Cloning of candidate mechanosensitive ion channel genes and determining the functional and structural properties of the related proteins in Astacus leptodacylus

Cells respond to various mechanical stimuli like tension or osmotic pressure changes in the membrane. Mechanosensitive ion channels play an important role in mechanotransduction, which is the conversion of mechanical stimuli into electrical and chemical signals. Recently identified Piezo1 and Piezo2 channels are representative members of the mechanosensitive channels in eukaryotes. However, Piezo channels alone can not account for all kinds of mechanosensitivity modalities in the body since they are not expressed in some cell populations that respond to mechanical stimuli. TMC and TMEM63 were recently reported to be mechanosensitive in various species. This thesis aimed to clone mRNAs of the orthologues of those channels in the crayfish, Astacus leptodactylus. After cloning the putative mRNAs, molecular properties of the cloned channels were investigated. Three-dimensional structures of the related protein sequences were calculated and some functional parts were identified. To be used in the functional experiments, the cloned mRNAs were synthesized and transferred to plasmid vectors and expressed in HEK293T cell lines. Mechanosensitivity was monitored by acquiring the emission signal from a calcium sensitive dye (Fluo-4 AM) in response to various mechanical stimuli. Two mRNAs coding TMC and TMEM63 orthologues were de novo cloned. Molecular calculations illustrated that they both can assemble into a transmembrane ion channel. Functional studies indicated that TMEM63 could be a mechanosensitive ion channel both in situ and in a heterologous system.