Inactivation Kinetics and Mechanical Gating of Piezo1 Ion Channels Depend on Subdomains within the Cap

Amanda H Lewis, Jörg Grandl, Amanda H Lewis, Jörg Grandl

Abstract

Piezo1 ion channels are activated by mechanical stimuli and mediate the sensing of blood flow. Although cryo-electron microscopy (cryo-EM) structures have revealed the overall architecture of Piezo1, the precise domains involved in activation and subsequent inactivation have remained elusive. Here, we perform a targeted chimeric screen between Piezo1 and the closely related isoform Piezo2 and use electrophysiology to characterize their inactivation kinetics during mechanical stimulation. We identify three small subdomains within the extracellular cap that individually can confer the distinct kinetics of inactivation of Piezo2 onto Piezo1. We further show by cysteine crosslinking that conformational flexibility of these subdomains is required for mechanical activation to occur and that electrostatic interactions functionally couple the cap to the extensive blades, which have been proposed to function as sensors of membrane curvature and tension. This study provides a demonstration of internal gating motions involved in mechanotransduction by Piezo1.

Keywords: Piezo1; Piezo2; activation; gating; inactivation; mechanosensitive ion channel; mechanotransduction.

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. A Chimeric Screening Strategy Reveals…
Figure 1.. A Chimeric Screening Strategy Reveals Subdomains of Piezo1 Required for Slow Inactivation Kinetics
(A) Left: structural model of full-length trimeric mouse Piezo1 (PDB: 6B3R) viewed from the side (top) and from the top (bottom). Right: ribbon model of one monomer of the C-terminal extracellular domain (cap) and inner helix (IH) with subdomains containing distinct secondary structure highlighted according to the naming and color scheme in (B) is shown. Highlighted residues L2475/V2476 form a previously identified constriction site posited to be involved in inactivation (Zheng et al., 2019a), and K2479 confers voltage dependence of inactivation (Wu et al., 2017b). (B) Alignment of the cap (gray), IH (pink), and C-terminal domain (CTD) (blue) of mouse Piezo1 (wtP1; residues 2,214–2,457) and mouse Piezo2 (wtP2; residues 2,497–2,731). Poorly conserved regions of the cap with distinct secondary structure are colored and correspond to structural subdomains in (A). Residues L2475, V2476, and K2479 are highlighted in bold lettering. (C) Mechanical indentation protocol (8 mm; top), voltage protocol (middle), and representative whole-cell current (bottom) from a HEK293t-P1Ko cell transiently transfected with wtP1. Red lines indicate single exponential fits to the current decay; inset shows current responses at negative voltages at higher magnification. (D) Mean inactivation time constants from single exponential fits to currents from 4–18 individual cells transfected with P1, P2, and chimeric channels, as a function of voltage. (E) Mean inactivation time constants at +90 mV and −90 mV for P1, P2, and chimeric constructs. Each symbol represents one individual cell. Constructs P1_bP2 and P1_dP2 were non-functional (n.f.). All data are mean ± SEM. See also Figure S1.
Figure 2.. Cysteine Crosslinking of Two Subdomains…
Figure 2.. Cysteine Crosslinking of Two Subdomains at Base of Piezo1 Cap Prohibits Channel Gating
(A) Schematic depicting whole-cell recording setup with mechanical indentation stimulation and gravity perfusion. (B) Indentation stimulus protocol (5 μm, top), voltage protocol (middle), and representative whole-cell current (bottom) from a HEK293t-P1Ko cell transiently transfected with wtP1 in with (black) and without (gray) 10 mM DTT in the bath. (C and D) Magnification of indentation stimulus protocol and currents from (B) at −80 mV (C) and +80 mV (D). (E) Structural model of Piezo1 cap highlighting cysteine pair A2328C and P2382C. Colors indicate three subunits of Piezo1. (F) Indentation stimulus protocol (5 μm) and representative currents from a cell transfected with A2328C/P2382C at −80 mV (top) and +80 mV (bottom) with (black) and without (gray) 10 mM DTT in the bath. (G) Mean peak current from 8–11 individual cells, normalized to the average of the first four peak currents in DTT, for wtP1 and A2328C/P2382C, at −80 mV (top) and +80 mV (bottom). (H) Mean inactivation time constant (τ) from 8–11 individual cells for wtP1 and A2328C/P2382C at −80 mV (top) and +80 mV (bottom). All data are mean ± SEM. See also Figures S2 and S3.
Figure 3.. Cysteine Crosslinking between a Glutamate…
Figure 3.. Cysteine Crosslinking between a Glutamate Residue in the Piezo1 Cap and Two Arginine Residues in the Blade Prevents Channel Opening
(A) Structural model of Piezo1 cap highlighting cysteine pair R1762C and E2257C. Colors indicate three subunits of Piezo1. (B) Indentation stimulus protocol (3 μm) and representative currents from a cell transfected with R1762C/E2257C at −80 mV (top) and +80 mV (bottom) with (black) and without (gray) 10 mM DTT in the bath. (C) Mean peak current from 8–11 individual cells, normalized to the average of the first four peak currents in DTT, for wtP1 and R1762C/E2257C, at −80 mV (top) and +80 mV (bottom). (D) Mean inactivation time constant (τ) from 8–11 individual cells for wtP1 and R1762C/E2257C at −80 mV (top) and +80 mV (bottom). (E) Structural model of Piezo1 cap highlighting cysteine pair R1761C and E2257C. Colors indicate three subunits of Piezo1. (F) Indentation stimulus protocol (6 mm) and representative currents from a cell transfected with R1761C/E2257C at −80 mV (top) and +80 mV (bottom) with (black) and without (gray) 10 mM DTT in the bath. (G) Mean peak current from 9–11 individual cells, normalized to the average of the first four peak currents in DTT, for wtP1 and R1761C/E2257C, at −80 mV (top) and +80 mV (bottom). (H) Mean inactivation time constant (τ) from 9–11 individual cells for wtP1 and R1761C/E2257C at −80 mV (top) and +80 mV (bottom). All data are mean ± SEM. See also Figures S2, S4, and S5.
Figure 4.. Disruption of an Electrostatic Interaction…
Figure 4.. Disruption of an Electrostatic Interaction between the Blade and Cap of Piezo1 Promotes an Open State
(A) Structural model of Piezo1 (PDB: 6B3R) highlighting the region of a monomer of the blade (pink) and a different monomer of the cap (blue) with predicted salt bridge interactions and interacting residues shown as sticks. (B and C) Indentation protocol (B, 8 μm; C, 11 μm) designed to measure inactivation (top), voltage protocol (middle), and representative whole-cell current (bottom) from a HEK293t-P1Ko cell transiently transfected with wtP1 (B) and from a cell transfected with E2257K/E2258K/D2264K (C). (D) Mean inactivation time constants from single exponential fits to currents from 9–18 individual cells transfected with wtP1 (black circles) or E2257K/E2258K/D2264K (red squares), as a function of voltage. (E and F) Indentation protocol (E, 5 μm; F, 7 μm) designed to measure deactivation and representative whole-cell current from a HEK293t-P1ko cell transiently transfected with wtP1 and from a cell transfected with E2257K/E2258K/D2264K. Insets show negative voltages at higher magnification. (G) Mean fast deactivation time constants from single (−150 to −30 mV) or double (+30 to +150 mV) exponential fits to currents from 6–10 individual cells transfected with wtP1 (black circles) or E2257K/E2258K/D2264K (red squares), as a function of voltage. (H) Mean slow deactivation time constants from single (−150 to −30 mV) or double (+30 to +150 mV) exponential fits to currents from 6–10 individual cells transfected with wtP1 or E2257K/E2258K/D2264K, as a function of voltage. Dashed lines are inactivation time constants (as in D) for wtP1 (black) and E2257K/E2258K/D2264K (red), showing the overlap with the slow component of deactivation that emerges at positive potentials. (I–K) Two-step indentation recovery protocol (I, 3 μm; J, 3 μm; K, 9 μm) and representative whole-cell currents from HEK293t-P1ko cells transiently transfected with wtP1, wtP2, and E2257K/E2258K/D2264K. (L) Mean current recovery from 5–19 individual cells, calculated as peak current during the second pulse as a percentage of peak current during the first pulse. All data are mean ± SEM.

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