Localized force application reveals mechanically sensitive domains of Piezo1

ARTICLE Received 22 Feb 2016 | Accepted 16 Aug 2016 | Published 3 Oct 2016 DOI: 10.1038/ncomms12939 OPEN Localized force application reveals mechanically sensitive domains of Piezo1 Jason Wu1, Raman Goyal1 & Jörg Grandl1 Piezos are mechanically activated ion channels that function as sensors of touch and pressure in various cell types. However, the precise mechanism and structures mediating mechanical activation and subsequent inactivation have not yet been identified. Here we use magnetic nanoparticles as localized transducers of mechanical force in combination with pressure-clamp electrophysiology to identify mechanically sensitive domains important for activation and inactivation. 1 Duke University Medical Center, Department of Neurobiology, Durham, North Carolina 27710, USA. Correspondence and requests for materials should be addressed to J.G. (email: ). NATURE COMMUNICATIONS | 7:12939 | DOI: 10.1038/ncomms12939 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12939 P iezos are large (B2,500 aa.) proteins with 14–38 transmembrane domains that form mechanically activated ion channels1–4. They have been implicated in several biological processes involving mechanical sensing such as the sense of touch, proprioception and cardiovascular development5–10. Recent studies have demonstrated that lateral membrane tension is the physical stimulus that activates Piezo1, suggesting hydrophobic mismatch between the membrane bilayer and transmembrane domains as a possible mechanism of mechanical sensing11,12. However, Piezos are unrelated to other known ion channel families, and therefore, the precise mechanism that transduces mechanical force into pore opening (activation) and subsequently leads to pore closing (inactivation) is unknown. The macroscopic organization of Piezo1 has been revealed by cryo-electron microscopy, and a smaller (B230 aa.) extracellular domain of the C. elegans Piezo orthologue was resolved at the atomic level4,13. The C-terminal region contains the pore domain and is also highlighted by several disease-related single-point mutations that cause a slowing of inactivation kinetics2,14–16. However, further detailed links between structural domains and distinct modalities of channel function such as activation and inactivation have remained unresolved. Existing thermodynamic models of mechanical gating simplify channel structure to be homogenous and elastic and are thus limited in revealing structural features17. Here we aimed to advance our understanding of this concept, hypothesizing that specific structures within Piezos are highly sensitive to localized application of force, whereas others are less sensitive in comparison. We further reasoned that mechanical perturbation of such domains may induce changes in channel function. In this study, we introduce a method by which localized force is applied through magnetic nanoparticles to distinct domains of Piezo1. We identify two domains that are mechanically sensitive and affect pressure-dependent channel activation and inactivation. Results Localized force application by magnetic nanoparticles. To probe mechanical sensitivity of Piezo1 ion channels with submolecular resolution, we generated a highly localized pulling force to specific domains of Piezo1 by attaching superparamagnetic nanoparticles and exposing the complex to a magnetic field while measuring channel activity electrophysiologically. Specifically, we engineered constructs of Piezo1-IRES-EGFP that each contained a 13 amino-acid (aa.) bungarotoxin binding sequence (BBS) within a predicted extracellular domain, further referred to as Piezo1-BBS18. We first treated HEK293T cells expressing Piezo1-BBS constructs with biotinylated bungarotoxin, which binds to the BBS with high affinity (KdB15 nM)19. Next, we applied 75 nm diameter streptavidin-coated nanoparticles to the cells, which in turn bind the biotinylated bungarotoxin (KdB0.01 pM), linking the targeted domain to the nanoparticle (Fig. 1a). We reasoned that due to the comparatively large size, each Piezo1 channel can accommodate at most one single nanoparticle (Supplementary Fig. 1). To probe the specificity of nanoparticle labelling, we immunostained nanoparticles bound to cells transfected with Piezo1-BBS constructs or wild-type Piezo1, which does not contain any BBS, and compared their near-membrane fluorescence. All but two Piezo1-BBS constructs (those with BBS tags inserted at residue positions 1,201 and 2,075 (BBS-1201 and BBS-2075)) exhibited a fluorescence intensity that was at least two times higher as compared to the levels present on wild-type Piezo1 expressing cells and were used for further experiments (Fig. 1b; Supplementary Fig. 2). Then, to probe the efficiency of nanoparticle labelling, we labelled unoccupied binding sites of all Piezo1-BBS constructs with a fluorescently conjugated bungarotoxin either directly or after the 2 binding of nanoparticles. We observed for all constructs that prior nanoparticle labelling reduced fluorescence by 60–80% (Fig. 1c; Supplementary Fig. 3a,b). Finally, to probe for possible nanoparticle dissociation or internalization, we immunostained nanoparticle-labelled cells transfected with one representative construct (BBS-2422) at various time points after labelling. We observed consistent fluorescence intensity (1.04±0.08 a.u.) along the membrane over a time period of at least 1.5 h (Fig. 1d). We therefore concluded that labelling of Piezo1-BBS constructs with magnetic nanoparticles was overall sufficiently specific, efficient and stable to be used as localized force transducers. We next probed by pressure-clamp electrophysiology if Piezo1-BBS constructs retained normal mechanical sensitivity, peak current amplitudes and inactivation kinetics as compared with wild-type Piezo1 (Fig. 1e; Supplementary Fig. 4a,b). The majority of the constructs retained channel properties similar to wild-type Piezo1. Only two constructs (BBS-2343 and BBS-2356 within one domain were non-functional, and for three other domains, we only obtained constructs with attenuated pressure sensitivity (P50) (BBS-1070 and BBS-1758) or altered inactivation kinetics (BBS-1070 and BBS-2329). We accommodated the rightward shift in P50 for BBS-1758 with a higher ranged pressure-step protocol (  50 to  120 mm Hg). We decided to study these constructs despite their altered function, because we reasoned that they might still be informative if external pulling force further alters channel function. Altogether, we created eleven functional and accessible constructs, covering eight of the nine individual extracellular loops that were previously identified by affinity-tag accessibility experiments2. Finally, we engineered an electromagnetic coil (magnetic field BB40 mT) with an iron-nickel alloy core needle tapered to a o10 mm tip to generate a focused magnetic field gradient and positioned it 54.7±5.5 mm above the tip of the patch pipette (Fig. 1f; Supple (...truncated)