-
Methods in Molecular Biology (Clifton,... 2021Electrophysiology is an essential tool aiding the study of the functions and dysfunctions of electrically excitable cells and their networks. The patch clamp method is a... (Review)
Review
Electrophysiology is an essential tool aiding the study of the functions and dysfunctions of electrically excitable cells and their networks. The patch clamp method is a refined electrophysiological technique that can directly measure the membrane potential and/or the amount of current passing across the cell membrane. The patch clamp technique is also incredibly versatile and can be used in a variety of different configurations to study a range of properties, from spontaneous cell firing activity in native tissue to the activation and/or deactivation kinetics of individual channels expressed in recombinant cell lines. In this chapter we give an overview of patch clamping and how the different configurations can be set up and applied to electrophysiological research.
Topics: Animals; Cell Membrane; Electrophysiological Phenomena; Equipment Design; Humans; Ion Channels; Patch-Clamp Techniques
PubMed: 33119844
DOI: 10.1007/978-1-0716-0818-0_1 -
Science (New York, N.Y.) Aug 2023Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of...
Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING's channel activity is critical for these two processes. Thus, STING's interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.
Topics: Humans; Golgi Apparatus; Hydrogen-Ion Concentration; Inflammasomes; Ion Channels; Liposomes; Membrane Proteins; Microtubule-Associated Proteins; Protein Domains; Protons; HEK293 Cells
PubMed: 37535724
DOI: 10.1126/science.adf8974 -
Proceedings of the National Academy of... May 2023Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and...
Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.
Topics: Membrane Proteins; Cell Membrane; Ion Channels; Binding Sites
PubMed: 37098056
DOI: 10.1073/pnas.2302325120 -
Trends in Pharmacological Sciences Dec 2019Mechanotransduction has a key role in vascular development, physiology, and disease states. Piezo1 is a mechanosensitive (MS) nonselective cationic channel that occurs... (Review)
Review
Mechanotransduction has a key role in vascular development, physiology, and disease states. Piezo1 is a mechanosensitive (MS) nonselective cationic channel that occurs in endothelial and vascular smooth muscle cells. It is activated by shear stress associated with increases in local blood flow, as well as by cell membrane stretch upon elevation of blood pressure. Here, we briefly review the pharmacological modulators of Piezo and discuss current understanding of the role of Piezo1 in vascular mechanobiology and associated clinical disorders, such as atherosclerosis and hypertension. Ultimately, we believe that this research will help identify novel therapeutic strategies for the treatment of vascular diseases.
Topics: Animals; Cardiovascular Physiological Phenomena; Cardiovascular System; Humans; Ion Channels; Mechanotransduction, Cellular; Models, Molecular; Morphogenesis
PubMed: 31704174
DOI: 10.1016/j.tips.2019.10.002 -
Neuron Sep 2022Adult brain activities are generally believed to be dominated by chemical and electrical transduction mechanisms. However, the importance of mechanotransduction mediated...
Adult brain activities are generally believed to be dominated by chemical and electrical transduction mechanisms. However, the importance of mechanotransduction mediated by mechano-gated ion channels in brain functions is less appreciated. Here, we show that the mechano-gated Piezo1 channel is expressed in the exploratory processes of astrocytes and utilizes its mechanosensitivity to mediate mechanically evoked Ca responses and ATP release, establishing Piezo1-mediated mechano-chemo transduction in astrocytes. Piezo1 deletion in astrocytes causes a striking reduction of hippocampal volume and brain weight and severely impaired (but ATP-rescuable) adult neurogenesis in vivo, and it abolishes ATP-dependent potentiation of neural stem cell (NSC) proliferation in vitro. Piezo1-deficient mice show impaired hippocampal long-term potentiation (LTP) and learning and memory behaviors. By contrast, overexpression of Piezo1 in astrocytes sufficiently enhances mechanotransduction, LTP, and learning and memory performance. Thus, astrocytes utilize Piezo1-mediated mechanotransduction mechanisms to robustly regulate adult neurogenesis and cognitive functions, conceptually highlighting the importance of mechanotransduction in brain structure and function.
Topics: Adenosine Triphosphate; Animals; Astrocytes; Cognition; Ion Channels; Mechanotransduction, Cellular; Mice; Neurogenesis
PubMed: 35963237
DOI: 10.1016/j.neuron.2022.07.010 -
The Journal of Clinical Investigation Oct 2023All cells in the body are exposed to physical force in the form of tension, compression, gravity, shear stress, or pressure. Cells convert these mechanical cues into... (Review)
Review
All cells in the body are exposed to physical force in the form of tension, compression, gravity, shear stress, or pressure. Cells convert these mechanical cues into intracellular biochemical signals; this process is an inherent property of all cells and is essential for numerous cellular functions. A cell's ability to respond to force largely depends on the array of mechanical ion channels expressed on the cell surface. Altered mechanosensing impairs conscious senses, such as touch and hearing, and unconscious senses, like blood pressure regulation and gastrointestinal (GI) activity. The GI tract's ability to sense pressure changes and mechanical force is essential for regulating motility, but it also underlies pain originating in the GI tract. Recent identification of the mechanically activated ion channels Piezo1 and Piezo2 in the gut and the effects of abnormal ion channel regulation on cellular function indicate that these channels may play a pathogenic role in disease. Here, we discuss our current understanding of mechanically activated Piezo channels in the pathogenesis of pancreatic and GI diseases, including pancreatitis, diabetes mellitus, irritable bowel syndrome, GI tumors, and inflammatory bowel disease. We also describe how Piezo channels could be important targets for treating GI diseases.
Topics: Humans; Mechanotransduction, Cellular; Ion Channels; Cell Membrane; Gastrointestinal Diseases
PubMed: 37781915
DOI: 10.1172/JCI171955 -
Trends in Biochemical Sciences Jun 2021The evolutionarily conserved Piezo channel family, including Piezo1 and Piezo2 in mammals, serves as versatile mechanotransducers in various cell types and consequently... (Review)
Review
The evolutionarily conserved Piezo channel family, including Piezo1 and Piezo2 in mammals, serves as versatile mechanotransducers in various cell types and consequently governs fundamental pathophysiological processes ranging from vascular development to the sense of gentle touch and tactile pain. Piezo1/2 possess a unique 38-transmembrane (TM) helix topology and form a homotrimeric propeller-shaped structure comprising a central ion-conducting pore and three peripheral mechanosensing blades. The unusually curved TM region of the three blades shapes a signature nano-bowl configuration with potential to generate large in-plane membrane area expansion, which might confer exquisite mechanosensitivity to Piezo channels. Here, we review the current understanding of Piezo channels with a particular focus on their unique structural designs and elegant mechanogating mechanisms.
Topics: Animals; Ion Channel Gating; Ion Channels; Mechanotransduction, Cellular; Protein Domains
PubMed: 33610426
DOI: 10.1016/j.tibs.2021.01.008 -
Neurobiology of Disease Sep 2023Lysosomes are acidic intracellular organelles with autophagic functions that are critical for protein degradation and mitochondrial homeostasis, while abnormalities in... (Review)
Review
Lysosomes are acidic intracellular organelles with autophagic functions that are critical for protein degradation and mitochondrial homeostasis, while abnormalities in lysosomal physiological functions are closely associated with neurological disorders. Transmembrane protein 175 (TMEM175), an ion channel in the lysosomal membrane that is essential for maintaining lysosomal acidity, has been proven to coordinate with V-ATPase to modulate the luminal pH of the lysosome to assist the digestion of abnormal proteins and organelles. However, there is considerable controversy about the characteristics of TMEM175. In this review, we introduce the research progress on the structural, modulatory, and functional properties of TMEM175, followed by evidence of its relevance for neurological disorders. Finally, we discuss the potential value of TMEM175 as a therapeutic target in the hope of providing new directions for the treatment of neurodegenerative diseases.
Topics: Humans; Ion Channels; Neurodegenerative Diseases; Lysosomes; Autophagy; Potassium Channels
PubMed: 37524211
DOI: 10.1016/j.nbd.2023.106244 -
American Journal of Physiology. Cell... Mar 2023PIEZO1 and PIEZO2 are mechanosensitive cation channels that are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in... (Review)
Review
PIEZO1 and PIEZO2 are mechanosensitive cation channels that are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in multiple organ systems. Within the musculoskeletal system, PIEZO1 functions to maintain muscle and bone mass, sense tendon stretch, and regulate senescence and apoptosis in response to mechanical stimuli within cartilage and the intervertebral disc. PIEZO2 is essential for transducing pain and touch sensations as well as proprioception in the nervous system, which can affect musculoskeletal health. PIEZO1 and PIEZO2 have been shown to act both independently as well as synergistically in different cell types. Conditions that alter PIEZO channel mechanosensitivity, such as inflammation or genetic mutations, can have drastic effects on these functions. For this reason, therapeutic approaches for PIEZO-related disease focus on altering PIEZO1 and/or PIEZO2 activity in a controlled manner, either through inhibition with small molecules, or through dietary control and supplementation to maintain a healthy cell membrane composition. Although many opportunities to better understand PIEZO1 and PIEZO2 remain, the studies summarized in this review highlight how crucial PIEZO channels are to musculoskeletal health and point to promising possible avenues for their modulation as a therapeutic target.
Topics: Cell Membrane; Ion Channels; Mechanotransduction, Cellular; Muscles; Musculoskeletal System; Humans
PubMed: 36717101
DOI: 10.1152/ajpcell.00544.2022 -
Nature Sep 2019PIEZO1 is a mechanosensitive channel that converts applied force into electrical signals. Partial molecular structures show that PIEZO1 is a bowl-shaped trimer with...
PIEZO1 is a mechanosensitive channel that converts applied force into electrical signals. Partial molecular structures show that PIEZO1 is a bowl-shaped trimer with extended arms. Here we use cryo-electron microscopy to show that PIEZO1 adopts different degrees of curvature in lipid vesicles of different sizes. We also use high-speed atomic force microscopy to analyse the deformability of PIEZO1 under force in membranes on a mica surface, and show that PIEZO1 can be flattened reversibly into the membrane plane. By approximating the absolute force applied, we estimate a range of values for the mechanical spring constant of PIEZO1. Both methods of microscopy demonstrate that PIEZO1 can deform its shape towards a planar structure. This deformation could explain how lateral membrane tension can be converted into a conformation-dependent change in free energy to gate the PIEZO1 channel in response to mechanical perturbations.
Topics: Aluminum Silicates; Animals; Cryoelectron Microscopy; HEK293 Cells; Humans; Ion Channels; Liposomes; Mice; Microscopy, Atomic Force
PubMed: 31435018
DOI: 10.1038/s41586-019-1499-2