title: Localization of synaptic proteins involved in neurosecretion in different membrane microdomains.
abstract: A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.
authors.affiliation: CNR Institute of Neuroscience, Department of Medical Pharmacology, University of Milan, Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Taverna
authors.forename: Elena
authors.initials: E
authors.lastname: Saba
authors.forename: Anna
authors.initials: A
authors.lastname: Linetti
authors.forename: Renato
authors.initials: R
authors.lastname: Longhi
authors.forename: Andreas
authors.initials: A
authors.lastname: Jeromin
authors.forename: Marco
authors.initials: M
authors.lastname: Righi
authors.forename: Francesco
authors.initials: F
authors.lastname: Clementi
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rosa
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051580:Neuronal Calcium-Sensor Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009479:Neuropeptides / Q000378:metabolism; D009489:Neurosecretion / Q000502:physiology*; D009490:Neurosecretory Systems / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D016716:PC12 Cells; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D001920:Bradykinin; D020864:Calcium Channels, N-Type; D009419:Nerve Tissue Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D017830:Octoxynol; D050600:SNARE Proteins; D047392:beta-Cyclodextrins; C101464:frequenin calcium sensor proteins; C108732:methyl-beta-cyclodextrin