Schrittmacherkanäle im olfaktorischen Epithel der Maus

HCN channels hyperpolarization-activated and $\underline{c}$yclic $\underline{n}$ucleotide-gated channels) are membrane proteins participating in the generation of spontaneous rhythmic electrical activity in cellular networks. Therefore these channels are called pacemaker channels. This thesis describes for the first time, the expression pattern of four different HCN channel isoforms (HCN1 - HCN4) in the olfactory epithelium of the mouse on a subcellular level. The isoforms HCN1 and HCN2 are expressed in olfactory receptor neurons (ORN), more specifically in the dendrites and axons of the ORN. Strong labeling was particularly seen in the axon bundles. The HCN4 isoform was found almost exclusively in the axons of the ORN. Furthermore, the immunohistochemical stainings allowed to distinguish between two morphologically different axon bundles: small, tightly-packed axon bundles which express HCN 1, 2 and 4 to similar degrees, and large axon bundles in which predominantly HCN4 is expressed. Notably, HCN3 seems not to be expressed in the olfactory epithelium of the mouse. Specific shRNA molecules can be utilized to achieve a post-transcriptional downregulation of genes. To this end, $\underline{r}$ecombinant $\underline{a}deno-$\underline{a}$ssociated$\underline{v}$iruses (rAAV) were constructed allowing the gene transfer of shRNA-coding sequences. In transgenic cell lines which constitutively expressed specific HCN isoforms, a significant downregulation of HCN1 and HCN2 gene expression was achieved after infections with rAAV_shRNA constructs. Especially for HCN2, de novo protein biosynthesis was impaired almost completely. In a series of experiments, the transduction capability of rAAV for ORN was examined in vivo by virus-mediated eGFP expression. Both, ORN and supporting cells were successfully transduced by rAAV of serotype 2 and 5. In transduced cells, eGFP expression was very high and allowed to unequivocally identify the different cell types by their morphology. In summary, this thesis demonstrates that rAAV-mediated gene transfer is a versatile method that can be used both, to introduce genes into living organisms as well as to specifically knock down gene expression by rAAV_shRNA thereby supporting the ultimate goal to study a proteins’ function in vivo. As likely candidates, individual HCN isoforms might now be targeted as their subcellular expression pattern has been unraveled in the olfactory epithelium of the mouse

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines

Calcium ions (Ca2+) play a pivotal role in cellular physiology. Often Ca2+-dependent processes are studied in commonly available cell lines. To induce Ca2+ signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca2+ signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca2+ signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca2+ in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca2+ signals and Ca2+ oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca2+ signaling processes directly and (ii) these cell lines are suitable for calibrating Ca2+ biosensors in situ based on histamine receptor evoked responses