Ca2+ channel signaling complexes in hippocampal neurons

In the central nervous system voltage-gated Ca2+ channels participate in numerous important functions, including neurotransmitter release, regulation of gene expression, synaptic plasticity, learning and memory. The specificity of Ca2+ signaling in these multiple tasks is accomplished by the spatial segregation of different types of Ca2+ channels in different macro-molecular signaling complexes located in distinct neuronal compartments. Thus, molecular mechanisms must exist for the functional incorporation of Ca2+ channels into specific neuronal signaling complexes. The goal of this project is to elucidate such targeting mechanisms and to characterize the molecular interactions of Ca2+ channels with scaffolding proteins involved in the organization of specific neuronal signaling complexes. In the preceding project we have developed a neuronal expression system, molecular tools, and analysis techniques that enable us to describe the subcellular distribution of Ca2+ channels with high sensitivity and precision. Here we build on this expertise in that we will disrupt interactions of the Ca2+ channels a 1C isoform (CaV1.2) with several known interaction partners and analyze the effects on the targeting and function of the Ca2+ channel in cultured hippocampal neurons. The interaction partners to be studied include the Ca2+ channel beta subunit, AKAP79, and the PDZ protein NIL 16. The protein-protein interactions will be disrupted by deletion or mutation of putative interaction sequences in the Ca2+ channel, by overexpression of competing peptides and proteins, and by depletion of the interaction partners using the novel siRNA gene silencing approach. Further, the expression and presynaptic targeting of Ca2+ channel (CaV2.2) mutants causing human disease (familial hemiplegic migraine) will be examined. Effects on the localization of Ca2+ channels in specific neuronal compartments will be studied using high-resolution immunofluorescence labeling of epitope-tagged channels, and selected Ca2+-mediated signaling functions will be analyzed in parallel. Determining the mechanisms underlying the functional expression of Ca2+ channels in neuronal signaling complexes is of significant interest for molecular and cellular neuroscience and has important clinical implications. It helps to solve the functional domain structure of voltage-gated Ca2+ channels, to elucidate the mechanisms underlying the signaling specificity of Ca2+-mediated neuronal functions, to understand the pathophysiology of channelophathies, and may reveal potential modulators of Ca2+-dependent neuronal functions.

Voltage-activated calcium channels control important functions of nerve cells including synaptic transmission and plasticity. Consequently, dysfunctions of calcium channels cause neurological diseases ranging from migraine to epilepsy. To accomplish their neuronal functions calcium channels are incorporated in macromolecular signaling complexes localized in pre- and postsynaptic compartments. However, how these channels are incorporated into these molecular machines and how they functionally interact with other components is still elusive. Therefore the overall objective of this project was to understand the molecular mechanisms and signals that determine the functional incorporation of voltage-gated calcium channels into pre- or postsynaptic signaling complexes in neurons. Several auxiliary channel subunits and scaffolding proteins have been implicated in the regulation of membrane expression and functional organization within signaling complexes. If any of these proteins are involved in the targeting of calcium channels in hippocampal neurons, mutation of putative binding sequences in recombinant channels and depletion of specific scaffolding proteins in cultured hippocampal neurons will result in the altered subcellular localization of calcium channels or the associated signaling proteins. Three such putative protein-protein interactions have been examined: Using site-directed mutagenesis we tested the hypothesis as to whether an interaction with the adaptor protein AKAP79 is required for membrane expression of the calcium channel in hippocampal neurons. However, AKAP79 was not specifically colocalized with the channel nor did the mutation of the putative binding site alter the expression levels or targeting of the channel. Also contrary to expectations based on published data, the truncation of a C-terminal sequence, the PDZ ligand, did not result in an altered distribution of the channel. Moreover, pharmacological disruption of the AKAP and PSD-95 postsynaptic scaffold by NMDA receptor activation did not affect the normal distribution of the calcium channel in dendritic spines of hippocampal neurons. Together these results indicated that in the postsynaptic compartment of hippocampal neurons L-type calcium channels form signaling complexes separate from those of the glutamate receptors, and that these signaling complexes remain stable upon activity-induced remodeling of the synapse. In contrast, mutating the primary interaction domain of the calcium channel ß subunit caused a total loss of membrane incorporation, indicating that association of the ß subunit is essential for the functional expression of this channel. Examining the subcellular distribution of the four ß subunit isoforms in hippocampal neurons showed a great permissiveness of &apha; 1 -ß interactions. This analysis also revealed a hitherto unnoticed nuclear localization of the ß 4 subunit in young and electrically quiescent neuron. This interesting finding indicated a new function of the calcium channel in the nucleus which is being investigated in a follow-up project.