Research ArticleBiochemistry

Intracellular cavity of sensor domain controls allosteric gating of TRPA1 channel

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Science Signaling  23 Jan 2018:
Vol. 11, Issue 514, eaan8621
DOI: 10.1126/scisignal.aan8621
  • Fig. 1 Schematic of TRPA1 channel sensor domain.

    (A) Inner cavity (light blue funnel) formed by the lower part of the S1–S4 sensor domain and adjacent structures. S5-P-S6 refers to the central ion-conducting pore. The cavity regulates the gating of the channel (gray arrows) right in the center of the integrated nexus formed by the web of interactions between the transient receptor potential (TRP)–like domain (brown), pre-S1 helix (helix preceding S1), and S4-S5 linker (violet). Red asterisks indicate TRPA1-activating stimuli. (B) The positions of the cavity-facing polar residues. Amino acids are annotated by their single-letter abbreviation and residue number. (C) The HOLLOW script (53) was used for a “casting” of the inner cavity of TRPA1 (model constructed in this study; based on the TRPA1 3J9P structure and TRPP1 structure 5K47) and TRPV1 (5IRZ) by filling the voids with dummy atoms defined on a grid. The inner cavity of the sensor in TRP channels is predicted to be hydrated, and the extracellular part is tightly packed with bulky hydrophobic residues (16). Note the differences between the predicted hydration in TRPV1 and TRPA1 sensors. (D) Amino acid sequence conservation within the S2-S3 region of TRPA1, TRPV1, and TRPV2 proteins (222, 183, and 293 sequences) represented as sequence logo (54). Residues participating in phosphatidylcholine binding to TRPV1 (yellow triangles). (E) Sequence alignment of the human TRPP1 and TRPA1 channels used for the homology modeling. The identical, strongly conserved, and weakly conserved residues are denoted with asterisk, double dots, and single dot marks, respectively.

  • Fig. 2 Modeling the sensor domain of human TRPA1.

    (A) Model of the intracellular side of the S1–S4 sensor domain, which forms a hydrated cavity. The intracellular loop connecting helices S2 and S3 was modeled using sequence homology with TRPP1 (5K47, 5T4D, 5MKE, and 5MKF) as described in Materials and Methods. Residues His719, Asn722, Lys787, Lys796, Asp802, Asn805, Glu808, Arg852, and Lys989 were mutated in this study. (B) Schematic of electrostatic potential surrounding TRPA1 [Protein Data Bank (PDB) ID: 3J9P] and TRPV1 (PDB ID: 3J5P) structures was determined by means of visual molecular dynamics (41). Red mesh indicates a negative electrostatic potential in the selectivity filter and inside the pore. Compared to TRPV1, a notable feature of TRPA1 is that the positive electrostatic potential (blue mesh) permeates into the intracellular part of the S1–S4 sensor domain. (C) Model of the putative phosphatidylinositol 4,5-bisphosphate (PIP2) binding site on the S1–S4 sensor domain of TRPA1. Left: Homology model of the S1–S4 sensor domains of TRPA1. The negatively charged inositol trisphosphate head group of PIP2 contacts residues His719, Asn722, Lys787, Lys796, Arg852, and Lys989. Right: Four PIP2 molecules located in the four identical binding pockets (left) shown in the context of the template 3J9P structure completed with the model of the S1-S2 and S3-S4 linkers.

  • Fig. 3 Voltage-dependent gating of TRPA1 mutants.

    (A) Representative family of whole-cell currents recorded from human embryonic kidney (HEK) 293T cells transfected with either wild-type human TRPA1 channel (WT) or the indicated mutants elicited with a voltage step protocol consisting of 100-ms depolarizing pulses from −80 up to +200 mV in steps of 20 mV and a holding potential of −70 mV. The voltage protocol is shown in the top right panel. Bath solution contained 160 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, and 10 mM glucose (adjusted to pH 7.3 and 320 mosmol). The currents were recorded ~1 min after whole-cell formation. Steady-state currents were measured at the end of the pulses as indicated by colored symbols atop each record. (B to D) Average conductances obtained from recordings as in (A). Data are means ± SEM (n = 132 cells for wild-type and n = 6 to 30 cells for mutants from at least two independent transfections). The lines represent the best fit to a Boltzmann function for wild-type and mutant TRPA1 (gray and colored lines) using high-buffer (solid lines) or low-buffer intracellular solution [(LB-ICS); dashed lines]. (E) Deactivation kinetics of TRPA1 mutants. Averaged tail currents normalized to the maximum amplitude at +200 mV obtained as indicated by dashed box in (A, top left) for the wild-type channel. The average currents are shown with color bars indicating means ± SEM [number of cells indicated in (B) to (D)]. The gray lines with gray bars (SEM) represent the averaged tail currents obtained from data for wild-type TRPA1. Dashed lines indicate zero current. (F) The average time constants obtained from single exponential fits of tail currents. The asterisks indicate a significant difference from wild-type channels [**P < 0.001; n as in (B) to (D)]. (G) Summary of half-activation voltage (V50) and apparent number of gating charges (z) from experiments in (B) to (D). *P < 0.05; analysis of variance (ANOVA) on ranks followed by Dunn’s test versus WT.

  • Fig. 4 Mutations in the inner cavity of sensor module affect chemical-dependent gating of TRPA1.

    (A) Time course of average whole-cell currents induced by 100 μM allyl isothiocyanate (AITC) measured at +80 and −80 mV in HEK293T cells transfected with wild-type TRPA1 (open circles). Inset shows voltage-ramp protocol used for measuring currents. The cells were first exposed to the electrophilic agonist (AITC) in the absence of external Ca2+ using the bath solution containing 2 mM HEDTA. The agonist was then washed out for 10 s, and 2 mM Ca2+ was added to the extracellular solution as indicated above the current traces. Data are mean + SEM (open circles; n = 34 cells). In some cases, the error bars are smaller than the symbol. The dashed line represents the average currents obtained for WT using low-buffer intracellular solution (LB-ICS) (n = 11 cells). Zero current is indicated by the horizontal line. (B to F) Time course of average AITC-induced currents recorded from the indicated mutant channels using either high-buffer intracellular solution (circles) or LB-ICS (squares). Data are means + SEM (n = 6 to 13 cells). The average current for WT is overlaid as a gray line with gray bars indicating mean + SEM (high-buffer pipette solution) or dashed gray line representing the average current for WT obtained with LB-ICS. (G) Average whole-cell currents induced by 100 μM cinnamaldehyde (CA) in Ca2+-free solution and then exposed to 2 mM Ca2+ measured at +80 and −80 mV in WT. The application of CA and subsequent addition of 2 mM Ca2+ are indicated above. Data are mean + SEM (n = 45 cells). (H to L) Average currents recorded from mutant channels. The average current for the WT is shown as a gray line with bars indicating + SEM. Data are mean + SEM (n = 7 to 9 cells). In some cases, the error bars are smaller than the symbol.

  • Fig. 5 Mutations in internal sensor domain increase the sensitivity of TRPA1 and strengthen Ca2+-induced inactivation.

    (A to E) Time course of average cinnamaldehyde (100 μM)–induced currents recorded from HEK293T cells transfected with either wild-type TRPA1 channel (WT) or the indicated mutants. The overlaid gray line with gray bars represents the average currents + SEM obtained for wild-type TRPA1 as in Fig. 4G. Below each panel, average rectification of currents shown above (−current at −80 mV/current at +80 mV) plotted as a function of time. Colored symbols and lines with gray bars indicate means + SEM (n = 45 cells for WT and n = 7 to 9 cells for mutants). (F) Bottom view of the inner cavity of TRPA1 sensor domain. (G) Representative current traces from E788I in response to voltage step protocol shown in Fig. 3A recorded in control extracellular solution containing 1 mM Ca2+ (left; indicated above as 1 mM [Ca2+]o) or in Ca2+-free bath solution (right; indicated above as 0 mM [Ca2+]o). Bottom: Averaged tail currents recorded from cells expressing E788I (lines with yellow and pink bars indicating means ± SEM; n = 20 and 16 cells, respectively) and WT (superimposed gray lines with gray bars indicating means ± SEM; n = 132 and 10 cells, respectively). (H to I) Average conductances obtained from HEK293T cells transfected with the indicated mutants compared with WT human TRPA1 channel. The currents elicited with a voltage step protocol were measured at the end of the pulses as indicated by colored circles atop the traces in (G). The average conductance obtained from the WT in control extracellular solution (n = 132 cells) is shown as a gray line. Data are means ± SEM for WT measured in Ca2+-free bath solution (open circles; n = 10 cells) and for N805A and Y799A recorded in control extracellular solution containing 1 mM Ca2+ (colored circles; n = 15 and 13 cells, respectively). Solid lines are best fits to a Boltzmann function. (J to L) Time course of average CA-induced currents through indicated mutants measured at +80 and −80 mV. The average current for the WT is shown as a gray line with bars indicating means ± SEM (n = 45 cells for WT and n = 8 cells for each of the mutants). Bottom: Mean rectification ratio for the cells shown above (colored lines with gray bars indicating means + SEM) plotted against time.

  • Fig. 6 Proposed mechanism for regulation of human TRPA1 by the inner cavity of sensor domain.

    The diagram of the transmembrane part of the TRPA1 channel only shows two of the four subunits for clarity. The sensor domains (S1–S4; green) are connected through the S4-S5 linkers (lilac) to the pore domains (S5 and S6; gray). The TRP-like domain (brown) interacts with the S4-S5 linker through hydrophobic interactions. The inner cavities of the sensor domains are shown as a light blue cone for the wild-type channel. Sodium and calcium ions (blue and red circles) are indicated. (A) In the absence of external Ca2+, the cavity can be occupied by a phospholipid (PIP2; lipids with yellow and red phosphate head groups) to enable proper gating at negative membrane potentials. (B and C) When the mutations (K787A, H719A, N722A/I, and R852A) prevent the stabilization of the sensor by phospholipids (indicated with a white cone), the channel tends to be closed (B), whereas mutations (E788A and E808A) with putative beneficial effects on phosphoinositide binding (indicated with a dark blue cone) cause an increase in currents in the absence of external Ca2+ (C). (D) Any alteration to the polarity balance deep in the cavity causes a Ca2+-dependent block of currents elicited by electrophilic agonists.

  • Fig. 7 Sequestering or reducing membrane PIP2 inhibits voltage- and cinnamaldehyde-induced TRPA1 currents.

    (A to D) Top: Average conductances obtained from HEK293T cells transfected with the indicated protein combinations measured in control bath solution containing 160 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, and 10 mM glucose (adjusted to pH 7.3 and 320 mosmol). The solid lines of the WT (gray) or E808A mutant (brown) are the best fits to a Boltzmann function, as described in Materials and Methods and shown in Fig. 3 (B and D). Data are means ± SEM (n = 7 to 13 cells from at least two independent transfections). Bottom: The time course of average currents induced by cinnamaldehyde (100 μM) and by external Ca2+ (2 mM) measured at +80 and −80 mV, as described in Fig. 4G. The average current through TRPA1 expressed alone is overlaid as a gray line, with bars indicating SEM (n = 45 cells) for comparison. A schematic model of the mechanism is indicated for each “pipmodulin” above. PIP2 molecules are indicated as lipids with yellow and red phosphate head groups. Data are means ± SEM (n = 7 to 11 cells from at least two independent transfections). Bottom: Average rectification of currents shown above expressed as absolute values of the amplitudes of inward currents at −80 mV divided by outward currents at +80 mV and plotted as a function of time. The average rectification of TRPA1 expressed alone is overlaid as a gray line with gray bars indicating SEM (n = 45 cells) for comparison. MARCKS, myristoylated alanine-rich C-kinase substrate; GAP43, growth-associated protein 43. (E) The same experiments described in (A) to (D) performed with TRPA1 coexpressed with voltage-sensitive lipid 5-phosphatase from Danio rerio (Dr-VSP). The voltage step protocol was preceded by a 2-s depolarization to +80 mV. Colored symbols and lines with gray bars indicate average ± SEM (n = 7 to 9 cells).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/514/eaan8621/DC1

    Fig. S1. Mutations in the inner cavity of the sensor module affect chemical-dependent gating of TRPA1.

    Fig. S2. Average rectification of whole-cell currents through the mutant channels.

    Fig. S3. Structural comparison with TRPV2 and a central role for Tyr726.

    Fig. S4. LB-ICS containing Ca2+ abolishes the inhibitory effects of MARCKS and mutant GAP43.

    Fig. S5. Polar residues in the sensor cavity regulate the activity of TRPA1 under physiological temperatures.

  • Supplementary Materials for:

    Intracellular cavity of sensor domain controls allosteric gating of TRPA1 channel

    Lucie Zimova, Viktor Sinica, Anna Kadkova, Lenka Vyklicka, Vlastimil Zima, Ivan Barvik, Viktorie Vlachova*

    *Corresponding author. Email: viktorie.vlachova{at}fgu.cas.cz

    This PDF file includes:

    • Fig. S1. Mutations in the inner cavity of the sensor module affect chemical-dependent gating of TRPA1.
    • Fig. S2. Average rectification of whole-cell currents through the mutant channels.
    • Fig. S3. Structural comparison with TRPV2 and a central role for Tyr726.
    • Fig. S4. LB-ICS containing Ca2+ abolishes the inhibitory effects of MARCKS and mutant GAP43.
    • Fig. S5. Polar residues in the sensor cavity regulate the activity of TRPA1 under physiological temperatures.

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    Citation: L. Zimova, V. Sinica, A. Kadkova, L. Vyklicka, V. Zima, I. Barvik, V. Vlachova, Intracellular cavity of sensor domain controls allosteric gating of TRPA1 channel. Sci. Signal. 11, eaan8621 (2018).

    © 2018 American Association for the Advancement of Science

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