Ion Channel Membrane Preparations
Background of Ion Channel
Ion channels are found in practically all living cells. More than 400 potential ion channels have been found by sequencing the human genome, but only a few of these have been cloned and functionally verified. The extensive tissue distribution of ion channels, as well as the wide range of physiological repercussions of their opening and closing, make ion-channeltargeted drug discovery an intriguing prospect. Despite the fact that certain essential medications are already in clinical use, ion channels as a class remain underexploited in drug development, and many existing treatments are poorly selective and have considerable side effects or effectiveness.
Application of Ion Channel Membrane Preparations
Drug development has been transformed by the introduction of membrane ligand binding assays and high-throughput screening. Ligand binding assays allow us to see how a ligand or a new synthetic molecule interacts with a target protein and assess affinity and kinetics of those interactions, while HTS helps to automate research and shorten development time.
Published Data
| Paper Title | Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing |
| Journal | Nature Communications |
| Published | 2021 |
| Abstract | During immune responses to pathogens and injury, macrophages carry out a variety of tasks within tissues, but the molecular mechanisms by which the physical characteristics of the tissue control macrophage behavior are less well understood. Here, we investigate the function of the mechanically activated cation channel Piezo1 in the polarization of macrophages and the detection of stiffness in the microenvironment. Researchers demonstrate that Piezo1-deficient macrophages exhibit decreased inflammation and improved wound healing responses. Additionally, Ca2+ influx is dependent on Piezo1, modulated by soluble signals, and enhanced on stiff substrates, as shown by macrophages expressing the transgenic Ca2+ reporter, Salsa6f. Additionally, Piezo1 is necessary for changes in macrophage function that are stiffness-dependent, both in vitro and in vivo in response to the subcutaneous implantation of biomaterials. Finally, we demonstrate that Piezo1-actin positive feedback drives macrophage activation. Our research shows that Piezo1 is a mechanical stiffness sensor in macrophages, and that its activity. |
| Result |
As expected, BMDMs expressed Salsa6f in the cytosol but not the nucleus. Salsa6f+ BMDMs were treated with Yoda1 in the presence of Ca2+ to confirm Piezo1 channel function. Yoda1 increased cytosolic Ca2+ dose-dependently, as measured by G/R ratios. Depletion of Piezo1 by siRNA inhibited Yoda1-induced Ca2+ influx, validating reduced ion channel expression. siPiezo1 prevented the increase in Ca2+ events induced by IFN/LPS in siControl-treated BMDMs. Piezo1 mediates macrophage Ca2+ influx in response to acute inflammatory stimuli. Long-term exposure to inflammatory and healing activation stimuli also altered Ca2+ events. TIRF microscopy revealed higher Ca2+ activity at the membrane in inflammatory macrophages. TIRF data show that millisecond-scale local and transient Ca2+ events are prominent in inflammatory activated macrophages. Piezo1 mediates Ca2+ signals in response to soluble inflammatory stimuli.
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Fig.1 Regulation of Ca2+ influx by Piezo1 channels in macrophages.1,2
References
- Atcha, Hamza, et al. "Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing." Nature communications 12.1 (2021): 3256.
- Image retrieved from Figure 2 " Regulation of Ca2+ influx by Piezo1 channels in macrophages. " Atcha, et al. 2021, used under CC BY 4.0. The original image was modified by extracting and the title was changed to " Representation of the basic structural components of an ion channel.".
