Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation

Abstract

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.

Figure 1. BacNa_V CTD affects function, Related to Figure S1 and Table S1

Functional comparison of: A, Na_VAe1, Na_VSp1, Na_VAe1Δ_CTD, and Na_VSp1Δ_CTD; B, Na_VAe1_SpCTD and Na_VSp1_AeCTD; C, Na_VAe1_SpNeck and Na_VSp1_AeCC; and D, Na_VAe1Δ_CC and Na_VSp1Δ_CC. Cartoons depict two BacNa_V subunits. Voltage-sensor domain (VSD), pore domain (PD), Neck, and Coiled-coil (CC) are labeled. Na_VAe1 and Na_VSp1 elements are blue and orange, respectively. E, Voltage protocols for Na_VAe1 (top), Na_VSp1 (bottom), and chimeras. F, Na_VSp1_AeCTD/GGG exemplar currents. G-H, Voltage-dependent activation curves for: G, Na_VAe1, Na_VAe1_SpCTD, Na_VAe1_SpNeck, Na_VAe1Δ_CTD, and Na_VAe1Δ_CC, and H, Na_VSp1, Na_VSp1_AeCC, Na_VSp1Δ_CTD, and Na_VSp1Δ_CC. I, Western blot of the total lysate and surface-biotinylated fraction for the indicated constructs probed using the specified antibodies. See also Figure S1.

Figure 2. CTD chimeras alter Na_VSp1 voltage dependence, Related to Figures S1 and S2 and Table S1 and Table S2

A, Sequence alignment of Na_VAe1, Alkalimnicola erlichii (Shaya et al., 2014; Shaya et al., 2011), Na_VSp1, Silicibacter pomeroyi (Koishi et al., 2004; Shaya et al., 2011); Na_VAb1, Alcanivorax borkumensis (Shaya et al., 2011); Na_VBh1 (NaChBac), Bacillus halodurans (Ren et al., 2001); Na_VAb, Arcobacter butzleri (Payandeh et al., 2011); Na_VMs, Magnetococcus sp. (McCusker et al., 2012); and Na_VPz, Paracoccus zeaxanthinifaciens (Koishi et al., 2004) indicated regions. S6, Neck, Coiled-coil ‘a’–‘d’ positions, and 3G mutation site are indicated. B, NaSp1, Na_VSp1_BhCTD, Na_VSp1_MsCTD, Na_VSp1_Ab1CTD, Na_VSp1_PzCTD, and Na_VSp1_AbCTD exemplar currents and voltage protocol. Cartoons depict two channel subunits. C, Activation curves for ‘B’. D, Temperature dependence of Na_VSp1 activation. E, V_1/2 temperature dependence for the indicated channels. Lines show linear fit.

Figure 3. BacNa_V neck disruption structural outcomes, Related to Figures S3 and S4, and Table S3

Cartoons of two subunits of A, Na_VAe1p-3G (dark blue) and B, Na_VAe1p-7G (firebrick) structures. Grey dashes indicate regions lacking electron density. Residues defining the electron density limits are indicated. Selectivity filter is orange. C, Superposition of Na_VAe1p (orange) (Shaya et al., 2014), Na_VAe1p-3G (dark blue), and Na_VAe1p-7G (firebrick). Angle shows Na_VAe1p-7G coiled-coil displacement. Selectivity filter ion positions are shown. D, S6-CTD sequences for the indicated channels. Polyglycine positions are red. Regions lacking electron density are grey. Coiled-coil ‘a–d’ repeat is orange. E, and F, CD spectra for the indicated proteins at 4°C. G, Thermal denaturation curves for the indicated proteins.

Figure 4. Na_VAe1p EPR studies, Related to Figure S5

A, S6-CTD region sequences. Spin label positions, polyglycine substitutions and, Coiled-coil ‘a–d’ repeat are highlighted green, red, and orange, respectively. Na_VAe1p-3G residues lacking electron density are grey. B, Na_VAe1p cartoon of two subunits and spin label sites. Pore domain, Neck and Coiled-coil are slate, sand, and orange, respectively, Red and black boxes denote Neck and coiled-coil spin-label positions, respectively. Panels show DEER decays, distance distributions, and CW spectra. Asterisks indicate the new Na_VAe1p-3G long-range distances.

Figure 5. Na_VAe1p CTD structure, Related to Figures S6 and S7 and Table S3

A, Na_VAe1 sequence. Neck and Coiled-coil ‘a’–‘d’ core residues, His245 and Glu256 are highlighted. B, Na_VAe1p CTD cartoon showing core residues. Neck ion, ${\text{Cl}}_{\mathrm{N}}^{-}$, and Coiled-coil ion, ${\text{Cl}}_{\text{CC}}^{-}$ are indicated. S6, Neck and Coiled-coil are slate, sand, and orange, respectively C, Na_VAe1p CTD π-stack, ‘a’ and ‘d’ layers, and Coiled-coil ion site packing geometries. D–E, π-stack ion binding site D, Side and E, top views. His245, Trp246, and Neck ion, ${\text{Cl}}_{\mathrm{N}}^{-}$, are indicated F, Coiled–coil ion binding site. Glu257, Ala260, Arg264, and Coiled-coil ion, ${\text{Cl}}_{\text{CC}}^{-}$ are indicated. F, Na_VAe1_H245G, Na_VAe1_W246G, and Na_VAe1_H245G/W246G exemplar currents and voltage-dependence of activation and inactivation. Colors are as in Figure 4.

Figure 6. BacNa_V CTD couples to the intracellular gate, Related to Table S4

A, Na_VSp1, Na_VSp1_GGG, Na_VSp1_M220A, and Na_VSp1_M220A/GGG exemplar currents. B, Activation curves from ‘A’. C, Na_VSp1 S4 mutant sites. D, Na_VSp1 R1–R4 and R1–R4/GGG mutant exemplar currents. E, Voltage-dependent activation curves for the indicated mutants. Na_VSp1 (tan dashes) and Na_VSp1_GGG (black dashes) curves are shown for reference.

Figure 7. Function and structural conservation of VGIC superfamily CTD four-helix bundles, Related to Table S5

A, BacNa_V gating is coupled to a Neck unfolding transition. Purple circles indicate Neck and Coiled-coil ions. B, Na_VAe1p (left) and TRPA1 (Paulsen et al., 2015) (right) cartoon diagrams CTD four-helix bundle is indicated. C, Na_VAe1p and TRPA1 coiled-coil superposition. TRPA1 shading indicates regions corresponding to the Na_VAe1p neck (light grey) and coiled-coil (dark grey). D, Comparison of Na_VAe1p (left) and TRPA1 (right) CTD cores. TRPA1 CTD buried hydrophilic residues are Gln1047 and Gln1061. E, Na_VAe1p and TRPA1 CTD superhelix radii comparison. Colors are as in ‘C’. See also Table S5.