Localization of synaptic proteins involved in neurosecretion in different membrane microdomains.
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.
Taverna
Elena
E
CNR Institute of Neuroscience, Department of Medical Pharmacology, University of Milan, Milan, Italy.
Saba
Elena
E
Linetti
Anna
A
Longhi
Renato
R
Jeromin
Andreas
A
Righi
Marco
M
Clementi
Francesco
F
Rosa
Patrizia
P
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
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