Localization of Calcium Channels at the Calyx of Held

Abstract

The Calyx of Held is a giant synapse in the auditory pathway. Due to its large size and its geometry it makes an excellent model system for studying the biophysical properties of synaptic transmission. Previous studies have demonstrated that the release rate of synaptic vesicles is governed by the distance between single synaptic vesicles and calcium channels. Therefore knowing the localization of presynaptic calcium channels is essential to understand the coupling of synaptic vesicle release with calcium influx into the calyx. Prior studies have attempted to characterize calcium channel distribution in the Calyx, however results have been inconclusive in providing an accurate picture. In order to conclusively localize calcium channels we are exploring several possibilities to stain calcium channels for fluorescence and electron microscopy studies. The first approach is to express genetically tagged P/Q-type calcium channels in the Calyx utilizing an adenovirus expression system. We have tried an eGFP-tagged P/Q-type calcium channel and found that the eGFP accumulates in the nuclei of the cells indicating cleavage of the tags. The tag also reduced calcium currents twofold compared to untagged channel. Our second approach was to screen several constructs with HA epitope tags inserted into different positions on the P/Q-type calcium channel. We found that extracellular tags frequently impair channel function. However we did obtain constructs which showed current upon expression in HEK cells. Unfortunately accessibility of the tag for the antibody is impaired in neurons. Intracellular tags are accesible in primary neurons and behave similar to untagged channel electrophysiologically, but the staining is difficult to interpret because tagged overexpressed channels accumulate in intracellular compartments. In parallel experiments we have screened several antibodies against P/Q-type calcium channels aiming to find one that gives clean staining for high-resolution microscopy. Funding: LSHM-CT-1005-019055.