How do cells respond to mechanical stimulation? We know that mechanosensitive ion channels (MSCs) are in all cells in all kingdoms, yet our biophysical understanding of how these channels function as transducers in the eukaryotic world lags in comparison to voltage and ligand-gated channels. Over the past decade a wealth of information derived from genetics and electrophysiological analysis of mechanical transduction has emerged from model systems such as C. elegans and bacteria. But the biophysics of mechanical gating at the molecular level in eukaryotic systems, such as direct measures of the forces needed to open channels have been confined to measuring endogenous channel activity rather than cloned channels. Two years ago a new channel family called Piezo was identified that is mechanically gated and has many, if not all, the properties of the endogenous channels we and others have studied for years. The Piezo family with nearly 2500 residues and 25-32 transmembrane domains bears no relationship to other channels (such as TRPs). They activate with membrane tension and inactivate with time, and much in parallel with endogenous channels, inactivation can be lost changing the channels from phasic to tonic sensors. The discovery of this channel family provided us with new opportunities to answer specific biophysical questions related to the gating of mechanical channels. The importance of this channel is underscored by the recent study showing single mutations to Piezo1 in erythrocytes leads to a condition called Xerocytosis, an inability to regulate cell volume. This laboratory has a long history of studying MSCs and their integration into cell mechanics. We discovered MSCs, developed the standard methods of study, the software and instrumentation to study them, characterized their biophysics, found the specific inhibitors and demonstrated their relevance to disease. We will study Piezo channels using electrophysiology at single and multichannel resolution in cells and reconstituted liposomes, generic expression systems and differentiated cells, light microscopy, molecular biology and mathematical analysis. The basic aims include ?Characterize ionic selectivity for monovalents and divalents. ?Characterize inactivation/activation using kinetic analysis of mutant channels. ?Characterize the cytoskeletal and membrane proteins that are involved in the cooperative loss of inactivation and responsible for creating mechanical domains. ?Visualize the mechanical domain structures in cells using high resolution light microscopy. ?Measure stress in patches addressing bilayer stress using dynamic capacitance and calibrated bacterial MSC gating standards, and cortical stress using high resolution optical microscopy and genetically coded FRET based probes of stress in specific cytoskeleton proteins.

Public Health Relevance

Mechanosensitive ion channels (MSCs) are responsible for touch, hearing, the regulation of blood pressure and many other physiological functions. The forces that reach MSCs are regulated by the cell's cytoskeleton, its extracellular matrix and the lipid bilayer. We will study MscS and how forces reach them and modulate their activity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL054887-18
Application #
8506728
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Krull, Holly
Project Start
1995-07-01
Project End
2017-05-31
Budget Start
2013-07-15
Budget End
2014-05-31
Support Year
18
Fiscal Year
2013
Total Cost
$722,441
Indirect Cost
$244,884
Name
State University of New York at Buffalo
Department
Physiology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Bae, Chilman; Suchyna, Thomas M; Ziegler, Lynn et al. (2016) Human PIEZO1 Ion Channel Functions as a Split Protein. PLoS One 11:e0151289
Cox, Charles D; Bae, Chilman; Ziegler, Lynn et al. (2016) Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension. Nat Commun 7:10366
Peng, Anthony W; Gnanasambandam, Radhakrishnan; Sachs, Frederick et al. (2016) Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer. J Neurosci 36:2945-56
Wang, Jinli; Ma, Yina; Sachs, Frederick et al. (2016) GsMTx4-D is a cardioprotectant against myocardial infarction during ischemia and reperfusion. J Mol Cell Cardiol 98:83-94
Maneshi, Mohammad Mehdi; Sachs, Frederick; Hua, Susan Z (2015) A Threshold Shear Force for Calcium Influx in an Astrocyte Model of Traumatic Brain Injury. J Neurotrauma 32:1020-9
Yang, Chao; Zhang, Xiaohan; Guo, Yichen et al. (2015) Mechanical dynamics in live cells and fluorescence-based force/tension sensors. Biochim Biophys Acta 1853:1889-904
Markin, V S; Sachs, F (2015) Free Volume in Membranes: Viscosity or Tension? Open J Biophys 5:80-83
Sachs, Frederick; Sivaselvan, Mettupalayam V (2015) Cell volume control in three dimensions: Water movement without solute movement. J Gen Physiol 145:373-80
Nishizawa, Kazuhisa; Nishizawa, Manami; Gnanasambandam, Radhakrishnan et al. (2015) Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations. Biochim Biophys Acta 1848:2767-78
Bae, Chilman; Sachs, Frederick; Gottlieb, Philip A (2015) Protonation of the human PIEZO1 ion channel stabilizes inactivation. J Biol Chem 290:5167-73

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