Philip A Gottlieb

University at Buffalo, State University of New York | SUNY Buffalo · Department of Physiology and Biophysics

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensiti...

Piezo1 is a eukaryotic cation-selective mechanosensitive ion channel. To understand channel function in vivo, we first need to analyze and compare the response in the whole cell and the patch. In patches, Piezo1 inactivates and the current is fit well by a 3-state model with a single pressure-dependent rate. However, repeated stimulation led to an...

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibit...

Significance Familial xerocytosis in humans, which causes dehydration of red blood cells and hemolytic anemia, was traced to mutations in the mechanosensitive ion channel, PIEZO1. The mutations slowed inactivation and introduced a pronounced latency for activation. Loss of inactivation and increased latency for activation could modify groups of cha...

Background: Mechanically gated PIEZO channels lead to an influx of cations, activation of additional Ca2+ channels, and cell depolarization. This study aimed to investigate PIEZO2's role in breast cancer. Methods: The clinical relevance of PIEZO2 expression in breast cancer patient was analyzed in a publicly available dataset. Utilizing PIEZO2 o...

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichm...

PIEZO proteins are large eukaryotic mechanically-gated channels that function as homotrimers. The basic PIEZO1 structure has been elucidated by CryoEM and it assembles into a protein–lipid dome. A curved lipid region allows for the transition to the lipid bilayer from the dome (footprint). Gating PIEZO1 is mediated by bilayer tension that induces a...

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink throug...

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichm...

PIEZO1 and PIEZO2 are mechanosensitive channels (MSCs) important for cellular function and mutations in them lead to human disorders. We examined how functional heteromers form between subunits of PIEZO1 using the mutants E2117K, E2117D, and E2117A. Homomers of E2117K do not conduct. E2117A homomers have low conductance with rapid inactivation, and...

Two mutations of E2117 produce lower unitary conductance. We replaced the glutamic acid with either cysteine (A) or serine (B) and measured the single channel currents. Cell-attached patch recordings at -100 mV in a potassium bath. The amplitude histogram shows that both homomeric channels have a low conductance similar to E2117A. For E2117C, the s...

First order characterization of ion selectivity through E2117A and E2117D. (A) is the current for E2117A with either LiCl or K-gluconate in the pipette compared to KCl. Cell-attached patch recordings performed at -100 mV in a high potassium bath. (B) is from E2117D using the same pipette solutions. Li+ used to evaluate selectivity. In wild-type, K+...

Log-likelihood values for titration analysis of E2117A and E2117D cotransfections. Fig 7 shows titration of various fractions of the two mutants performed with a total of 300 channels. Those same data were reanalyzed using either 200 (two top panels) or 400 (two bottom panels) channels to observe the dependence of the fit on channel number density....

Spatial distribution of heteromeric and homomeric channels in a cotransfected cell. Left Panels—E2117D internally labeled with EGFP and E2117A labeled with mCherry. The channels’ functional behavior in a patch is unaffected by the presence of the labels [33]. Top panel is a single cell and bottom panel is a cluster of cells. Imaging Z-stacks Right...

Standard deviation errors to the kinetic rate constants modeled in Fig 3D. (TIF)

Abstract Traumatic brain injury (TBI) elevates Abeta (Aβ) peptides in the brain and cerebral spinal fluid. Aβ peptides are amphipathic molecules that can modulate membrane mechanics. Because the mechanosensitive cation channel PIEZO1 is gated by membrane tension and curvature, it prompted us to test the effects of Aβ on PIEZO1. Using precision flui...

Supplemental Digital Content is available in the text

Traumatic brain injury (TBI) elevates Abeta (Aβ) peptide concentrations in the brain and cerebral spinal fluid. The response of cells to these forces and how Aβ peptides modulate cellular function is unclear. We examined the effect of Aβ peptides on PIEZO1, and show that the monomerized form inhibits channel function at fM to pM concentrations. Ena...

Cholesterol’s effect on PIEZO1 channels.

Microfluidics is an interdisciplinary field intersecting many areas in engineering. Utilizing a combination of physics, chemistry, biology, and biotechnology, along with practical applications for designing devices that use low volumes of fluids to achieve high-throughput screening, is a major goal in microfluidics. Microfluidic approaches allow th...

Enterochromaffin (EC) cells are the primary mechanosensors of the gastrointestinal (GI) epithelium. In response to mechanical stimuli EC cells release serotonin (5-hydroxytryptamine; 5-HT). The molecular details of EC cell mechanosensitivity are poorly understood. Recently, our group found that human and mouse EC cells express the mechanosensitive...

Mechanical transducers appear throughout cell biology and are used to convert mechanical stress into chemical or electrical signals that allow the cell to respond to environmental changes. In the past six years, a eukaryotic mechanical channel family with two members, Piezo1 and Piezo2, has been identified. Piezo1 was shown to be a cation-selective...

Piezo channels are eukaryotic, cation-selective mechanosensitive channels (MSCs), which show rapid activation and voltage-dependent inactivation. The kinetics of these channels are largely consistent across multiple cell types and different stimulation paradigms with some minor variability. No accessory subunits that associate with Piezo channels h...

Key points: The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown. In the present study, we show, for the first...

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional...

Ct-EGFP protein expressed alone shows diffuse, homogenous, labeling of cytoplasmic and nuclear regions with no indication of plasma membrane labeling. Middle section images taken from two groups of cells. (A) shows two cells expressing higher concentration of Ct-EGFP with cytoplasmic and nuclear distribution. (B) Shows two cells with lower expressi...

Nt-mCherry protein expressed alone labels internal and surface membranes, but does not enter the nucleus. Two representative cells are shown (A and B) with image sections near the glass coverslip and in the middle of the cell. Many punctae form on the surface and dense staining occurs in the ER and at the ends of cell protrusions. In addition, most...

TREK1 and MscL proteins do not colocalize. Optical sections (0.25 μm thick) showing DIC and fluorescent images of a representative HEK cell expressing TREK1-mCherry and MscL-EGFP bicistronic vector. A section near the glass coverslip and in the middle of the cell are shown. EGFP images at 525 nm (green) and mCherry image at 680 nm (orange). TREK1 p...

Ct-EGFP and Nt-mCherry coexpression causes Ct proteins to relocate to Nt regions. While not complete colocalization, most of the Ct protein is localized to Nt regions, and no Ct protein no longer goes to the nucleus. Single representative cell showing image sections near glass coverslip and near the middle of the cell. Cells expressing both halves...

Eukaryotic PIEZO1 ion channels are cation selective and open with membrane tension. The channel is further characterized by voltage dependent inactivation and a conductance of ∼45 pS. We mutated the glutamic acid at position 2117 to an aspartic acid and this increased channel conductance to 72 pS. The mutations E2117A or E2117K lowered channel cond...

Supplementary Figures 1-6

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the chan...

PIEZO1 is a recently cloned eukaryotic cation-selective channel that opens with mechanical force. We found that extracellular protonation inhibits channel activation by ≈90% by increased occupancy in the closed or the inactivated state. Titration between pH 6.3 and 8.3 exhibited a pK of ≈6.9. The steepness of the titration data suggests positive co...

A 21 year old male student presented in 1980 as an Olympic athlete with a 12 year history of jaundice, pallor, and darkened urine induced by the atraumatic exercise of swimming (1). Physical examination at that time was remarkable only for moderate scleral icterus without hepatosplenomegaly. Hematological examination revealed moderate macrocytosis...

Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, wit...

Piezo1 is a eukaryotic mechanosensitive cation channel. We characterized its permeability to monovalent and divalent cations. The i-V relationships for 150 mM Na+, K+ and Cs+ ions were similar and they showed non-linearity (for 150 mM K+, the conductance was 36 pS at −40 mV and 47 pS at −100 mV). This similarity implies that the ions permeate witho...

We created clones that covalently linked a fluorescent protein to PIEZO1 in order to image PIEZO1 on the cell surface. Inserting mCherry1 in internal positions of the PIEZO1 protein, we found a construct at position 1591 that yielded a protein whose channel kinetics were nearly identical to wild type. Through cotransfection, we imaged PIEZO1 (mCher...

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The do...

A method and apparatus for measuring changes in cell volume generally includes introducing cells into a chamber having a volume between 2 and 100 times the volume of the introduced cell. A first electrically conductive extracellular fluid is introduced into the chamber and a current is applied. The voltage induced by said current flow is measured....

Piezo1 is a cation selective channel isolated from eukaryotes which shows voltage dependent inactivation. Xerocytosis is a condition that causes hemolytic anemia of red blood cells associated with either of two individual mutations in the gene encoding Piezo1. In this work we have incorporated both mutations together into the Piezo1 gene and charac...

Piezo1 is a cation selective channel in eukaryotes, and Xerocytosis is a disease of red cells that has been mapped to mutations in the gene encoding Piezo1. We introduced these mutations into a cloned human Pieoz1 and measured their effects on the channel kinetics. Substituting arginine for methionine at position 2225 (M2225R), or a hisitidine for...

Intracellular Ca(2+) signals control the development and regeneration of spinal axons downstream of chemical guidance cues, but little is known about the roles of mechanical cues in axon guidance. Here we show that transient receptor potential canonical 1 (TRPC1) subunits assemble mechanosensitive (MS) channels on Xenopus neuronal growth cones that...

Piezo ion channels have been found to be essential for mechanical responses in cells. These channels were first shown to exist in Neuro2A cells, and the gene was identified by siRNAs that diminished the mechanical response. Piezo channels are approximately 2500 amino acids long, have between 24–32 transmembrane regions, and appear to assemble into...

Piezo proteins have been shown to form large ion channels that serve a sensory function in fruitflies. The findings help to explain how Piezos convert mechanical force into biological signals. See Article p.176 & Letter p.209

Piezo1 channels gate with mechanical stress in the membrane and gating involves both activation and inactivation. In HEK293 cells transfected with Piezo1 and subjected to pressure stimuli, cell-attached patch recordings showed that the inactivation rate slowed as extracellular divalent ions were reduced. With >1mM Mg+2, activation had no measurable...

GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata folded into an inhibitory cysteine knot and it selectively affects gating of some mechanosensitive channels. Here we report the effects of cytoplasmic GsMTx4 on the two bacterial channels, MscS and MscL, in giant Escherichia coli spheroplasts. In excised inside-out pat...

GsMTx4, a peptide inhibitor for mechanosensitive ion channels (MSCs), promoted neurite outgrowth from PC12 cells in the presence of NGF in a dose-dependent manner between 5 and 100 microM peptide. Enhanced neurite growth required >12 h of peptide exposure in cells grown with NGF. Adsorption of GsMTx4 to serum proteins in the media lowered the free...

Cells respond to a hyposmotic challenge by swelling and then returning toward the resting volume, a process known as the regulatory volume decrease or RVD. The sensors for this process have been proposed to include cationic mechanosensitive ion channels that are opened by membrane tension. We tested this hypothesis using a microfluidic device to me...

A. Changes in Fluo-3 fluorescence intensity in (n) individual human SS red cells 210 sec after deoxygenation (*, P<10–9), and in (n) individual Berkeley sickle mouse red cells 300 sec after deoxygenation (*, P<10–7). 1 µM GsMTx-4 was absent or present as indicated. B. Change in Fluo-3 fluorescence intensity in (n) individual SAD sickle mouse red ce...

Lysophosphatidic acid activates a cation-permeable channel in human SS red cells. A. On-cell patch recording from a human SS red cell before (upper trace) and after exposure to 5 µM lysophosphatidic acid (LPA, lower trace). Symmetrical bath and pipette solutions contained (in mM) 150 Na+ methanesulfonate, 10 Na EDTA, and 10 Na HEPES, pH 7.4. −Vp =...

Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS poly...

The spider peptide GsMTx4, at saturating concentration of 5 muM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is...

GsMTx4, the only known specific inhibitor for mechanically gated ion channels, is 34 amino acid long and adopts an ICK motif. The peptide when applied extracellularly inhibits nonselective MSCs. Understanding the relationship peptide's structure and mechanism using mutagenesis has been slowed due to a lack of a specific target. In this work we show...

Deoxygenation of sickle erythrocytes activates a cation permeability (Psickle) leading to elevated [Ca2+]i and subsequent KCa channel activation. The resulting erythrocyte volume decrease is believed to accelerate deoxygenation-induced HbSS polymerization. Deoxygenation-activated currents with some properties of Psickle have been recorded from sick...

Stretch is an important regulator of atrial function. The functional effects of stretch on human atrium, however, are poorly understood. Thus, we characterized the stretch-induced force response in human atrium and evaluated the underlying cellular mechanisms. Isometric twitch force of human atrial trabeculae (n = 252) was recorded (37 degrees C, 1...

Mechanical load is an important regulator of cardiac force. Stretching human atrial and ventricular trabeculae elicited a biphasic force increase: an immediate increase (Frank-Starling mechanism) followed by a further slow increase (slow force response, SFR). In ventricle, the SFR was unaffected by AT- and ET-receptor antagonism, by inhibition of p...

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca(2+) permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using...

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca(2+) permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using...

Mechanosensitive ion channels (MSCs) are found in all types of cells ranging from Escherichia coli to morning glories to humans. They seem to fall into two families: those in specialized receptors, such as the hair cells of the cochlea, and those in cells not clearly differentiated for sensory duty. The physiological function of the channels in non...

In this paper, we have developed a simple and rapid method for the detection and identification of bacteria using a microfluidic lab-chip. The microfluidic chip utilizes impedance-based measurement to (1) detect cells and (2) identify them when used in conjunction with immobilized monoclonal antibodies. Bacteria in suspension passing through the mi...

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitionin...

Sensing the energy from mechanical inputs is ubiquitous--and perhaps the oldest form of biological energy transduction. However, the tools available to probe the mechanisms of transduction are far fewer than for the chemical and electric field sensitive transducers. The one pharmacological tool available for mechansensitive ion channels (MSCs) is a...

Volume 419, Antineoplastic, Immunogenic and Other Effects of the Tetrapeptide Tuftsin: A Natural Macrophage Activator pages 93–106, December 1983

Ca2+ signals are known to be important regulators of neurite outgrowth and steering. Here we show that inhibiting Ca2+ influx through stretch-activated channels using various compounds, including a highly specific peptide isolated from Grammostola spatulata spider venom (GsMTx4), strongly accelerates the rate of neurite extension on diverse substra...

Regulation of cell volume was one of the earliest evolutionary demands for life and remains a universal measure of cell metabolism. Since conventional methods to measure cell volume, such as microscopy, are complex and time-consuming, cell volume has not been used as the basis for cell-based screening. We have developed a microfabricated chip that...

The mdx mouse lacks dystrophin and is a model of human Duchenne muscular dystrophy. Single mdx muscle fibres were isolated and subjected to a series of stretched (eccentric) contractions while measuring intracellular calcium concentration ([Ca(2+)](i)) with fluo-3 and confocal microscopy. Following the stretched contractions there was a slow rise i...