Surface Traffic of Dendritic Ca_V1.2 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 ϳ20% 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 ϳ0.005 m 2 s Ϫ1 . Consistent with the mobile Ca V 1.2 fraction observed in FRAP, a ϳ30% 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 activitydependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.

Surface dynamics of voltage-gated ion channels

“…38 However, the use of the intracellular calcium transients induced by the opening of one, or small clusters of VGCC called sparklet, 136 as readout for channel localization over time is difficult, since the kinetic of intracellular calcium signals and dynamics of membrane proteins are very different. Direct measurement of calcium channel surface dynamics in the cell membrane has been made possible by well-defined fluorescent probes attached directly to the pore-forming subunit 40,64,119 or associated a2d-subunits. 96,147 Calcium channel dynamics differs along the neuronal membrane, but the majority of channels are confined in clusters along the membrane, as described for Ca V 1.2.…”

“…96,147 Calcium channel dynamics differs along the neuronal membrane, but the majority of channels are confined in clusters along the membrane, as described for Ca V 1.2. 40,64 In the axonal membrane, Ca V 2.1 and Ca V 2.2 channels are mobile, but confined in presynaptic-terminals. 119 Therefore, the contribution of channel mobility to calcium-mediated signaling is most likely a local variable within zones of confinement along dendrites and axons, 40,119 which are a few hundred nanometres in diameter.…”

“…As predicted by a modeling approach, activation of BK Ca channels could be dramatically altered by very small displacements of 10-50 nm. 35 Despite the numerous binding partners identified for BK Ca channels and VGCC, 36,50,85 the reported diffusion coefficients for both channels are in the range of 0.005-0.05 mm 2 /s 40,119,140 and could facilitate diffusion-driven displacements of BK Ca channels and VGCC for 10-30 nm within a physiologically relevant time interval of 10 ms. It should be noted however, that the ideas discussed here presume that action of individual VGCCs are independent from each other, as reported for the presynaptic-terminal of GABAergic neurons.…”

“…A dynamic distribution of SK channels has been not explored yet. Given that VGCC or NMDA receptors are mobile but confined in small clusters along the dendritic membrane, 40,60 one can assume that the availability of SK channels within the critical distance of 100-200 nm from VGCC might be achieved by lateral dynamics in subcellular compartments like synaptic spines.…”

“…84,109 Apart from this neuron-specific structure, most of the surface expressed voltage-gated ion channels are mobile within cellular membranes. 3,32,40,108,119,124 The existence of such surface mobility produces the potential to characterize molecular interactions of the observed molecule based merely on changes in its diffusion behavior. Given that kinetic properties of most ion channels depend not only on the pore-forming subunit, but to a large extent on the assembly and interaction with accessory subunits, one can assume that channel composition, and hence function, might be tuned by a dynamic assembly over time.…”