The Function and Regulation of Piezo Ion Channels Jason Wu,1 Amanda[13 TD$IF]H

TIBS 1305 No. of Pages 15 Series: Fresh Perspectives from Emerging Experts Review Touch, Tension, and Transduction – The Function and Regulation of Piezo Ion Channels Jason Wu,1 Amanda[13_TD$IF]H. Lewis,1 and Jörg Grandl1,* In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought Trends molecular carriers of an excitatory mechanically activated current found in many Piezo proteins were identified in 2010 cells. This discovery has opened the floodgates for studying a vast number of as the pore-forming subunits of excita- mechanotransduction processes. Over the past 6 years, groundbreaking tory mechanosensitive ion channels. research has identified Piezos as ion channels that sense light touch, proprio- Piezo ion channels play essential roles ception, and vascular blood flow, ruled out roles for Piezos in several other in diverse physiological processes ran- mechanotransduction processes, and revealed the basic structural and func- ging from regulation of red blood cell fi volume to sensation of gentle touch, tional properties of the channel. Here, we review these ndings and discuss the and are associated with a number of many aspects of Piezo function that remain mysterious, including how Piezos diseases. convert a variety of mechanical stimuli into channel activation and subsequent A recent medium-resolution structure inactivation, and what molecules and mechanisms modulate Piezo function. gives insight into the overall architec- ture of Piezo1, but does not give straight answers as to how the channel Piezo Proteins: True Mechanically Activated Ion Channels? transduces mechanical force into pore Piezo proteins are pore-forming subunits of ion channels that open in response to mechanical opening. stimuli, allowing positively charged ions, including calcium, to flow into the cell (Figure 1) [1]. Piezo orthologs have thus far been identified in numerous eukaryotes. Most vertebrates have two The function of Piezos, including the inactivation mechanism, can be modu- channel isoforms, Piezo1 and Piezo2, whereas Drosophila melanogaster has a single ortholog lated by many factors both intrinsic and (sharing equal homology to Piezo1 and Piezo2) that has also been confirmed to form a channel extrinsic to the channel. [1–3]. Genomic analysis predicts single Piezo orthologs in most lower organisms, including Caenorhabditis elegans, plants, and slime mold, although zebrafish have three and protozoa have up to six predicted isoforms [1,2,4–6]. No homologs have been identified in bacteria or yeast, suggesting the evolutionary need for a novel mechanosensor in higher organisms. Across species, Piezos are very large proteins (2521 and 2752 amino acids for human Piezo1 and human Piezo2, respectively) with numerous (>14) predicted transmembrane (TM) domains per subunit and, strikingly, no homology to other known proteins [1]. In near-physiological solutions, Piezos permeate cations with a single-channel conductance of approximately 29 pS and approximately 24 pS for mouse Piezo1 and mouse Piezo2, respectively [3,7]. In the presence of a constant stimulus, Piezo-mediated currents decay on a millisecond timescale 1Duke University Medical Center, due to a poorly understood mechanism, likely including channel inactivation (see Glossary), Department of Neurobiology, Durham, that is subject to genetic and physiological modulation (Box 1 and Figure 1). Piezos fulfill many NC 27710, USA requirements for true mechanically activated ion channels, as they are pore-forming subunits, confer mechanically activated currents when expressed in a heterologous system, and are *Correspondence: necessary for mechanical responses in many cells [8]. However, no one has yet demonstrated [email protected] (J. Grandl). Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tibs.2016.09.004 1 © 2016 Elsevier Ltd. All rights reserved. TIBS 1305 No. of Pages 15 (A) (B) Glossary Pipee Blunt probe Arthrogryposis: a family of disorders including distal arthrogryposis type 5, Negave pressure Indenon Gordon syndrome, and Marden– Walker syndrome. Patients with these + Piezo1 Na Na+ disorders all exhibit congenital joint Ca2+ 2+ Ca contractures (or abnormal stiffness of joints), but can be distinguished by other, specific symptoms. Channel inactivation: a process in which a channel initially opens in Cell-aached patch Whole-cell patch response to a stimulus but over time, despite the continued presence of 6 μm 0 mmHg the stimulus, ceases to conduct ions (closes). 0 μm Channelopathy: a disease caused (first contact) by dysfunction of an ion channel, –70 mmHg often resulting from a mutation in the channel gene. Congenital lymphatic dysplasia: a disease characterized by severe swelling, or lymphedema, in the 40 pA 1 nA limbs. Dehydrated hereditary 50 ms 50 ms xerocytosis: a disease characterized by dehydration of red blood cells, Pressure (mmHg) Displacement (μm) resulting in increased fragility of these 0 –10 –20 –30 –40 –50 –60 –70 0 1 2 3 4 5 6 cells and subsequent anemia. The 0 0 dehydrated cells have a cup shape, and are often referred to as –40 –1 stomatocytes. –2 Dorsal root ganglia neurons: –80 sensory neurons with afferents that Current (pA) Current (nA) –3 terminate in the spinal cord, and with –120 sensitivity to mechanical touch, temperature, and specific chemicals. (C) High-speed pressure clamp: an experimental device allowing precise Æ 8 μm poke ( 1 mmHg) and rapid ( 10 ms) control of pressure (both negative 2 and positive). It is used to mechanically stimulate a channel- +100 mV 1 containing membrane patch within a Current (nA) pipette during an electrophysiological –100 –50 50 100 recording (Figure 1). –100 mV –1 Voltage (mV) Hydrophobic mismatch: a difference in length between the 1 nA –2 hydrophobic segment of a protein 50 ms and the hydrophobic thickness of a membrane, a situation that results in energetically unfavorable exposure of Figure 1. Piezos Are Mechanically Activated Ion Channels. (A) Schematic of ‘stretch’ setup, in which negative hydrophobic protein residues to the suction is applied to a cell-attached patch with a high-speed pressure clamp through the patch pipette, stimulating only hydrophilic environment. those channels contained within the patch dome (above). Piezo1 peak current amplitudes initially rise with increasing Mechanically activated ion magnitudes of pressure before reaching saturation (middle). The pressure–response relationship can be fit with a sigmoidal channel: to fulfill this definition, a function to measure pressure sensitivity (below). Data are from J. Wu and J. Grandl, unpublished. (B) Schematic of ‘poke’ protein must form a channel, and setup depicting cell deformation by a blunt probe (typically a fire-polished glass pipette) during a whole-cell recording, which confer mechanically activated activates a larger population of channels throughout the cell (above). Piezo1 current amplitudes increase with increasing currents when expressed steps of displacement beginning a few micrometers beyond first contact of the probe with the cell membrane. From these heterologously. Further, to be experiments, a current–displacement curve can be generated. Typically, currents do not plateau before cell rupture (below). considered a mechanosensor in vivo, Data are from A. Lewis and J. Grandl, unpublished. (C) Voltage step protocol with a single ‘poke’ displacement during each the protein must additionally be step (left). A family of currents from a single cell illustrates the voltage dependence of channel inactivation, with severely expressed in mechanosensory cells, slowed decay times at positive voltages (middle). An I–V curve plotted from peak current amplitudes reveals a reversal and be necessary and sufficient for potential near 0 mV, demonstrating cationic nonselectivity (right). Data are from[4_TD$IF]A. Lewis and[5_TD$IF]J. Grandl, unpublished. 2 Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy TIBS 1305 No. of Pages 15 Box 1. Inactivation versus Adaptation mechanically activated currents in In the presence of a constant stimulus, Piezo currents decay with a characteristic time constant that could arise from two those cells. distinct mechanisms:[12_TD$IF] inactivation and adaptation (Figure I). Inactivation refers to a process where after initial response Mechanotransduction: the process and decay to a given stimulus, a further increase in stimulus intensity is not sufficient to elicit an increase in open by which a mechanical stimulus is probability; the stimulus must be completely removed and channels must deactivate in a time-dependent manner and transduced into biological signals return to a basal state, where they are available for new stimulation. By contrast, adaptation is a process where after an within a cell. initial response and decay to a given stimulus, a further increase in stimulus intensity increases open probability, even if no Membrane tension: the lateral (in- time is given for channels to recover. plane) force (N/m) in a membrane bilayer. Membrane tension has been demonstrated to be an activating For Piezo1, the contribution of adaptation was tested using the ‘stretch’ assay: after currents decayed in response to a stimulus for Piezo1 [44,45]. moderate pressure stimulus, only a small amount of additional current was elicited upon a step to a stronger pressure, Merkel cell: specialized skin cell which is consistent with inactivation as the process driving the loss of current [67]. Moreover, after removal of a stimulus, type that is sensitive to light both Piezo1

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