Fundamental survival feature of organisms is an adequate response to the environmental signal. Cells sense extracellular signals via membrane proteins, which transfer signals inside the cell through second messengers. Mechanosensitive ion channels are the key transductors of mechanical signals to ion flux. Calcium ions are important second messengers involved in many different cellular processes, and are therefore highly conserved through evolution. As a part of this master’s thesis, we aimed to study expression, activity and localization of mechanosensitive calcium channel. Experimental methods included molecular cloning in bacterial cells, transfection of mammalian HEK293T cells, monitoring the intesity of signals after ultrasound or calcium ionophore stimulation with a luminometer, and fluorescence confocal microscopy for localization imaging. We prepared gene constructs expressing rat mechanosensitive TRPV6 ion channel of TRP superfamily and CorA magnesium ion transporter from fungus Aspergillus niger. Genes for TRPV6 or CorA were tagged at C-terminus with hemagglutinin tag, self-cleaving T2A peptide and fluorescent protein TagBFP. Ultrasound was tested as a mechanical signal for activation. Ultrasound stimulation opens channels and enables the influx of calcium ions. Calcium ions bind calcium-binding proteins inside the cell and trigger expression of genes under calcium-dependent promotor, such as reporter protein luciferase and NFAT promotor. With calcium ionophore, we proved the functionality of reporter system, as there was an elevated cell response. Cells expressing TRPV6 synthesized more luciferase than cells expressing pcDNA3. In contrast, cells expressing CorA attenuated the response. Ultrasound stimulation results showed that TRPV6 does not contribute to ultrasound cell sensitivity, as the response was comparable to cells transfected with the empty pcDNA3 plasmid. As before, CorA response to ultrasound was attenuated. Fluorescence confocal microscopy showed TRPV6 located in cell membrane, endosomes and around the nucleus. CorA is observed in cellular organelles. Cells transfected with TRPV6 ion channel or CorA transporter were as sensitive to ultrasound stimulation as untransfected, control cells. These should be the subject of further research because ultrasound as a non-invasive tool for cell and deep tissue activation is gaining more recognition and potential applications in synthetic biology, diagnostics and therapy.
|
