title: Peptidomics of a single identified neuron reveals diversity of multiple neuropeptides with convergent actions on cellular excitability.
abstract: In contrast to classical transmitters, the detailed structures and cellular and synaptic actions of neuropeptides are less well described. Peptide mass profiling of single identified neurons of the mollusc Lymnaea stagnalis indicated the presence of 17 abundant neuropeptides in the cardiorespiratory neuron, visceral dorsal 1 (VD1), and a subset of 14 peptides in its electrically coupled counterpart, right parietal dorsal 2. Altogether, based on this and previous work, we showed that the high number of peptides arises from the expression and processing of four distinct peptide precursor proteins, including a novel one. Second, we established a variety of posttranslational modifications of the generated peptides, including phosphorylation, disulphide linkage, glycosylation, hydroxylation, N-terminal pyroglutamylation, and C-terminal amidation. Specific synapses between VD1 and its muscle targets were formed, and their synaptic physiology was investigated. Whole-cell voltage-clamp analysis of dissociated heart muscle cells revealed, as tested for a selection of representative family members and their modifications, that the peptides of VD1 exhibit convergent activation of a high-voltage-activated Ca current. Moreover, the differentially glycosylated and hydroxylated alpha2 peptides were more potent than the unmodified alpha2 peptide in enhancing these currents. Together, this study is the first to demonstrate that single neurons exhibit such a complex pattern of peptide gene expression, precursor processing, and differential peptide modifications along with a remarkable degree of convergence of neuromodulatory actions. This study thus underscores the importance of a detailed mass spectrometric analysis of neuronal peptide content and peptide modifications related to neuromodulatory function.
authors.affiliation: Department of Molecular and Cellular Neurobiology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands. connie.jimenez@falw.vu.nl
authors.forename: Connie R
authors.initials: CR
authors.lastname: Jiménez
authors.forename: Sabine
authors.initials: S
authors.lastname: Spijker
authors.forename: Simone
authors.initials: S
authors.lastname: de Schipper
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Lodder
authors.forename: Cornelis Kees
authors.initials: CK
authors.lastname: Janse
authors.forename: Wijnand P M
authors.initials: WP
authors.lastname: Geraerts
authors.forename: Jan
authors.initials: J
authors.lastname: van Minnen
authors.forename: Naweed I
authors.initials: NI
authors.lastname: Syed
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: KaWan
authors.initials: K
authors.lastname: Li
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000502:physiology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002478:Cells, Cultured / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D018920:Coculture Techniques; D017952:Ganglia, Invertebrate / Q000166:cytology; D015870:Gene Expression; D006031:Glycosylation; D006900:Hydroxylation; D017136:Ion Transport / Q000187:drug effects; D008195:Lymnaea / Q000737:chemistry* / Q000166:cytology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D032383:Myocytes, Cardiac / Q000502:physiology; D009474:Neurons / Q000737:chemistry* / Q000502:physiology; D009479:Neuropeptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011498:Protein Precursors / Q000032:analysis; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D012333:RNA, Messenger / Q000032:analysis; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009479:Neuropeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; D011498:Protein Precursors; D012333:RNA, Messenger; C041307:small cardioactive peptide B