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The article presents a study examining the turnover and surface traffic of Cav1.2 calcium channels in the dendrites of mouse and rat hippocampal neurons through fluorescence recovery after photobleaching (FRAP) technique. The study found a major immobile and a minor mobile population of membrane-expressed Cav1.2 calcium channels in the dendrities. The study also recommends a model for the existence of the channels inside and outside of clusters in a dynamic equilibrium.

Auxiliary β subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca2+ channels in heterologous cells. In brain, all four β isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of β subunits and their role in membrane expression of Cav1.2 α1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous β subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged β subunits demonstrated that all β subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Cav1.2, thus indicating a great promiscuity in α1-β interactions. In contrast, restricted axonal targeting of β1 and weak colocalization of β4b with Cav1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Cav1.2 was strongly enhanced by all β subunits in an isoform-specific manner. Conversely, mutating the α-interaction domain of Cav1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a β subunit with the α-interaction domain is absolutely essential for membrane expression of α1 subunits, as well as for the subcellular localization of β subunits, which by themselves possess little or no targeting properties. [ABSTRACT FROM AUTHOR]

Sequences in the cytoplasmic II–III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Δ1 R756-L1139, Δ2 K737-A1001) including the synprint protein–protein interaction domain. Here we expressed green fluorescent protein (GFP)-α1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-α1B but divergent from the somatodendritic α1C-HA (CaV1.2) channel type, the splice variants GFP-α1B-Δ1 and GFP-α1B-Δ2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-α1B-Δ1 and significantly reduced for GFP-α1B-Δ2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II–III loop of α1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein–protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission. [ABSTRACT FROM AUTHOR]

In central nervous system neurons L-type Ca2+ channels are involved in developmental processes, the integration and conduction of postsynaptic electric activity and synaptic plasticity. However, little is known about the channel isoforms underlying each of these functions or about the exact localization and targeting properties of the major L-type channel isoform α1C (Cav1.2) in neurons. We addressed these questions using high-resolution immunofluorescence analysis of the endogenous α1C and epitope-tagged recombinant channel isoforms expressed in mouse hippocampal neurons. Endogenous α1C and surface-expressed hemagglutinin (HA)tagged α1C-HA were localized in small clusters distributed between the axon initial segment and the apical branches of the dendritic tree. The average cluster size was estimated to be eight channels per α1C-HA cluster. Analysis of the subcellular localization of α1C-HA clusters relative to known synaptic markers suggested the existence of two distinct populations of α1C clusters, extrasynaptic and synaptic, the latter associated with glutamatergic synapses in dendritic spines. Both glutamatergic and GABAergic neurons expressed α1C in the soma and dendrites. In contrast to the N-type channel GFP-α1B, GFP-α1C was excluded from distal axons and nerve terminals of mature neurons. In developing neurons, however, α1C and α1C-HA were robustly expressed in the growth cone, indicating that specific targeting properties of neuronal compartments change during differentiation. Synaptic and extrasynaptic localizations of α1C correspond to putative roles of L-type Ca2+ currents in synaptic modulation and in the propagation of dendritic Ca2+ spikes, respectively. The transiently expressed α1C in the growth cone may be involved in neurite extension and axonal pathfinding. [ABSTRACT FROM AUTHOR]

Abstract In the central nervous system small conductance Ca2+ -activated K+ (SK) channels are important for generating the medium/slow afterhyperpolarization seen after single or trains of action potentials. Three SK channel isoforms (SK1,-2,-3) are differentially distributed throughout the brain, but little is known about their specific expression in particular neuronal compartments. In the hippocampus SK3 was found in the neuropil, predominantly in the terminal field of the mossy fibres and in fine varicose fibres, but excluded from the pyramidal and granule cell layers. Because this expression pattern suggested a presynaptic localization, we examined the subcellular distribution of SK3 in cultured hippocampal neurons using high-resolution immunofluorescence analysis. SK3 was localized in a punctate, synaptic pattern. The SK3 clusters were precisely colocalized with the presynaptic marker synapsin and at close range (0.4–0.5 µm) from NMDA-receptors and PSD-95. This arrangement is consistent with a localization of SK3 in the presynaptic nerve terminal, but not restricted to the synaptic membrane proper. In agreement with the increasing expression of SK3 during early postnatal development in vivo , the fraction of synapses containing SK3 increased from 14% to 57% over a six-week culture period. SK3-containing synapses were equally observed on spiny, glutamatergic and smooth GABAergic neurons. In contrast to its close association with NMDA-receptors and PSD-95, SK3 was rarely associated with GABAA -receptor clusters. Thus, SK3 is a presynaptic channel in excitatory hippocampal synapses, with no preference for glutamatergic or GABAergic postsynaptic neurons, and is probably involved in regulating neurotransmitter release. [ABSTRACT FROM AUTHOR]

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser- 1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser- 1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes. [ABSTRACT FROM AUTHOR]