mechanosensitivity by overexpressed
- Ectopically expressed mechanosensitive ion channels MscS and P3:FAStm:TRPC1 were used to enhance sensitivity of mammalian cells to ultrasound stimulation.
- Membrane localization of the mechanosensitive channel TRPC1 was improved by fusing it with a FAS transmembrane domain, which also led to increased sensitivity to ultrasound stimulation.
The detailed mechanism of mechanosensing is not known, however most mechanosensitive
receptors respond to mechanical stimuli through
opening of the channel pore and allowing calcium ions to enter the cell
We chose to test two mechanosensitive channels: human nonspecific cation channel TRPC1
and bacterial channel MscS, previously described as important receptors involved
in the response to mechanical stimulation in humans and bacteria
Transient receptor potential channel 1 (TRPC1) is a human non-specific cation
channel located at the plasma membrane. It has been previously reported as
broadly expressed in human tissues where it functions as a store-operating
The second channel that we selected is the bacterial mechanosensitive channel
MscS. Its role is to mediate turgor regulation in bacteria and is
activated by changes in osmotic pressure
Localization and expression
The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane helices (3aA lower) and the MscS channel with three transmembrane helices (3aA upper) were expressed in HEK293T cells (3aD). MscS was detected as the 31 kDa band. TRPC1 was observed at 60 kDa, which was lower than expected. We observed that the membrane localization in HEK293 was more evident for MscS (3aB) rather than TRPC1 (3aC).
To improve membrane localization of TRPC1 we fused a FAS
transmembrane domain to TRPC1 (4aA), since the
transmembrane FAS domain has been very efficient in Jerala lab for the membrane
In addition to the improved membrane localization, the FAS transmembrane domain linked to the TRPC1 presents another advantage. The TRPC1 is an ion channel with six transmembrane helices, therefore both the N- and the C-terminus of the protein are orientated towards the interior of the cell. By addition of the FAS transmembrane domain, the N-terminus of P3:FAStm:TRPC1 chimera (where P3 stands for coiled coil) is exposed in the extracellular space and could interact with different proteins from outside the cell via the N-terminal tag. We reasoned that this interaction could be used to achieve a higher sensitivity to mechanical stimuli.
After we showed that the selected ion channels MscS, TRPC1 and P3:FAStm:TRPC1 are expressed in HEK293 and localized at the plasma membrane, we further tested their function as mechanosensors by exposing them to ultrasound stimulation.
Ultrasound stimulation offers potentially remarkable advantages over the
majority of external stimuli used for targeted cell stimulation.
Optogenetics, as another
promising approach to cell stimulation, requires invasive surgery to
implement optical fibers connected to the source of light – LED or laser
For mechanical stimulation of cells with ultrasound we designed our own unique experimental setup, which included the ultrasound device MODUSON that we constructed and connected to the unfocused transducer Olympus V318-SU and a 3D printed support for a transducer to fix it at a defined position relative to the cells. Stimulation conditions were optimized for our cell line and experimental setup. To measure the changes of free calcium ion concentration we stained cells with two fluorescent dyes Fura Red and Fluo-4. The combination of these two dyes enabled us to present changes in the calcium ion concentration as a ratio of the fluorescence intensity at two wavelengths, which was superior to the intensity based measurements, since it is independent of photobleaching and dye sequestration.
Fura Red and Fluo-4 are visible light-excitable dyes used for ratiometric measurement of calcium ions which excitation maximum is at 488 nm. While Fluo-4 exhibits an increase in fluorescence emission at 515 nm upon binding of calcium ions, fluorescence emission at 655 nm of Fura Red decreases once the indicator binds calcium ions. By calculating the ratio of fluorescence emission intensities captured at 488 nm exaction (where the difference of fluorescence between the bound and free indicator is at its maximum), we could observe changes in intracellular calcium concentrations in real time.
We followed changes of calcium concentration after ultrasound stimulation in real time using ratiometric confocal microscopy. For processing of data we developed our software CaPTURE, which automatically calculated the ratio between fluorescence intensities of FuraRed and Fluo-4 and presented the data as image and calculated values.
We showed that by expressing the MscS channel cells gained sensitivity for ultrasound stimulation in comparison to non-transfected cells (5a and 6a). Influx of calcium ions was observed at a lower rate in the case of ectopically expressed TRPC1 (data not shown), probably due to its poor membrane localization.
Fusion of the FAS transmembrane domain to TRPC1 did not only improve its membrane localization, but also significantly enhanced its sensitivity to ultrasound stimulation (7a and 8a), suggesting the importance of membrane localization in the function of mechanosensors.
In order to observe mechanostimulation of cells with ectopically expressed mechanoreceptors we had to use high-power ultrasound. However, we tested that the cells nevertheless did not lose the viability by ultrasound stimulation. Our next challenge was to further improve sensitivity of cells to respond to lower power ultrasound as this would avoid stimulation of any endogenous channels and limit stimulation only to the engineered.