New and neglected Ca channel subunit splice variants

The overwhelming majority of genes in our organism can make blueprints for more than one end product. This process is called alternative splicing and expands the repertoire of possible protein products severalfold. Also voltage-gated calcium channels use this mechanism. These ion channels are important to release the essential messenger calcium from inside cells or let it enter the cells in a controlled way. The calcium in turn is then for example responsible for muscle contraction or synaptic transmission in neurons. Here we investigate in particular two alternative splice variants of calcium channels that are found in the retina und are indispensable for vision. These variants putatively have particular influence on the properties of calcium currents in photoreceptors, the light-sensitive cells of the retina, and neurons downstream, socalled bipolar cells, that relay the light information. How and why precisely these variants fulfill these tasks in the retina shall be investigated in this study. To this end, gene activity of the variants will be measured in purified photoreceptors and bipolar cells to confirm the contribution of these variants in the respective cell types. Furthermore, the variants will be detected on the protein level via mass spectrometry (in collaboration with Dr. Marcel Kwiatkowski in the lab of Prof. Kathrin Thedieck, Department of Biochemistry, LFUniversity of Innsbruck) and their association with the cell membrane will be determined in cell culture. Functional assessment of the variants properties will be conducted in cell culture. Taken together, the gained understanding shall give a more detailed picture of how calcium currents are tuned to the requirements of the respective cell types of the retina. This knowledge is intended to form a new basis for an even more detailed diagnosis and more precise treatments of retinal disorders in the future.

The vast majority of genes in our organism can create blueprints for more than one end product. This process is called alternative splicing and expands the repertoire possible protein products. In this project we particularly investigated two alternative splicing variants of voltage-gated calcium channels which are present in the retina and are essential for vision. These variants were expected to affect the properties of calcium currents in photoreceptors, i.e. the light-sensitive cells of the retina, and downstream nerve cells, so-called bipolar cells that take over the transmission of light information. How and why exactly these variants fulfil these tasks in the retina was a matter of investigation in this study. Gene activity measurements of the variants in purified photoreceptors and bipolar cells confirmed their involvement in the respective cell types. The study was complemented by functional analyses in heterologous expressions systems. We also considered available structural data to elucidate potential mechanisms underlying the observed characteristics. With the description of a novel N-terminus in one of the variants we expanded the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features provide new angles on the way their subunits modulate calcium channels at the plasma membrane.