Role of the Ca2+ channel alpha2delta1 in hippocampal neurons

In the central nervous system voltage-gated calcium channels play important and diverse roles in the synaptic transmission of electrical signals, in the integration and modulation of these signals, and in the transduction of membrane depolarization into intracellular signals. To accomplish these diverse functions neurons express a variety of calcium channel complexes which are composed of a pore forming alpha1 subunit and the auxiliary alpha2/delta, beta, and gamma subunits. Whereas knock-out mice were instrumental for investigating the roles of the beta and gamma subunits, no such mouse model system is available for the alpha2/delta-1 subunit. Therefore, little is known about specific functions of this calium channel subunit in differentiated cells such as nerve cells. This is critical, because alpha2/delta-1 is an important target for the anticonvulsant drugs gabapentin and pregabalin, two highly effective treatments for neuropathic pain and epilepsy. The goal of this project is to elucidate the role of the calcium channel alpha2/delta-1 subunit in cultured hippocampal neurons by a loss-of-function approach, i.e. RNA interference. In particular I propose to study the functional consequences of alpha2delta-1 depletion on neuronal differentiation, channel targeting, synaptic transmission, and calcium channel subunit composition. In the preceding project I personally established a neuronal expression system, analysis techniques, and molecular tools, including an siRNA technique to efficiently silence alpha2/delta-1 expression in various cell types. Quantitative RT-PCR and single cell RT-PCR will be used to demonstrate the efficacy of the siRNA depletion of alpha2/delta-1 and whether this leads to a compensation by other isoforms or to a change in the calcium channel subunit composition. The effects of alpha2/delta-1 depletion on differentiation, synaptogenesis, and pre-and postsynaptic channel targeting in hippocampal neurons will be analyzed using high-resolution immunofluorescence labeling of endogenous proteins and epitope-tagged recombinant calcium channels. Possible effects on synaptic function will be studied with a fluorescent synaptic vesicle fusion assay. Which neuornal functions require the alpha2/delta-1 calcium channel subunit, and does it contribute to the correct targeting of specific calcium channels into pre- and postsynaptic compartments, are eminent questions of cellular neurosciences. Elucidating the functions of alpha2/delta-1 in neurons will be an important step forward in understanding the mechanisms of calcium-dependent signal transduction in the normal and diseased nervous system.

Role of the calcium channel alpha-2/delta-1 subunit in hippocampal neurons: Voltage-activated calcium channels are the major cellular proteins regulating the entry of the second messenger calcium into excitable cells. In nerve cells calcium channels are regulators of important functions including synaptic transmission and plasticity and are thus involved in learning and memory. As a consequence genetic defects of the individual calcium channels cause a number of human diseases, for example arrhythmia of the heart, migraine, or epilepsy. Calcium channels are not single isolated units but consist of up to four subunits. Alpha-1 is the largest subunit and contains the calcium conducting pore and the so-called voltage sensor. The other three accessory or auxiliary subunits are termed alpha- 2/delta, beta, and gamma. Their general role is to assist the alpha-1 subunit in its primary function that is allowing calcium to flow into the cell. While it has long been shown that the auxiliary alpha-2/delta subunit regulates membrane expression and the overall properties of calcium channels, surprisingly little was known on its specific functions in native cells of muscle, heart, and brain. Therefore the overall goal of this project was to understand the role of the alpha-2/delta subunit in differentiated nerve cells such as cultured hippocampal neurons. If this subunit indeed is involved in important cellular functions, interfering with its expression will result in altered cellular functions that can be experimentally tested. To investigate this issue several methodological and scientific aspects had to be addressed: First, we developed the molecular tools to knock-down the expression of alpha-2/delta that were initially tested in a cardiac muscle cell model. Combining electrophysiological measurements on muscle cells with a newly adapted mathematical model of all currents of a cardiac muscle cell we could show that the alpha- 2/delta subunit determines the shape of the cardiac action potential and the intracellular calcium concentration. It was thus found to be actually necessary for normal heart function. Second, we made the interesting observation that, in contrast to its role in muscle, alpha-2/delta had strikingly different effects on neuronal calcium channels. These discrepancies in alpha-2/delta functions led us to a new model of the role of auxiliary calcium channel subunits in excitable cells that will be ultimately tested in future studies. Third, in a newly established side branch of the project we could gain insight into the mechanisms how neuronal calcium channel beta and alpha-1 subunits cooperate in regulating the localization and the membrane expression of the channel complex. Interestingly, analysis of the distribution of the four beta subunits in hippocampal neurons showed a great redundancy in alpha-1 and beta interactions. However, preventing alpha-1 and beta interactions caused a total loss of membrane incorporation, indicating that association of the beta subunit is essential for the functional expression of this channel. These analyses also provided novel insights into the mechanisms how channels are transported into dendritic spines, which may be more widely applicable to postsynaptic proteins. Finally, we developed new methods for the analysis of the localization of synaptic proteins and for the quantification of the calcium channel subunit repertoire of different brain regions and single types of neurons.