Zhang, X (reprint author), Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Neurosci, 320 Yue Yang Rd, Shanghai 200031, Peoples R China,firstname.lastname@example.org
Stimulus-induced exocytosis of large dense-core vesicles (LDCVs) leads to discharge of neuropeptides and fusion of LDCV membranes with the plasma membrane. However, the contribution of LDCVs to the properties of the neuronal membrane remains largely unclear. The present study found that LDCVs were associated with multiple receptors, channels and signaling molecules, suggesting that neuronal sensitivity is modulated by an LDCV-mediated mechanism. Liquid chromatography-mass spectrometry combined with immunoblotting of subcellular fractions identified 298 proteins in LDCV membranes purified from the dorsal spinal cord, including G-protein-coupled receptors, G-proteins and other signaling molecules, ion channels and trafficking-related proteins. Morphological assays showed that delta-opioid receptor 1 (DOR1), beta 2 adrenergic receptor (AR), G(alpha i2), voltage-gated calcium channel alpha 2 delta 1 subunit and P2X purinoceptor 2 were localized in substance P (SP)-positive LDCVs in small-diameter dorsal root ganglion neurons, whereas beta 1 AR, Wnt receptor frizzled 8 and dishevelled 1 were present in SP-negative LDCVs. Furthermore, DOR1/G(alpha i2)/G(beta 1 gamma 5)/phospholipase C beta 2 complexes were associated with LDCVs. Blockade of the DOR1/G(alpha i2) interaction largely abolished the LDCV localization of G(alpha i2) and impaired stimulation-induced surface expression of G(alpha i2). Thus, LDCVs serve as carriers of receptors, ion channels and preassembled receptor signaling complexes, enabling a rapid, activity-dependent modulation of neuronal sensitivity.