Structural basis for conductance through TRIC cation channels

Abstract

Mammalian TRICs function as K⁺-permeable cation channels that provide counter ions for Ca²⁺ handling in intracellular stores. Here we describe the structures of two prokaryotic homologues, archaeal SaTRIC and bacterial CpTRIC, showing that TRIC channels are symmetrical trimers with transmembrane pores through each protomer. Each pore holds a string of water molecules centred at kinked helices in two inverted-repeat triple-helix bundles (THBs). The pores are locked in a closed state by a hydrogen bond network at the C terminus of the THBs, which is lost when the pores assume an open conformation. The transition between the open and close states seems to be mediated by cation binding to conserved residues along the three-fold axis. Electrophysiology and mutagenesis studies show that prokaryotic TRICs have similar functional properties to those of mammalian TRICs and implicate the three-fold axis in the allosteric regulation of the channel.

Figure 1. Sequence analysis and characterization for the TRIC proteins.

(a) Structure-based sequence alignment of prokaryotic TRICs from SaTRIC, bacterial CpTRIC, and human TRIC-A and TRIC-B. The structures of both SaTRIC and CpTRIC have been used to restrict sequence gaps to inter-helical segments. Superior coils define extents of the helical segments; boxes are drawn for the highly conserved GGG signature motifs; red letters mark residues in the prokaryotic TRIC that are involved in the ion conduction pathway; and the coloured inferior bar encodes ConSurf sequence variability for the prokaryotic TRIC of 100 non-redundant proteins (top) and eukaryotic TRIC of 169 non-redundant proteins (bottom). (b) Crosslinking of purified SaTRIC and CpTRIC in detergent micelles. The purified proteins were incubated with increasing amounts of glutaraldehyde, and samples were analysed by SDS–PAGE. SaTRIC showed gradually increasing crosslinked trimeric species with increasing glutaraldehyde, whereas CpTRIC mainly maintained a trimeric state, even in the absence of glutaraldehyde and the presence of SDS. MW, molecular weight. (c) Electron density map of SaTRIC at 1.6 Å resolution. The initial phases were determined at 3.1 Å by Se-SAD, and further extended to 1.6 Å into the type 2a crystal (with a space group of P6₃) by molecular replacement. A section of the experimental map is superimposed onto the SaTRIC model as refined at 1.6 Å resolution, contours are at 2.0σ.

Figure 2. Crystal structure of prokaryotic TRICs.

(a) Ribbon drawing of the SaTRIC trimer, as viewed from outside of the membrane. The colouring is spectral for each protomer, from dark blue at its N terminus to red at its C terminus. (b,c) Electrostatic potential at the solvent accessible contact surfaces as viewed from the extracellular side (b), as in a, and from the intracellular side (c), 180° from b. The contour level is at ±7 kT e⁻¹. Red is for negative potential and blue is for positive potential. (d,e) Ribbon drawing of the SaTRIC protomer, coloured as in a. Side chains of conserved residues F20 and F106 are shown as red stick. Top view (d), as viewed from outside of the membrane; front view (e), looking towards the three-fold axis from within the lipid bilayer, 90° rotation from d. Membrane boundaries were calculated by the Orientations of Proteins in Membranes (OPM) server. (f) Membrane topology diagram for prokaryotic TRICs; TM_1–3 constitutes N-THB and TM_4–6 constitutes C-THB. Spectral colouring is as in e. (g) Superimposition of the trimeric structure of SaTRIC (type 2a) and CpTRIC (Se-Met). SaTRIC protomers are coloured as in a, and CpTRIC protomers are coloured in grey. (h) Superimposition of the protomer structure of SaTRIC (type 2a) and CpTRIC (Se-Met). Stereo view of the superimposed C_α backbones, viewed as in e and coloured as in g.

Figure 3. The ion conduction pathway of prokaryotic TRIC.

(a) The pore-lining surface as computed by the programme HOLE is drawn into a ribbon model of the SaTRIC structure (type 2a). We used a simple van der Waals surface for the protein and the programme default probe radius of 1.15 Å. The pore at radius below 1.15 Å is shown in red and that above 2.3 Å is shown in purple, and the intermediate zone is in green. A yellow line through the channel marks the calculated centre line of the pore. (b) Stereo view of the C_α backbone of SaTRIC, oriented as in Fig. 2e. Two conserved residues, F20 (TM₁) and F106 (TM₄), were shown in red stick. Water molecules observed within the ion conduction pathway are shown as red sphere. Density contours are shown for the water molecules. (c) Ribbon drawing of SaTRIC protomer as in a, but with N-THB (TM_1–3) in salmon, C-THB (TM_4–6) in green and TM₇ in grey. The observed water molecules are shown as b. (d,e) Cross-section through the SaTRIC (type 2a). The models are viewed as c; surface conservation is shown in d and electrostatic potential is shown in e. (f) Ribbon drawing of C-THB in the locked state of SaTRIC type 2a. The inter-helices network of D99–R139–D140–Y155 is indicated. The GGG motif (TM₅) is shown in magenta. (g) Ribbon drawing of N-THB, in its unlocked state and oriented as for the C-THB. The corresponding symmetry mates are shown for comparison. The GGG motif (TM₂) is shown in magenta. (h) The superimposed N-THB and C-THB, coloured as in f,g. All membrane boundaries were calculated as Fig. 2e.

Figure 4. Characteristics of the ion conduction pathway.

(a) Ribbon diagram of SaTRIC protomer, with the kinked TM₂ (in salmon) and TM₅ (in green) helices, oriented as in Fig. 2e. (b) The kinked TM₂ (in salmon) and TM₅ (in green), with red stars marking the GGG motifs. (c) Hydrogen-bonding patterns for the kinked helices and associated water molecules from the ion conduction pathway. The GGG motif of TM₂ is in orange and GGG motif of TM₅ is in light green. Density contours are shown for the water molecules. (d) Ribbon diagram of SaTRIC protomer, viewed as in a and coloured by sequence conservation as in Fig. 1b. Two conserved residues, F20 (TM₁) and F106 (TM₄), are shown as stick. (e) A zoom view of the GGG motifs in the kinked TM₂ and TM₅, coloured as in d. Backbone-coordinated water molecules are shown as in c.

Figure 5. Ionic conductance measurements of the prokaryotic TRIC.

(a) Representative traces of single SaTRIC currents recorded from planar lipid bilayers at different voltages (210 mM KCl in both trans and cis solutions). (b) Single SaTRIC channel current–voltage relationship. Data are presented as mean±s.e.m. (n=6 for each point). (c) Relative cation permeability for SaTRIC, n=4 for each point. (d) Sub-conductance levels of the SaTRIC, as in asymmetrical solutions, but here with 210 mM KCl in cis chamber and 810 mM in trans chamber. (e) Representative current traces of single SaTRIC channels at +20 mV (210 mM KCl in both trans and cis solutions) from wild-type and mutant proteins. (f) Representative current traces of single SaTRIC channel at the asymmetrical solutions (210 mM KCl in cis and 810 mM KCl in trans chambers) from wild-type and mutant proteins. Interestingly, SaTRIC D99A mutant displays a downward current, whereas wild type and other mutants show an upward current, suggesting SaTRIC D99A is permeable to Cl⁻. (g) The intra-facial gate of the C-THB as locked by a complex H-bond interaction network involving residues tested by mutation. (h) Current amplitude for the wild-type and mutants of single SaTRIC channel at +20 mV holding potentials, n=4 for each group. (i) Open probabilities for the wild-type and mutants of single SaTRIC channel at +20 mV holding potentials (same condition as in e).

Figure 6. Gating of prokaryotic TRIC.

(a) Cross-section through the SaTRIC in closed state (type 2a); N-THB in salmon, C-THB in green and TM₇ in grey. The superimposed pore drawing is coloured as in Fig. 3a. (b) Cross-section through the SaTRIC in an open state (type 3); N-THB in violet, C-THB in cyan and TM₇ in grey. The superimposed pore drawing is coloured as in Fig. 3a. (c) The superimposition of SaTRIC structures in different states: closed state (type 2a) versus open state (type 3), coloured as in a and b. (d) The superimposition of closed- and open-state structures at the GGG-kinked helices. TM₂ above and TM₅ below. Colouring is as in a,b. (e) Zoom-up view for the superimposed C-THBs: locked state (type 2a) versus unlocked state (type 3), coloured as in a and b. The surrounding residues of the intra-facial gate are shown.

Figure 7. Ion binding and modulation of prokaryotic TRIC.

(a) Ribbon diagram of the SaTRIC trimer, coloured as in Fig. 1a but viewed from the cytosolic side. The conserved N144 residues along the three-fold axis of SaTRIC trimer (type 2a) are shown as stick, and bound Na⁺ ions (purple) and water molecules (red) are shown as spheres. (b) Corresponding molecular surface for a coloured by sequence conservation as in Fig. 1b. (c) A zoom view of the N144, Na⁺ ions and water molecules in the structure of SaTRIC (type 2a). Water molecules are shown as red spheres and bound Na⁺ ions are shown as purple spheres. Asn144 O_δ1 to Na⁺1, distance=2.4 Å; Na⁺1 to water molecules, distance=2.4 Å; Asn144 O_δ1 to Na⁺2, distance=2.5 Å; and Na⁺2 to water molecules, distance=2.2 Å. Density contours are shown for both water molecules and Na⁺ ions. (d) A zoom view of the N144, Mg²⁺ ions and water molecules in the structure of SaTRIC (type 2b). Water molecules as in c, and Mg²⁺ is shown as green sphere. Asn144 O_δ1 to Mg²⁺, distance=2.1 Å; and Mg²⁺ to water molecules, distance=2.3 Å. Density contours are shown for both water molecules and Mg²⁺ ion. (e) Close-up of bottom view of the N144 in the Na⁺ bound structure of SaTRIC (type 2a). Bond distances are indicated. (f) Close-up of bottom view of the N144 in the ion-free structure of SaTRIC (type 3). Bond distances are indicated.