A hypothetical molecular mechanism for TRPV1 activation that invokes rotation of an S6 asparagine

Abstract

The transient receptor potential channel vanilloid type 1 (TRPV1) is activated by a variety of endogenous and exogenous stimuli and is involved in nociception and body temperature regulation. Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes that occur in the pore domain and result in ionic conduction have not yet been identified. Here we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves rotation of a conserved asparagine in S6 from a position facing the S4-S5 linker toward the pore. This rotation is associated with hydration of the pore and dehydration of the four peripheral cavities located between each S6 and S4-S5 linker. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, evaluate newly available structures, and reexamine previously reported water accessibility and mutagenesis experiments. These analyses provide several independent lines of evidence to support our hypothesis. Finally, we show that our proposed molecular mechanism is compatible with the prevailing theory that the selectivity filter acts as a secondary gate in TRPV1.

Figure 1.

A possible molecular mechanism for TRPV1 activation. The channel is shown in gray; water in the pore and peripheral cavities in cyan and blue, respectively; the conserved asparagine N676 in S6 in green; the membrane in purple (extra and intra denote extra- and intracellular solutions). In the capsaicin-bound closed state, the peripheral cavities host several water molecules, while the pore is partially dehydrated; N676 projects its side chain toward the peripheral cavities. Upon application of an activating stimulus, the peripheral cavities dehydrate, an event that correlates with the rotation of N676 toward the pore. The presence of a hydrophilic side chain inside the pore promotes its hydration and hence permeability to ions.

Figure 2.

π-helical segment in the S6 helix of TRP channels. (A) Cartoon representation of the TRPV1 pore domain (Cao et al., 2013b). The S6 helices are colored in green, and the rest of the pore domain in white. The inset shows the π-helical segment and the π-bulge located on the Y671 residue (highlighted with an arrow). The atoms are colored by element type: C in green, N in blue, O in red, and H in white. (B) Schematic representation of an α-helix (right) and an α-helix with a π-bulge (left). Note that the residues facing S5 in the α-helix are lining the pore when the π-bulge is introduced. (C) Per residue twist angle in the S6 helices of the available TRP structures estimated using HELANAL (Bansal et al., 2000). The structures with the π-bulge are shown in red (TRPV1, TRPV3, TRPV5, TRPV6, TRPA1, TRPM2, TRPM4, TRPM7, TRPC4, TRPP2, NOMPC, TRPML1, and TRPML3), and without the π-bulge in blue (TRPV2, TRPV3, TRPV4, TRPV5, TRPV6, TRPM8, TRPC3, and TRPP2). The structures in which S6 is distorted and cannot be assigned to either the α-helix or the α-helix with π-bulge are shown in green (TRPC3, TRPC6, and TRPP2). For the PDB codes of the structures, see Table 1. The twist angle is 99.4° and 86.6° for idealized α-helix and π-helix, respectively (highlighted by dashed black lines).

Figure 3.

Conserved asparagine adopts a range of conformations from pointing toward the pore to pointing toward the S4–S5 linker. (A) Sequence logos and per-position hydrophobicity plot of the S6 helix across the TRP family (numbering based on the TRPV1 sequence; Palovcak et al., 2015). In the sequence logo, the height of each residue at a given position is proportional to its frequency at this position, and the height of the overall stack of residues decreases linearly with Shannon entropy (Crooks et al., 2004). Aromatic residues (F, W, Y) are shown in orange; hydrophobic not aromatic (A, C, I, L, M, V) in black; hydrophilic (N, Q, S, T) in green; positively (H, K, R) and negatively (D, E) charged in blue and red, respectively; and G and P in purple. Note that in a few cases, the conserved asparagine can be substituted by a serine (TRPML channels) or glutamine (TRPN-like channel NOMPC). In the hydrophobicity plot, positions with positive $\Delta G$ are hydrophilic, and those with negative $\Delta G$ are hydrophobic. The error bars represent SD. The red shaded area highlights the residues lining the central cavity (between Y671 and I679). (B) Cartoon representation of the TRPV1 pore domain (Cao et al., 2013b). The S6 helices are shown in green, and the rest of the pore domain in white. The π-bulge and the neighboring conserved asparagine are colored in red and orange, respectively. (C) Orientation of the conserved asparagine with respect to the pore in different TRP structures (Cao et al., 2013b; Liao et al., 2013; Barad et al., 2015; Paulsen et al., 2015; Gao et al., 2016; Huynh et al., 2016; Saotome et al., 2016; Shen et al., 2016; Zubcevic et al., 2016, 2018; Chen et al., 2017; Grieben et al., 2017; Guo et al., 2017; Hirschi et al., 2017; Jin et al., 2017; Schmiege et al., 2017; Singh et al., 2017, 2018a,b; Winkler et al., 2017; Zhou et al., 2017; Autzen et al., 2018; Deng et al., 2018; Duan et al., 2018a,b; Fan et al., 2018; Hughes et al., 2018b,a; Hulse et al., 2018; McGoldrick et al., 2018; Su et al., 2018a,b; Vinayagam et al., 2018; Yin et al., 2018; Zhang et al., 2018; Zheng et al., 2018). The side chain position in the plane perpendicular to the pore axis is shown: the Cα-atom is represented as a black dot and is located at (0,0), and the terminal Cγ-atom (Cδ and Oγ in NOMPC and TRPML channels, respectively) is shown as a colored symbol (a different one for each TRP channel). The x-axis is aligned with the vector connecting the Cα-atom and the center of the pore. The red shaded area highlights the pore region. The three insets show the pore domains of the TRPV1 capsaicin-bound structure (1), the TRPV6 open structure (2), and TRPV2 (3): the conserved asparagine points toward the S4–S5 linker in TRPV2, points toward the center of the pore in the TRPV6 open structure, and lies just outside the pore in the TRPV1 capsaicin-bound structure. In TRPV6 and TRPV2 (the two extreme cases), the black arrows highlight the direction of the asparagine rotation between the conformations inside and outside the pore.

Figure 4.

Two alternative conformations of the S6 helix in the TRPV1 closed and open states. (A) Coupling between N676 and Y671. The top left panel shows Y671 orientation (the cosine of the angle between the Cα-Cγ vector and the pore axis) and the π-bulge position (the difference between the two distances: I672 carbonyl oxygen–N676 amine hydrogen and Y671 carbonyl oxygen–N676 amine hydrogen; positive values indicate the location of the π-bulge on I672, while negative ones indicate that the π-bulge is on Y671). Only two conformations are observed: open state (1), Y671 is perpendicular to the pore axis, and the π-bulge is on I672; and closed state (2), Y671 is parallel to the pore axis, and the π-bulge is on Y671. The bottom left panel shows the distance between N676 carboxamide carbon and I672 carbonyl oxygen and the π-bulge position. The energetically most favorable conformations are open state (1), a hydrogen bond between N676 carboxamide and I672 carbonyl oxygen is present, and the π-bulge is on I672; and closed state (2), the hydrogen bond between N676 carboxamide and I672 carbonyl oxygen is absent, and the π-bulge is on Y671. The right panel shows the cartoon representations of the two conformations. Y672 is colored in purple, the π-bulge in red, and N676 in orange. The dashed lines show the network of hydrogen bonds. (B) A change of Y671 orientation results in a displacement of the pore helix. The top panel shows the conformations of Y671 and of the adjacent pore helix in the open (left) and closed (right) states. The bottom panel shows the superposition between the pore helix in the open (solid) and closed (transparent) states.

Figure 5.

Accessibility of the S6 residues to the intracellular solution in the TRPV1 open and closed states. The open and closed states data are shown in orange and blue, respectively. The three top panels show the computational results, while the bottom panel shows the experimental data extracted and digitalized from (Salazar et al., 2009). In the simulations, water accessibility is calculated as the overlap between the atomic density of a given S6 residue and that of the intracellular water (the values shown correspond to a sum over all four channel subunits). The open state data correspond to the results obtained for the capsaicin-bound (CAP-bound) open state (Kasimova et al., 2018); the closed state data correspond to the results obtained for the capsaicin-bound closed state and the apo closed state (Liao et al., 2013; Kasimova et al., 2018). The box plots report median and interquartile range with the whiskers representing the SD. The threshold for water accessibility was chosen to be zero. In the experiments, accessibility to the intracellular solution is measured as an effect on current after application of a thiol-modifying reagent. The dashed lines highlight the hypothetical upper gate according to Salazar et al. (2009) and I679 (the gate at the level of the S6 bundle crossing).

Figure 6.

Mutagenesis of S6 residues lining the peripheral cavities. The residues mutated by Susankova et al. (2007) are shown in magenta, the conserved asparagine in orange, and the S6 helices and the rest of the pore domain in green and white, respectively. The peripheral cavity is highlighted with a blue circle.

Figure 7.

Structurally homologous and evolutionarily related families of voltage-gated sodium and calcium channels show a conserved asparagine in S6 and the presence of four hydrated cavities between S6 and the S4–S5 linker. (A) Sequence logos and per-position hydrophobicity plots of the S6 helix for voltage-gated sodium and calcium channels (Nav/Cav), bacterial voltage-gated sodium channels (NavBac), and TRP channels (TRP; Palovcak et al., 2015; Kasimova et al., 2016). In the sequence logos, the height of each residue at a given position is proportional to its frequency at this position, and the height of the overall stack of residues decreases linearly with Shannon entropy (Crooks et al., 2004). Aromatic residues (F, W, Y) are shown in orange; hydrophobic not aromatic (A, C, I, L, M, V) in black; hydrophilic (N, Q, S, T) in green; positively (H, K, R) and negatively (D, E) charged in blue and red, respectively; and G and P in purple. In the hydrophobicity plots, positions with positive $\Delta G$ are hydrophilic, and those with negative $\Delta G$ are hydrophobic. The error bars represent SD. The red shaded area highlights the residues lining the central cavity (between Y671 and I679). (B) Orientation of the conserved asparagine with respect to the pore in different Nav and Cav structures (Payandeh et al., 2011, 2012; Zhang et al., 2012; Tsai et al., 2013; Tang et al., 2014, 2016; Ahuja et al., 2015; Wu et al., 2015, 2016; Lenaeus et al., 2017; Shen et al., 2017; Sula et al., 2017; Yan et al., 2017; Irie et al., 2018). The side chain position in the plane perpendicular to the pore axis is shown: the Cα-atom is represented as a black dot and is located at (0,0), and the terminal Cγ-atom (Nζ in one of the NavPas subunits where the conserved asparagine is substituted for a lysine) is shown as colored symbols (different for each Nav/Cav channel). The x-axis is aligned with the vector connecting the Cα-atom and the center of the pore. The red shaded area highlights the pore region. The two insets show the pore domains of the NavMs structure (1, top and side views) and of the NavPas structure (2, top view). The conserved asparagine points toward the S4–S5 linker in NavMs and toward the pore in NavPas. In NavMs, the water molecules inside the cavity located between the S6 helix and the S4–S5 linker are shown in blue.