Surface traffic of dendritic Ca V1.2 calcium channels in hippocampal neurons

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 V1.2 L- L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca V1.2 L- 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 V1.2 L- in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca V1.2 L--SEP) demonstrated~20% recovery within 2 min without reappearance of clusters. Pulse-chase labeling showed that membrane-expressed Ca V1.2 L--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 V1.2 L-s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca V1.2 L-s show highly confined mobility with diffusion coefficients of~0.005μm 2 s -1. Consistent with the mobile Ca V1.2 L- 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 V1.2 L-s maintains stable calcium channel complexes involved in activitydependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations. © 2011 the authors.