Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor

Figures

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  Fig. 1. Main chain mutagenesis in a voltage-gated potassium channel voltage-sensor. a Structure of the voltage-sensing domain (VSD) of the Kv1.2/2.1 chimera (pdb 2R9R). The S4-helix is in pink with side-chains for conserved Arg and Lys residues shown as sticks. Right, sequence alignments of the S4 sequence. Residues mutated in Shaker are shown in red. The α-helical (red) and 310-helical (blue) regions in the Kv1.2/2.1 structure are shown above the alignment. b Structures of amino acids (aa) and α-hydroxy acids (ah): Vah hydroxyl-isovaleric acid, Lah 2-hydroxy-4-methylpentanoic acid, Fah 3-phenyl lactic acid, Iah 2-hydroxy-3-methylpentanoic acid. Right, these are ligated to the tRNAs (pdb 2ZNI) for nonsense suppression. Shaker cRNA containing a UAG stop codon at the suppression site is co-injected with an orthogonal amber (TAG) suppressor tRNA chemically ligated to an amino acid or α-hydroxy acid (red)

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  Fig. 2. Hydrolysis of α-hydroxy encoded channels. Western blot of crude membrane preps of oocytes injected as labeled. WT channels cannot be hydrolyzed by base (NaOH, see Methods), but V363Vah and V369Vah are cleaved at the ester bond with base addition. Full-length channels contain a conserved glycosylation site (G), producing a two-band pattern. Channels are detected with a C-terminal FLAG tag

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  Fig. 3. Functional nonequivalence of ester substitutions in the S4 segment. a Conductance–voltage (G–V) relationships for amino (WT, black) and hydroxyl acids (red) encoded at the indicated sites in S4. Solid lines represent Boltzmann fits to the data obtained with 5 mV steps. Insets show representative potassium current traces elicited with depolarizations from −80mV to +60 mV in 10 mV steps. Full-size currents and pdCpA-negative control traces can be found in Supplementary Figure 2. Scale bars equal 5 μA and 50 ms for current size and duration, respectively. b Free energy change on channel gating by site. All data shown in a, b are mean ± standard deviation from 3 to 13 oocytes. Data were repeated from at least two separate batches of cRNA and suppressor tRNA

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  Fig. 4. A backbone ester mutation between R3 and R4 alters the movement of S4. a Exemplar traces showing gating currents and capacitance in response to membrane depolarizations for Shaker W434F and W434F/V369TAG co-injected with valine acylated tRNA (top) as well as W434F/V363TAG and W434F/V369TAG co-injected with valine α-hydroxy tRNA (bottom). For each trace, voltages between −150 and 0 mV are displayed, with the exception of V369Vah/W434F, wherein −150 to +50 mV is shown. Scale bars denote 3 µA (Y-axis) and 5 ms (X-axis). b Q-V relationship for V363Vah/W434F (red) compared to either the W434F or the V363Val/W434F control. Gating Q was extracted from traces by subtracting the linear capacitance from the integrated signal from the OFF currents39, Supplementary Figure 5. c Same as in b, but comparing Q-V relationships between V369Vah/W434F and either W434F or the V369Val/W434F control. All data shown in b, c are mean ± standard deviation from four to eight oocytes. Note the shallowing of the curve generated by the ester mutation at position 369, as well as the two visible components of gating charge

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  Fig. 5. Simulations identify secondary structure sampling at the S4 transition region. a Helical content of the S4 segment for WT, V363Vah, and V369Vah shown on top, middle, and bottom panels, respectively. Main-chain structure at V363 and V369 highlighted by red boxes. Top, WT S4 maintains the α-helical (white) and 310 elements at the N- and C-termini, and highlights helical sampling at V369—the transition point. Middle, V363Vah recapitulates WT S4 structural elements. Bottom, V369Vah displays a localized loss of helical structure. b Representative snapshot images from the simulations for WT, 363Vah (left), and 369Vah. Main-chain structure is represented by the transparent cartoon overlay and main-chain H-bonds are indicated by red and green lines