title: Surface traffic of dendritic CaV1.2 calcium channels in hippocampal neurons.
abstract: In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal Ca(V)1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca(V)1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of Ca(V)1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca(V)1.2-SEP) demonstrated ∼20% recovery within 2 min without reappearance of clusters. Pulse-chase labeling showed that membrane-expressed Ca(V)1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered Ca(V)1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca(V)1.2s show highly confined mobility with diffusion coefficients of ∼0.005 μm² s⁻¹. Consistent with the mobile Ca(V)1.2 fraction observed in FRAP, a ∼30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic Ca(V)1.2s maintains stable calcium channel complexes involved in activity-dependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.
authors.affiliation: Department of Physiology and Medical Physics, Medical University of Innsbruck, 6020 Innsbruck, Austria and Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany.
authors.forename: Valentina
authors.initials: V
authors.lastname: Di Biase
authors.forename: Petronel
authors.initials: P
authors.lastname: Tuluc
authors.forename: Marta
authors.initials: M
authors.lastname: Campiglio
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Obermair
authors.forename: Martin
authors.initials: M
authors.lastname: Heine
authors.forename: Bernhard E
authors.initials: BE
authors.lastname: Flucher
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism*; D004058:Diffusion; D034281:Dynamins / Q000037:antagonists & inhibitors; D004622:Embryo, Mammalian; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism; D005260:Female; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D006835:Hydrazones / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020746:Calcium Channels, L-Type; D006835:Hydrazones; C419385:L-type calcium channel alpha(1C); C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D034281:Dynamins; D011188:Potassium