Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain

Fig. 1. Kv3.1a full-length structure showing alternative T1 arrangement compared to Kv1.2/2.1 and electrostatic ATM/T1 interactions. a Density maps of the human Kv3.1a channel, coloured by chain. TM region is illustrated by dashed lines. b Superposition of a single chain of Kv3.1a (dark blue cartoon) with one protomer of the Kv1.2-2.1 paddle chimera (pdb: 6EBK, shown in yellow cartoon representation), aligning the S4/S5 linker and pore domain. c Cytoplasmic view of the T1 domain of Kv3.1a superposed on the Kv1.2/2.1 structure (yellow cartoon) using the same alignment as in (b). Angle indicates rotational displacement measured between a vector defined by cα atoms of G16/H22 in Kv3.1 and G41/Q47 in Kv1.2-2.1, respectively. d Superposition of an individual T1 protomer (residues 8–120, dark blue) from Kv3.1 with the respective region of the Kv1.2-2.1 protomer (yellow). e Detailed view of the ATM/T1 contact region. T1 is shown as semi-transparent surface representation/cartoon and the ATM motif C-terminal from S6T is shown as red cartoon. f Close-up view of the inset from e, illustrating electrostatic contacts between acidic residues in α5 of the N-terminal T1 domain and a cluster of lysines…

Fig. 2. Electrostatic surface properties and location of disease mutations. a Electrostatic surface representation of the Kv3.1a tetramer with a single protomer shown as cartoon, highlighting the relative arrangement of VSD (blue), S4/S5 linker (magenta) and α6 (teal) of the T1 domain. Lipids are shown in brown space-fill representation. The surface is coloured by electrostatic potential (red, −5 kT e−1; blue, +5 kT e−1). b Enlarged view of the Kv3.1 tetramer in full surface representation, highlighting electrostatic attraction between negatively charged α5 and α6 in T1 (red surface areas) and positively charged ATM (blue surface areas). Two lipid-binding sites are shown: site I between S4 and S4/S5 linker and site II at the interface between VSD and PD. c Slab view of (a) with insets 1. highlighting residues involved in lipid interactions at site I, 2. the interfacial Zn2+ binding site in T1 and 3. the lower constriction in T1 with D81 as major player for negative surface potential and local K+ ion abundance. d Kv3.1a protomer in cartoon representation with cα atoms of residues implicated in disease mutations shown as spheres. Lipids are shown in stick representation.

Fig. 3. Voltage sensor domain arrangement and pore properties of Kv3.1a. a Cartoon representation of the VSD S1–S4 (S3 is omitted for clarity) in Kv31a, showing the relative positioning of the gating charges R1–R4 (blue labelled) in S4 (shown in yellow) with respect to conserved residues (F256 and E249) of the hydrophobic charge transfer centre (CTC), located in S2 (shown in light green). Blue circles highlight voltage-sensing gating residues located above the position of conserved F256 of the CTC, which is represented by the dotted red line. b Cartoon representation of the VSD arrangement in Kv1.2-2.1 (pdb 6EBK), highlighting that all four voltage-sensing charges R1–R4 (blue circles) are located above F233 compared to only three (R1–R3) in Kv3.1a shown in panel (a). c Plot of the Kv3.1a pore radius, as determined by MOLE77. Red dotted line marks the radius of hydrated K+. d Sequence of S4 residues for Kv3.1a (top) and Kv1.2-2.1 (bottom) with the main voltage-sensing R/Q residues within the conserved arginine-rich motif highlighted in blue. Blue boxes encircle voltage-sensing arginines which are located above the CTC. e Cross-membrane view of the Kv3.1a pore domain and selectivity…

Fig. 4. The α6 helix of the Kv3.1 T1 domain forms a second cuff below the S4/S5 linker, stabilized by inter- and intrasubunit interactions. a Cartoon representation of the Kv1.2-2.1 tetramer (light grey) with one protomer highlighted in dark grey. S4/S5 linker is represented in magenta, S6 is coloured in pale yellow and α6 of the T1 domain is shown in aquamarine blue. b Cartoon representation of the Kv3.1a tetramer, illustrating the unique orientation of α6 (teal blue) of the T1 domain. Colour-coding of other elements is the same as in (a). c Inset from b with an enlarged view of the α6 helix and its interactions with residues in the S4/S5 linker (magenta) and the lower gate formed by S6T (pale yellow). d Cytoplasmic view of the tetrameric “α6 gating cuff” (shown as teal cartoon), located below the gating machinery formed by S4/S5 (magenta) and S6T (yellow). Only helix α6 of T1 is shown for clarity. e Inset from d, detailing inter-subunit interactions between α6 helices from adjacent subunits and interactions between α6 and S6T. Zn2+ ions are shown as grey spheres.

Fig. 5. A cluster of Kv3-specific alanines allows three-way interactions between α6, S4/S5 linker and S6T and location of Kv3.1-specific methionines in S6T. a Cytoplasmic view onto S6T (pale yellow cartoon) of the lower gate in Kv3.1a, highlighting the close proximity to neighbouring helices S4/S5 and α6 of the T1 domain, which is possible due to the compact side chains of A115 and A118 (α6), A340 (S4/S5 linker) and A446 and A448 (S6T). The close contact allows the formation of a stabilizing salt bridge between D120 in α6 and K449 in S6T. b Rotated view from a, highlighting the abundance of Kv3-specific methionines in S6T (M430, M441, M447) and α6 (M107). S4/S5 linker is omitted for clarity. Red frame indicates the location variant M441L associated with EPM7. c Illustration of the hydrophobic surface created by aromatic S6T residues F439, Y442, and Y443 (displayed as semi-transparent surfaces) which cradles the S4/S5 linker (magenta) close to the PVP motif (green sticks) and the α6 helix in the T1 domain. F345 in S5 (slate blue) and M430 (lime green) from adjacent subunits form a sulfo-aromatic interaction. d Sequence alignment of the S6/S6T (left) and S4/S5 linker (right) regions of…

Fig. 6. Mutational and electrophysiological analyses of key residues involved in functional inter-domain interactions between α6, S4/S5 linker and S6T. a Normalized G–V curves (G/Gmax) of Kv3.1a WT and the indicated mutants. The symbols and error bars represent mean ± SEM (n = 9–19 oocytes), and the solid lines are the best fits assuming a first-order Boltzmann equation (see the “Methods” section). b Scatter graphs comparing of V0.5 values from WT and several Ala mutants. Each symbol represents a measurement from an individual oocyte, and the overlaid horizontal line represents the mean value. In each group, n = 9-19 oocytes. P values are indicated on the graph. c Normalized G–V curves for the indicated α6 mutations as described for panel (a). In each group, n = 10-11 oocytes. d Scatter graph for the indicated α6 mutations as described for panel (b). In each group, n = 10–11 oocytes. e Left: Overlay of representative whole-oocyte currents from Kv3.1a WT and the mutant H336A at 50 mV. These currents were evoked by the voltage protocol shown in the inset. Centre: Overlay of representative whole-oocyte currents from Kv3.1a WT and the mutant H336A while the repolarizing voltage is 0 mV. From…

Fig. 7. MD simulations of S4 voltage sensor movement during activation/deactivation reveal a correlation with E116/K449 salt bridge stability. a Cartoon representation of the S4–S5/S6T contact region in Kv3.1a, showing the positions of the R326/R332 H-bond and the salt bridge E116A/K449 between α6 in the T1 domain and S6T. b Comparison of the Z-position of the R4 dating charge relative to the CTC (boxplot) and the stability of the E116/K440 (violet bars) for each subunit under activating conditions (+300 mV). Data calculated from 750 ns (12,000 frames) following the movement of R4 Arginine (SI Fig. S15). The salt-bridge was assumed to form in frames with distance between the heavy atoms of the residues being <3.3 Å. Boxes span the interquartile range and whiskers extend up to 1.5 times this range. c, d Comparison of the Z-position of the R4 gating charge relative to the CTC (boxplot) and the stability of the E116/K440 (violet bars) for each subunit under deactivating conditions (−650 mV) from two independent trajectories. Data calculated from 750 ns (12,000 frames) following the movement of R4 arginine. The salt-bridge was assumed to form in frames with distance between the heavy atoms…

Fig. 8. Architecture of the turret domain of Kv3.1a and its interaction with the S1/S2 linker of the adjacent VSD. a Overview of the S5-PH linker region (“turret region”, shown in shades of blue) and the interface between pore helix PH (green cartoon) and S1/S2 linker of the VSD (surface representation and pink cartoon). b Inset I from a: Detailed view of histidines H381 and H383 (bold blue sticks) in the turret domain and their interactions with Q409 in S6 (pale yellow) and Y407 (orange sticks) near the selectivity filter of the pore domain. c Sequence alignments of the S5-PH linker (left) and the S1/S2 linker region (right) for human Kv3 family members and other human T1-containing Kv channels, highlighting the enlarged turret domain unique to the Kv3 subfamily and the location of conserved Kv3-specific histidines. Conserved residues are represented in bold letters. Coloured regions follow colour-coding in panels a, b, d. d Inset II from a: detailed view of the PD/VSD interface formed by residues at the N-terminal end of the S1/S2 linker of the VSD unique to Kv3 subfamily (pink ribbon) and their interactions with residues in the transition zone between the turret domain (light cyan)…

Fig. 9. Lipid binding site near the turret domain of Kv3.1a resembles known drug binding sites in other channels. a Kv3.1a residues in the pore domain (S6 in yellow, PH in green) involved in hydrophobic interactions with the fatty acid tails (black) of the lipid bound to the groove at the PD/VSD interface. Residues from the nearby S1/S2 linker are coloured in pink. K+ ions are represented as purple spheres. b Binding site for agonist AM-0883 (white) between S6 (yellow) and PH (green) of TRPC6 (pdb: 6UZ8).