Priest MF et al. (2021), Tracking the movement of discrete gating charge...

XB-ART-58623

Elife 2021 Nov 15;10. doi: 10.7554/eLife.58148.

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Tracking the movement of discrete gating charges in a voltage-gated potassium channel.

Priest MF , Lee EE , Bezanilla F .

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Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP).

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Species referenced: Xenopus laevis
Genes referenced: mapt
GO keywords: voltage-gated ion channel activity [+]

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	Figure 1. The basic principles of qBBr gating charge tracking in time.(A) Chemical structure of monobromo(trimethylammonio)bimane, a small positively charged (blue) fluorophore with the ability to conjugate to cysteine through a bromide group (yellow). (B) Size comparison of qBBr-Cys (top) to an arginine (bottom) (blue, nitrogen; red, oxygen; sulfur, yellow; gray, carbon). (C) Cartoon schematic of qBBr-tryptophan distance-based quenching with representative fluorescence data for unquenching (top), no effect (center), and quenching (bottom).Figure 1—figure supplement 1. Single molecule fluorescence trace simulations in comparison to macroscopic fluorescence traces.Simulated fluorescence traces at the single molecule scale (left) and the summed macroscopic fluorescence (right) for the tryptophan placement in an unquenching position (A), when tryptophan is at the peak of the barrier but cannot be detected (B), when tryptophan is at the peak of the barrier in the case of unlimited bandwidth (C) and placement in a quenching position (D).
	Figure 2. qBBr mimics a native gating charge.(A) Representative gating current traces of Shaker R1C-qBBr (top), R2C-qBBr (middle), and Shaker W434F (bottom). (B) Normalized charge (Q) versus voltage (V) QV curves of R1C (top, red circles, n = 4), R1C-qBBr (top, black circles, n = 4), R2C (bottom, red circles, n = 5), and R2C-qBBr (bottom, black circles, n = 3), compared to Shaker W434F (blue triangles both, n = 3). Data are shown as mean ± standard error of the mean (SEM). (C) Representative fluorescence traces of R1C-qBBr (top) and R2C-qBBr (bottom). In all figures, membrane potentials during the pulse are: brown, + 80 mV; red, +40 mV; orange, 0 mV; green, −40 mV; blue, −80 mV; black, −120 mV; purple, −160 mV. (D) Structure of an active state voltage sensor showing R1C-qBBr (stick model) with the position of the in silico mutation 454 from a tryptophan (left, green) to an alanine (right) (PDB: 3LUT) (Chen et al., 2010). In all Shaker structures, transmembrane domains (S1–S6) are colored: S1, white; S2, yellow; S3, red; S4, blue; S5 and S6, gray. (E) Representative fluorescence traces for R1C-qBBr:W454A (top) and R1C-qBBr:W454F (bottom). Figure 2—source data 1. Source data for normalized QVs in Figure 2B.Figure 2—figure supplement 1. R1C-qBBr:W454A and R2C-qBBr fluorescence signals are not produced by endogenous tryptophans or tyrosines.Representative fluorescence traces of (A) R1C-qBBr:W454A;W289F, (B) R1C-qBBr:W454A;Y323F, and (C) R2C-qBBr W289F;Y323F. In all panels, membrane potentials during the pulse are: brown, + 80 mV; red, +40 mV; orange, 0 mV; green, −40 mV; blue, −80 mV; black, −120 mV; purple, −160 mV. (D) Structure showing the positions of endogenous amino acids from A to C and the positions of R1 and R2 (PDB: 3LUT).
	Figure 3. An exogenously substituted tryptophan (E247W) produces voltage-dependent fluorescence changes in R1C-qBBr:W454A.(A) Homology structure of Shaker construct, R1C-qBBr:W454A;E247W demonstrating the tryptophan placement within the protein. (B) A representative activation family of fluorescence traces. (C) A normalized ΔF/F0 fluorescence versus voltage curve. Normalization was performed for each oocyte separately (n = 4). (D) A single exponential τ of fluorescence upon activation (n = 3). Data are shown as mean ± standard error of the mean (SEM). Figure 3—source data 1. Source data for normalized ΔF/F0 versus voltage in Figure 3C and single exponential τact of fluorescence in Figure 3D.
	Figure 4. Activation pathway of R1C-qBBr:W454A mapped by several individually substituted tryptophans.(A) Representative qBBr traces at +80 mV. As qBBr moves closer to a W, the W quenches (green) the qBBr fluorescence (Y415W and W454). When it moves further away from a W, the qBBr fluorescence is unquenched (pink; F244W, E247W, I320W, and T326W). Some W mutations have no effect (black; 289W, F290W, and L294W). (B) A summary structure of activation of R1C-qBBr, with side (left) and extracellular (right) views of the voltage-sensing domain (VSD). The activation pathway based on the W quenching/unquenching for R1C-qBBr is marked by a gray arrow.
	Figure 5. Activation pathway of R2C-qBBr mapped by several individually substituted tryptophans.(A) Representative qBBr traces at +80 mV. As qBBr moves closer to a W, the W quenches (green) the qBBr fluorescence (T326W, Y415W, and F416W). When it moves further away from a W, the qBBr fluorescence is unquenched (pink; I241W, F244W, I287W, and L294W). Residue W289 (black) does not affect the fluorescence. (B) A summary structure of activation of R2C-qBBr with side (left) and extracellular (right) views of the voltage-sensing domain (VSD). The activation pathway based on the W quenching/unquenching for R2C-qBBr is marked by a gray arrow.Figure 5—figure supplement 1. Effect of L294W in both R1C-qBBr:W454A and R2C-qBBr.Representative fluorescence traces of qBBr labeled (A) R1C-qBBr:W454A (black) and R1C-qBBr:W454A;L294W (red) at +80 mV. (B) R2C-qBBr (black) and R2C-qBBr;L294W (red) at +40 mV.
	Figure 6. Voltage-sensing domain (VSD) deactivation transitions are followed by qBBr.(A) Activation (purple) and deactivation (red) protocols. Shading indicates where gating and fluorescence were measured for (B–D). (B) Comparison of activation (purple triangles) and deactivation (red circles) QVs for R1C-qBBr:W454A;E247W (left, activation n = 4, deactivation n = 9), R1C-qBBr:W454A;I320W (center, activation n = 4, deactivation n = 5), and R1C-qBBr:W454A;T326W (right, activation n = 5, deactivation n = 5). (C) As in (B), but a comparison of activation and deactivation FVs, rather than QVs. (D) Comparison of single exponential τact (red circles, n = 3) to τdea (purple triangles, n = 4) of R1C-qBBr:W454A;E247W (left). Weighted τact (red circles, n = 3) to τdea (purple triangles, n = 5) of R1C-qBBr:W454A;I320W (center). Weighted τact (red circles, n = 3) to τdea (purple triangles, n = 3) of R1C-qBBr:W454A;T326W (right). Data are shown as mean ± standard error of the mean (SEM). Figure 6—source data 1. Source data for normalized QVs in Figure 6A, normalized FVs in Figure 6B, and single or weighted τ of fluorescence in Figure 6C.Figure 6—figure supplement 1. The voltage-sensing domain (VSD) deactivation path of R2C-qBBr differs from that of activation.(A) Activation (purple) and deactivation (red) protocols. Shading indicates where gating and fluorescence were measured for (B–D). (B) Comparison of activation (purple triangles) and deactivation (red circles) QVs for R2C-qBBr:F244W (left, act n = 4,deact n = 4), R2C-qBBr:I287W (center, act n = 4, deact n = 5), and R2C-qBBr:Y415W (right, act n = 3, deact n = 3). (C) As in (B), but a comparison of activation and deactivation FVs, rather than QVs. (D) Comparison of single exponential τact (red circles, n = 6) to τdea (purple triangles, n = 3) of R2C-qBBr:F244W (left), weighted τact (red circles, n = 8) to τdea (purple triangles, n = 4) of R2C-qBBr:I287W (center), and weighted τact (red circles, n = 4) to τdea (purple triangles, n = 6) of R2C-qBBr:Y415W (right). Data are shown as mean ± standard error of the mean (SEM). Figure 6—figure supplement 1—source data 1. Source data for normalized QVs in Figure 6—figure supplement 1A, normalized FVs in Figure 6—figure supplement 1B, and single or weighted τ of fluorescence in Figure 6—figure supplement 1C.
	Figure 6—figure supplement 2. R1C-qBBr fluorescence visualizes the relaxed state.(A) A relaxation pulse protocol. (B) Comparison of activation (purple, triangles) and derelaxation (black, diamonds) QVs for R1C-qBBr:W454A;E247W (left, activation n = 4, derelaxation n = 4) and R1C-qBBr:W454A;T326W (right, activation n = 5, derelaxation n = 4). (C) Inverted FVnormalized and FVnormalized for activation and derelaxation for R1C-qBBr:W454A;E247W (left, activation n = 8, derelaxation n = 6) and R1C-qBBr:W454A;T326W (right, activation n = 5, derelaxation n = 3). Data are shown as mean ± standard error of the mean (SEM). Figure 6—figure supplement 2—source data 1. Source data for normalized QVs in Figure 6—figure supplement 2B and normalized FVs in Figure 6—figure supplement 2C.
	Figure 7. R1C–qBBr interaction with F290 provides the basis of the Cole–Moore shift.(A) A family of fluorescence traces for R1C-qBBr:W454A;F290W from a pulse protocol as in (6A). Note the slow fluorescence response when hyperpolarized to −160 mV (purple) and the residual signals during depolarizations. (B) Two pulse protocols that induce a Cole–Moore shift: a variable voltage pulse (−80 to −160 mV, every −20 mV) before a depolarization (top) or a prolonged hyperpolarization pulse (5, 1000, and 3000 ms) before depolarization (bottom) and the resulting Cole–Moore shifts of representative Shaker ionic currents (right). (C) Representative normalized fluorescence traces for R1C-qBBr:W454A;F290W using the pulse protocol from (B, bottom),with a −160 mV prepulse with a variable duration for 80 ms (red), 1 s (blue), and 5 s (green). (D) Comparison of the τweighted of the change in qBBr fluorescence in (C) (80 ms pulses, n = 4; 1 s pulses, n = 5; 5 s pulses, n = 4). Data are shown as mean ± standard error of the mean (SEM). Figure 7—source data 1. Source data for weighted fluorescence τ in Figure 7D.Figure 7—figure supplement 1. R1C-qBBr:W454A;F290W hyperpolarization-induced gating charge and fluorescence signal time course.Data are taken from a pulse from −120 to −160 mV. The integrated gating current of all four charges moving through the electric field (gating charge, Q, black) is more rapid than the hyperpolarization-induced fluorescence signal of R1C-qBBr:W454A;F290W, shown as the single exponential fit to the inverted fluorescence signal (purple). The fluorescence signal only follows the movement of the most extracellular gating charge (R1C-qBBr). Traces are aligned to each other, but the fluorescence signal is still slower than the gating charge.
	Figure 8. Voltage-sensitive membrane protein CiVSP is interrogable by qBBr.(A) CiVSP structure (PDB: 4G7V) with qBBr attached at R1C and highlighting residue Y206. Transmembrane domains (S1–S4) are colored with S1, green; S2, blue; S3, orange; S4, red. (B) Representative fluorescence traces of CiVSP R217Q R1C-qBBr (top) and CiVSP R217Q R1C-qBBr:Y206A (bottom). (C) Normalized QV curves comparing CiVSP R217Q R1C-qBBr (red circles, n = 4) and CiVSP R217Q R1C-qBBr:Y206A (blue squares, n = 3). Data are shown as mean ± standard error of the mean (SEM). Figure 8—source data 1. Source data for normalized QV in Figure 8C.
	Figure 9. Fluorescence kinetics of qBBr.(A) Comparison of the single exponential tau of fluorescence of R1C-qBBr (red squares) to the gating ττfast (black triangles) and the gating τslow (open circles) obtained from a double exponential fit to the gating charge (n = 4). (B) As in A, but for R1C-qBBr:W454A;E247W (n = 4). Figure 9—source data 1. Source data for fluorescence and gating τ in Figures 9A and 9B.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase