multiple neurotransmitters to expand their range of their regulatory control. Despite the discovery of neurotransmitter co-release in the 1970s, the regulation of co-release at the molecular level is relatively poorly understood. Motivated by the sympathetic control of blood pressure in health and disease, we study the regulation of norepinephrine and ATP co-release in perivascular nerves, and the role of the relatively recently discovered Vesicular Nucleotide Transporter,  VNUT, in the filling of synaptic vesicles with ATP. We found that VNUT and its norepinephrine-transporting counterpart, the Vesicular Monoamine Transporter (VMAT), largely localize to separate pools of vesicles, which led us to attempt to identify co-markers of the pool of ATP-containing vesicles and to understand the regulation of trafficking of VNUT. Using a combination of tissue and cell culture models, immunolabelling studies suggest that VNUT largely anti-colocalizes with traditional vesicle markers. Bioinformatics analyses revealed that mammalian VNUT lacks typical motifs to be directed to synaptic vesicles, and complementary chimeric analyses of EGFP::VNUT fusion proteins indicates that the C-terminal of mammalian VNUT both contains an endoplasmic reticulum (ER) retention motif and lacks vesicle-targeting motifs. Using AlpahFol2, an AI-based protein structure predictor, we identified additional Golgi retention motifs in the N-terminal, cytosolic portion of VNUT. Given that diverse experiments show the VNUT is involved in the secretion of ATP from cells, , our novel insights on the trafficking of VNUT suggest a novel mechanism of vesicle cargo filling that occurs in early vesicle maturation.